AU2011202966B2 - Expression enhancing intron sequences - Google Patents

Abstract

The invention relates to methods for the identification and use of introns with gene expression enhancing properties. The teaching of this invention enables the identification of introns causing intron-mediated enhancement (IME) of gene expression. The invention furthermore relates to recombinant expression construct and vectors comprising said IME-nitrons operably linked with a promoter sequence and a nucleic acid sequence. The present invention also relates to transgenic plants and plant cells transformed with these recombinant expression constructs or vectors, to cultures, parts or propagation material derived there from, and to the use of same for the preparation of foodstuffs, animal feeds, seed, pharmaceuticals or fine chemicals, to improve plant biomass, yield, or provide desirable phenotypes.

Description

Pool Section 29 Regulation 3.2(2) AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: Expression enhancing intron sequences The following statement is a full description of this invention, including the best method of performing it known to us: P11 1AHAU/0710 Expression enhancing intron sequences FIELD OF THE INVENTION The invention relates to methods for the identification and use of introns with gene ex pression enhancing properties. The teaching of this invention enables the identification 5 of introns causing intron-medlated enhancement (IME) of gene expression. The inven tion furthermore relates to recombinant expression construct and vectors comprising said IME-introns operably linked with a promoter sequence and a nucleic acid se quence. The present invention also relates to transgenic plants and plant cells trans formed with these recombinant expression constructs or vectors, to cultures, parts or 10 propagation material derived there from, and to the use of same for the preparation of foodstuffs, animal feeds, seed, pharmaceuticals or fine chemicals, to improve plant biomass, yield, or provide desirable phenotypes. BACKGROUND OF THE INVENTION The aim of plant biotechnology is the generation of plants with advantageous novel 15 properties, such as pest and disease resistance, resistance to environmental stress (e.g., drought), improved qualities (e.g., high yield), or for the production of certain chemicals or pharmaceuticals. Appropriate gene expression rates play an important role in order to obtain the desired phenotypes. The gene expression rate is mainly modulated by the promoter, additional DNA sequence located in the 5' untranscribed 20 and 5' untranslated region and the terminator sequences of a given gene. Promoters are the portion of DNA sequences located at the 5' end a gene which contains signals for RNA polymerases to begin transcription so that a protein synthesis can then pro ceed. Regulatory DNA sequences positioned in the 5' untranscribed region modulate gene expression in response to specific biotic (eg. pathogen infection) or abiotic (e.g. 25 salt-, heat-,. drought-stress) stimuli. Furthermore, other so called "enhancer' sequences have been Identified that elevate the expression level of nearby located genes in a po sition and orientation independent manner. Beside the elements located on the untranscribed regions of a gene (e.g. promoter, 30 enhancer), it Is documented in a broad range of organisms (e.g. nematodes, insects, mammals and plants) that some introns have gene expression enhancing properties. In plants, the inclusion of some introns in gene constructs leads to increased mRNA and protein accumulation relative to constructs lacking the intron. This effect has been termed "intron mediated enhancement (IME) of gene expression (Mascarenhas at at., 35 (1990) Plant Moi. Biol. 15:913-920). Introns known to stimulate expression in plants have been identified in maize genes (e.g. tubAl, Adhl, Shl, Ubl1 (Jeon et al. (2000) Plant Physiol. 123:1005-1014; Callis et al. (1987) Genes Dev. 1:1183-1200; Vasil at al. (1989) Plant Physiol 91:1575-1579; Christiansen at al. (1992) Plant Mol. Biol. 18:675 689]) and in rice genes (e.g. saff, tpi [McElroy st al. (1990) Plant Cell 2: 163-171; Xu at 40 al. (1994) Plant Physiol 106:459-467]). Similarly, introns from dicotyledonous plant genes like those from petunia (e.g. rbcS), potato (e.g. st-sl) and from Arabidopsis thalana (e.g. abq3 and pat1) have been found to elevate gene expression rates (Dean at al. (1989) Plant Cell 1:201-208; Leon at al. (1991) Plant Phyisiol. 95:968-972; Norris et al. (1993) Plant Mol Biol 21:895-906; Rose and Last (1997) Plant J 11:455-464). It 45 has been shown that deletions or mutations within the splice sites of an intron reduce gene expression, indicating that splicing might be needed for IME (Mascarenhas at al.

(1990) Plant Mol Biol 15:913-920; Clancy and Hannah (2002) Plant Physiol 130:918 929). However, that splicing per se is not required for a certain IME in dicotyledonous plants has been shown by point mutations within the splice sites of the pat1 gene from A.thaliana (Rose and Beliakoff (2000) Plant Physiol 122:535-542). 5 Enhancement of gene expression by introns is not a general phenomenon because some intron insertions into recombinant expression cassettes fail to enhance expression (e.g. introns from dicot genes (rbcS gene from pea, phaseolin gene from bean and the st/s-1 gene from Solanum tuberosum) and introns from maize genes (adhl gene the ninth 10 intron, hsp8l gene-the first intron)) (Chee et al. (1986) Gene 41:47-57; Kuhlemeier et a. (1988) Mol Gen Genet 212:405-411; Mascarenhas et al (1990) Plant Mol Biol 15:913 920; Sinibaldi and Mettler (1992) In WE Cohn, K Moldave, eds, Progress in Nucleic Acid Research and Molecular Biology, Vol 42. Academic Press, New York, pp 229-257; Vancanneyt et al 1990 Mol Gen Gent 220:245-250). Therefore, not each intron can be 15 employed in order to manipulate the gene expression level of alien genes or endogenous genes in transgenic plants. What characteristics or specific sequence features must be present in an intron sequence in order to enhance the expression rate of a given gene is not known in the prior art and therefore from the prior art it is not possible to predict whether a given plant intron, when used heterologously, will cause IME. 20 The introduction of a foreign gene into a new plant host does not always result in a high expression of the incoming gene. Furthermore, if dealing with complex traits, it is sometimes necessary to modulate several genes with spatially or temporarily different expression pattern. Introns can principally provide such modulation. However multiple 25 use of the same intron in one plant has shown to exhibit disadvantages. In those cases it is necessary to have a collection of basic control elements for the construction of appropriate recombinant DNA elements. However, the available collection of introns with expression enhancing properties is limited and alternatives are needed. 30 Thus, there is still a growing demand for basic control elements including promoters, regulatory sequences (e.g., inducible elements, enhancers) or intron sequences that have an impact on gene expression rates. It is therefore an objective of the present invention, to provide further examples of introns with expression enhancing properties. 35 This objective is achieved by the methods and new introns provided within this invention. 2 SUMMARY OF THE INVENTION The invention relates to a recombinant DNA expression construct comprising at least one promoter sequence functioning in plants cells, at least one nucleic acid sequence and at least one intron selected from the group consisting of the sequences described by SEQ 5 ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22, and functional equivalents thereof, wherein said promoter sequence and at least one of said intron sequences are functionally linked to said nucleic acid sequence and wherein said intron is heterologous to said nucleic acid sequence or to said promoter sequence. 10 Furthermore, the invention preferably relates to recombinant expression constructs comprising at least one promoter sequence functioning in plants cells, at least one nucleic acid sequence and at least one functional equivalents of an intron described by any of sequences SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22, wherein said functional equivalent comprises the functional elements of an 1 5 intron and is characterized by a) a sequence having at least 50 consecutive base pairs of the intron sequence described by any of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, or b) having an identity of at least 80% over a sequence of at least 95 consecutive nucleic 20 acid base pairs to a sequences described by any of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, or c) hybridizing under high stringent conditions with a nucleic acid fragment of at least 50 consecutive base pairs of a nucleic acid molecule described by any of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, 25 wherein said promoter sequence and at least one of said intron sequences are functionally linked to said nucleic acid sequence and wherein said intron is heterologous to said nucleic acid sequence or to said promoter sequence. More preferably the intron is described by SEQ ID No: 2. 30 A preferred aspect of the invention relates to a method for identifying an intron with expression enhancing properties in plants comprising selecting an intron from a plant genome, wherein said intron is characterized by at least the following features 1) an intron length shorter than 1,000 base pairs, and 3 ll) presence of a 5' splice site comprising the dinucleotide sequence 5'-GT-3' (SEQ ID NO: 78), and Ill) presence of a 3' splice site comprising the trinucleotide sequence 5'-CAG-3' (SEQ ID NO: 79), and 5 IV) presence of a branch point resembling the consensus sequence 5'-CURAY-3' (SEQ ID NO:75) upstream of the 3'splice site, and V) an adenine plus thymine content of at least 40% over 100 nucleotides downstream from the 5' splice site, and VI) an adenine plus thymine content of at least 50% over 100 nucleotides upstream 10 from the 3' splice site, and VI) an adenine plus thymine content of at least 50%, and a thymine content of at least 30% over the entire intron. In another preferred embodiment, the invention relates to a method for enriching the 15 number of introns with expression enhancing properties in plants in a population of plant introns to a percentage of at least 50% of said population, said method comprising selecting introns from said population, wherein said introns are characterized by at least the following features I) an intron length shorter than 1,000 base pairs, and 20 II) presence of a 5' splice site comprising the dinucleotide sequence 5'-GT-3' (SEQ ID NO: 78), and Ill) presence of a 3' splice site comprising the trinucleotide sequence 5'-CAG-3' (SEQ ID NO: 79), and IV) presence of a branch point resembling the consensus sequence 5'-CURAY-3' 25 (SEQ ID NO:75) upstream of the 3'splice site, and V) an adenine plus thymine content of at least 40% over 100 nucleotides downstream from the 5' splice site, and VI) an adenine plus thymine content of at least 50% over 100 nucleotides upstream from the 3' splice site, and 30 VII) an adenine plus thymine content of at least 50%, and a thymine content of at least 30% over the entire intron. Preferably, the population of plant introns chosen for the enrichment of introns with gene expression enhancing properties in plants comprises substantially all introns of a plant 4 genome represented in a genomic DNA sequence database or a plant genomic DNA library. In a preferred embodiment, the intron with gene expression enhancing properties in 5 plants ("IME-intron") is selected by the method of the invention for identifying IME-introns or the method of the invention for enriching the number of IME-introns in a population of plant introns. Preferably, said intron is selected from the group consisting of introns located between two protein encoding exons or introns located within the 5' untranslated region of the corresponding gene. 10 In a particularly preferred embodiment, the IME-intron is identified or enriched by one of the inventive methods from a group or population of genes representing the 10% fraction of genes with the highest expression rate in a gene expression analysis experiment performed using a plant cell, plant tissue or a whole plant. 15 The invention furthermore relates to a method wherein the gene sequence information used for the identification or enrichment of IME-introns is present in a DNA sequence database and the selection steps for identifying or enriching said introns are performed using an automated process, preferably by using a computer device and an algorithm 20 that defines the instructions needed for accomplishing the selection steps for identifying or enriching said introns. Additionally, the invention relates to computer algorithm that defines the instructions needed for accomplishing the selection steps for identifying or enriching IME-introns from 25 a plant genome or a population of introns selected from the group consisting of introns located between two protein encoding exons, and/or introns located within the 5' untranslated region of the corresponding gene and/or introns located in the DNA sequences of genes representing the 10% fraction of genes with the highest expression rate in a gene expression analysis experiment performed using a plant cell, plant tissue 30 and/or a whole plant. The invention also relates to the computer device or data storage device comprising an algorithm as described above. 5 In a preferred embodiment, the invention relates to methods for isolating, providing or producing IME-introns comprising the steps of performing an identification or enrichment of IME-introns as described above and providing the sequence information of said IME introns identified or enriched, and providing the physical nucleotide sequence of said 5 identified or enriched introns and evaluating the gene expression enhancing properties of the isolated introns in an in vivo or in vitro expression experiment, and isolating the IME introns from the population of introns tested in the in vivo or in vitro expression experiment, Preferably, the evaluation of the gene expression enhancing properties of the IME-intron is done in a plant cell and wherein IME-intron enhances the expression of 10 a given nucleic acid at least twofold. In another embodiment, the recombinant DNA expression construct of the invention further contains one or more additional regulatory sequences functionally linked to promoter. Those regulatory sequences can be selected from the group consisting of heat 15 shock responsive-, anaerobic responsive-, pathogen responsive- drought responsive-, low temperature responsive-, ABA responsive-elements, 5' untranslated gene region, 3' untranslated gene region, transcription terminators, polyadenylation signals and enhancers. 20. The nucleic acid sequence of the inventive recombinant DNA expression construct may result in the expression of a protein and/or sense, antisense or double-stranded RNA encoded by said nucleic acid sequence, In another embodiment, the nucleotide sequence encoding the transgenic expression 25 construct of the invention is double-stranded. In yet another embodiment, the nucleotide sequence encoding the transgenic expression construct of the invention is single stranded, In yet another alternative embodiment of the invention, the recombinant expression construct comprises a nucleic acid sequence encoding for a selectable marker protein, a 30 screenable marker protein, a anabolic active protein, a catabolic active protein, a biotic or abiotic stress resistance protein, a male sterility protein or a protein affecting plant agronomic characteristics. The invention relates furthermore to vectors containing a transgenic expression construct of the invention. Additionally, the invention relates to transgenic cells or trans 5a genic non-human-organisms like bacteria, fungi, yeasts or plants comprising an ex pression vector containing a transgenic expression construct of the invention. In a pre ferred embodiment, the transgenic cell or transgenic non-human organism transformed with an expression construct of the invention is a monocotyledonous plant or is derived 5 from such a plant. in a yet more preferred embodiment, the monocotyledonous plant is selected from the group consisting of the genera Hordeum, Avena, Secale, Tricum, Sorghum, Zea, Saccherum, and Oryza. Further embodiments of the invention relate to cell cultures, parts or propagation material derived from non-humanorganisms like bacteria, fungi, yeasts and/or plants, preferably monocotyledonous plants, most pref 10 erably plants selected from the group consisting of the genera Hordeum, Avena, So cale, Tridcum, Sorghum, Zea, Saccharum, and Oryza, transformed with the inventive vectors or containing the inventive recombinant expression constructs. The invention furthermore relates to a method for providing an expression cassette for 15 enhanced expression of a nucleic acid sequence in a plant or a plant cell, comprising the step of functionally linking at least one sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 to said nucleic adid sequence. 20 The invention further relates to a method for enhancing the expression of a nucleic acid sequence in a plant or a plant cell, comprising functionally linking at least one se quence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14,15, 16, 17, 18,19, 20, 21 and 22 to said nucleic acid sequence. 25 An additional embodiment of the invention relates to a method a) for providing an expression cassette for enhanced expression of a nucleic acid se quence in a plant or a plant cell, or b) for enhancing the expression of a nucleic acid sequence in a plant or a plant cell said method comprising functionally linking at least one sequence selected from the 30 group consisting of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 to said nucleic acid sequence, wherein furthermore a promoter se quence functional in plants is linked to said nucleic acid sequence. Preferably, at least one sequence selected from the group consisting of SEQ ID NOs: 35 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 is linked to a nu cleic acid sequence by insertion into the plant genome via homologous recombination. Preferably, said homologous recombination is comprising at least the steps of a) providing in vivo or in vito a DNA construct comprising said intron flanked by se quences ("recombination substrate') allowing homologous recombination into a pre 40 existing expression cassette between the promoter and the nucleic acid of said ex pression cassette, and b) transforming a recipient plant cell comprising said cassette of step a) and regenerat ing a transgenic plant, wherein said intron has been inserted into the genome of said plant. Preferably, the site of integration into the genome of said plant is deter 45 mined by the DNA sequence of the recombination substrate of step a), wherein said sequence sharing sufficient homology (as defined herein) with said genomic target DNA sequence allowing the sequence specific integration via homologous recombi nation at said genomic target DNA locus. 6 In a preferred embodiment of the invention, said recipient plant or plant cell is a mono cotyledonous piant or plant cell, more preferably a plant or plant cell selected from the group consisting of the genera Hordeum, Avena, Secale, Triticum, Sorghum, Zea, Sac charum, and Otyza, most preferably a maize plant. 5 Preferably, the nucleic acid sequence to which one of the inventive intron is functionally linked, encodes for a selectable marker protein, a screenable marker protein, an ana bolic active protein, a catabolic active protein, a biotic or abiotic stress resistance pro tein, a male sterility protein or a protein affecting plant agronomic characteristics and/or 10 a sense, antisense, or double-stranded RNA. Additionally, the invention relate to the use of a transgenic organism of the invention or of cell cultures, parts of transgenic propagation material derived there from for the pro duction of foodstuffs, animal feeds, seeds, pharmaceuticals or fine chemicals. 15 The invention furthermore relates to a recombinant DNA expression construct compris ing a) at least one promoter sequence functioning in plants or plant cells, and b) at least one intron selected from the group of introns with expression enhancing 20 properties in plants or plant cells characterized by at least the following features 1) an intron length shorter than 1,000 base pairs, and II) presence of a 5' splice site comprising the dinucleotide sequence 5'-GT-3' (SEQ ID NO: 78), and ill) presence of a 3' splice site comprising the trinucleotide sequence 5'-CAG-3' 25 (SEQ ID NO: 79), and IV) presence of a branch point resembling the consensus sequence 5'-CURAY-3' (SEQ ID NO: 75) upstream of the 3'splice site, and V) an adenine plus thymine content of at least 40% over 100 nucleotides down stream from the 5' splice site, and 30 VI) an adenine plus thymine content of at least 50% over 100 nucleotides up stream from the 3' splice site, and VI1) an adenine plus thymine content of at least 55%, and a thymine content of at least 30% over the entire intron, and c) at least one nucleic acid sequence, wherein said promoter sequence and at least 35 one of said intron sequences are functionally linked to said nucleic acid sequence and wherein said intron is heterologous to said nucleic acid sequence and/or to said promoter sequence. BRIEF DESCRIPTION OF THE DRAWINGS 40 Fig. 1 Map of pBPSMM291 (SEQ ID NO: 109) This vector comprises the maize ubiquitin promoter, followed by the BPSL.1, then the GUSint ORF (including the potato invertase [PIV]2 intron to prevent bacterial expression), followed by nopaline synthase (NOS) terminator. This vector contains the attLI and attL2 sites to make it compatible with modification 45 via the Gateway@ cloning Technology from Invitrogen t m . This vector is based 7 on the pUC based expression vector pBPSMM267. The Xmal-Rsrl digested BPSI.1 PCR product was ligated into the Xmal-RsrilI digested pBPSMM267 to create pBPSMM291. The vectors pBPSMM293, pBPSMM294 and pBPSMM295 have been created accordingly (see table 6 and 1.6.1). 5 Fig. 2 Map of pBPSMM305 (SEQ ID NO:1 10) The expression vector pBPSMM305 comprises the maize lactate dehydro genase (LDH) promoter without intron driving expression of the GUSint ORF (including the potato invertase [PIV]2 intron to prevent bacterial expression), fol 10 lowed by the NOS terminator. This vector has been used to create the pUC based expression vectors pBPSJB041, pBPSJB042, pBPSJB043, pBPSJB044, pBPSJB045, pBPSJB046 and pBPSJB050 (see examples 2.3). Fig. 3 Map of pBPSMM350 (SEQ ID NO:111): 15 The vector pBPSMM350 comprises the maize ubiquitin promoter, followed by the BPSI.1, then the GUSint ORF (including the potato invertase [PIV]2 Intron to prevent bacterial expression), followed by ropaline synthase (NOS) terminator. The expression cassette has been transferred from the vector pBPSMM291 us ing the Gateway@ cloning Technology from Invitrogen Tm . The vectors 20 pBPSMM353, pBPSMM312 and pBPSMM310 have been created accordingly (see table 6 and example 1.6.2). Fig. 4 Map of pBPSLM139 (SEQ ID NO:112): The vector pBPSLM139 comprises the selectable marker expression cassette. 25 In order to produce the vectors pBPSL01 7 to pBPSLI023, Pmel/Pacl fragments have been isolated from the vectors pBPSJB-042, -043, -044, -045, 046 and 050 and cloned into the Pmel-Pacl digested pBPSLM130 (see example 2.3 and 2.4) 30 Fig. 5a-f: Computer algorithm for retrieving sequence information from NCBI genebank file. Fig. 6 Transgenic plants containing promoter constructs with BPSI.1 intron (all but pBPSLM229) or BPSI.5 intron (only pBPSLM229) were tested for GUS 35 expression at 5-leaf (A), flowering (B) and seed set (C) stages. Shown are examples of typical staining patterns obtained from at least 15 independent events. All samples were stained for 16 hours in GUS solution. Promoters in the constructs are: rice chloroplast protein 12 (Os.CP12; pBPSMM355), the maize hydroxyproline-rich glycoprotein (Zm.HRGP; pBPSMM370), the rice p-caffeoyl 40 CoA 3-0-methyltransferase (Os.CCoAMT1; pBPSMM358), the maize Globulin 1 promoter W64A (Zm.Glbl; EXS1025), the putative Rice H+-transporting ATP synthase promoter (Os.V-ATPase; pBPSMM369), Zm.LDH (pBPSMM357), the rice C-8,7 sterol isomerase promoter (Os.C8,7 Sl; pBPSMM366), the rice Late Embryogenesis Abundant Protein promoter (Os.Lea; pBPSMM371), and the 45 maize lactate dehydrogenase promoter (ZM.LDH; pBPSLM229).. 8 GENERAL DEFINITIONS It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, plant species or genera, constructs, and reagents described as such It must be noted that as used herein and in the appended claims, the singular 5 forms "a" and "the" include plural reference unless the context clearly dictates other wise. Thus, for example, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art. About: the term "about" is used herein to mean approximately, roughly, around, or in 10 the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent up or down (higher or lower). As used herein, the word "or" means any one member of a 15 particular list. Agrobacterium: refers to a soil-bome, Gram-negative, rod-shaped phytopathogenic bacterium which causes crown gall. The term "Agrobacterium" includes, but is not lim fled to, the strains Agrobacterium tumefaciens, (which typically causes crown gall in 20 infected plants), and Agrobacterium rhizogenes (which causes hairy root disease in infected host plants). Infection of a plant cell with Agrobacterium generally results In the production of opines (e.g., nopaline, agropine, octopine etc.) by the infected cell. Thus, Agrobacterium strains which cause production of nopaline (e.g., strain LBA4301, C58, A208) are referred to as "nopaline-type" Agrobacteria; Agrobacterium strains which 25 cause production of octopine (e.g., strain LBA4404, Ach5, B6) are referred to as "oc topine-type" Agrobacteria; and Agrobacterium strains which cause production of ag ropine (e.g., strain EHA105, EHA101, A281) are referred to as "agropine-type" Agro bacteria. 30 Algorithm: as used herein refers to the way computers process information, because a computer program is essentially an algorithm that tells the computer what specific steps to perform (in what specific order) in order to carry out a specified task, such as identification of coding regions of a set of genes. Thus, an algorithm can be considered to be any sequence of operations that can be performed by a computer system. Typi 35 cally, when an algorithm is associated with processing information, data is read from an input source or device, written to an output sink or device, and/or stored for further use. For any such computational process, the algorithm must be rigorously defined: speci fied in the way it applies in all possible circumstances that could arise. That is, any conditional steps must be systematically dealt with, case-by-case; the criteria for each 40 case must be clear (and computable). Because an algorithm is a precise list of precise steps, the order of computation will almost always be critical to the functioning of the algorithm. Instructions are usually assumed to be listed explicitly, and are described as starting 'from the top' and going 'down to the bottom', an idea that is described more formally by flow of control. In computer applications, a script is a computer program 45 that automates the sort of task that a user might otherwise do interactively at the key board. Languages that are largely used to write such scripts are called scripting lan guages. Many such languages are quite sophisticated, and have been used to write elaborate programs, which are often still called scripts even if they go well beyond 9 automating simple sequences of user tasks. Computer languages are created for vary ing purposes and tasks different kinds and styles of programming. Scripting program ming languages (commonly called scripting languages or script languages) are com puter programming languages designed for "scripting" the operation of a computer. 5 Early script languages were often called batch languages or job control languages. Examples for script languages are: ACS, ActionScript, Active Server Pages (ASP), AppleScript, Awk, BeanShell (scripting for Java), bash, Brain, CobolScript, csh, Cold Fusion, Dylan, Escapade (server side scripting), Euphoria, Groovy, Guile, Haskell, Hy 10 perTalk, ICI, IRC script, JavaScript, mlRC script, MS-DOS batch, Nwscript, Perl, PHP, Pike, ScriptBasic. Antisense: is understood to mean a nucleic acid having a sequence complementary to a target sequence, for example a messenger RNA (mRNA) As used herein, the terms "complementary" or "complementarity" are used in reference to nucleotide sequences 15 related by the base-pairing rules. For example, the sequence 5'-AGT-3' is complemen tary to the sequence 5'-ACT-3'. Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base 20 pairing rules. The degree of complementarity between nucleic acid strands has signifi cant effects on the efficiency and strength of hybridization between nucleic acid strands. Sense: is understood to mean a nucleic acid having a sequence that is homologous or identical to a target sequence, for example a sequence which is bound by a protein 25 factor of the spliceosome Bombarding, "bombardment and "biolistic bombardment': refer to the process of accel erating particles (microprojectiles) towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample 30 and/or entry of the particles into the target biological sample. Methods for biolistic bom bardment are known in the art (e.g., US 5,584,807, the contents of which are herein incorporated by reference), and are commercially available (e.g., the helium gas-driven microprojectile accelerator (PDS-1000/He) (BioRad). 35 Cell: refers to a single cell. The term "cells" refers to a population of cells. The popula tion may be a pure population comprising one cell type. Likewise, the population may comprise more than one cell type. In the present invention, there is no limit on the number of cell types that a cell population may comprise. The cells may be synchronize or not synchronized, preferably the cells are synchronized. 40 Chromosomal DNA or chromosomal DNA-sequence: is to be understood as the ge nomic DNA of the cellular nucleus independent from the cell cycle status. Chromoso mal DNA might therefore be organized in chromosomes or chromatids, they might be condensed or uncoiled, An insertion into the chromosomal DNA can be demonstrated 45 and analyzed by various methods known in the art like e.g., polymerase chain reaction 10 (PCR) analysis, Southern blot analysis, fluorescence in situ hybridization (FISH), and in situ PCR. Coding region or coding sequence (CDS): when used in reference to a gene refers to 5 the nucleotide sequences which encode the amino acids found in the nascent polypep tide as a result of translation of a mRNA molecule. The coding region is bounded, in eucaryotes, on the 5'-side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3'-side by one of the three triplets, which specify stop codons (i.e., TAA, TAG, TGA). 10 Complement of a nucleic acid sequence: as used herein refers to a nuceotide se quence whose nucleic acids show total complementarity to the nucleic acids of the nu cleic acid sequence. 15 Decile: when used in connection with statistical data is any of the 10 values that divide sorted data into 10 equal parts, so that each part represents 1/10th of the sample or population. Thus, the ist decile cuts off lowest 10% of data, the 9th decile cuts off low est 90% or the highest 10% of data. A quartile is any of the three values which divide the sorted data set into four equal parts, so that each part represents 1/4th of the sam 20 pIe or population (third quartile = upper quartile = cuts off highest 25% of data, or low est 75% = 75th percentile). A percentile is any of the 99 values that divide the sorted data into 100 equal parts, so that each part represents 1/100th of the sample or popu lation. Thus, the 1st percentile cuts off lowest 1% of data, the 98th percentile cuts off lowest 98% of data and the 25th percentile cuts off lowest 25% of data. 25 DNA databases: in the field of bioinformatics, a DNA sequence database is a large collection of DNA sequences stored on a computer. A database can include sequences from only one organism, or it can Include sequences from all organisms whose DNA has been sequenced. 30 Enrichment or enriching: when used in connection with the selection of inventive in trons refers to an increase in the success rate of identifying introns with gene expres sion enhancing properties within a population of introns (e.g. a population of introns representing all introns of a plant genome present in a genomic DNA sequence data 35 base). The enrichment is achieved by reducing the number of candidate introns by us ing the inventive method and the inventive selection criteria. If, as an example, the suc cess rate of identifying an intron with expression enhancing properties from a given population of introns - by using the herein described methods for measuring gene ex pression enhancement- is one out of ten analyzed Introns, enrichment has to be under 40 stood as an increase in the number of identified introns with gene expression enhanc ing properties -by using the inventive method- to at least five out of ten analyzed in trons. Therefore, the number of introns needed to be analyzed in order to identify one inventive intron is reduced to two introns by using the inventive method as a preselec tion or filtering process. 45 Evaluation of the expression enhancing properties: of an intron can be done using methods known in the art. For example, a candidate intron sequence whose gene ex 11 pression enhancing effect is to be determined can be inserted into the 5'UTR of a nu cleic acid sequence encoding for a reporter gene (e.g., a visible marker protein, a se lectable marker protein) under control of an appropriate promoter active in plants or plant cells to generate a reporter vector. The reporter vector and an identical control 5 reporter vector lacking the candidate intron can be introduced into a plant tissue using methods described herein, and the expression level of the reporter gene, in depend ence of the presence of the candidate intron, can be measured and compared (e.g., detecting the presence of encoded mRNA or encoded protein, or the activity of a pro tein encoded by the reporter gene). An intron with expression enhancing properties will 10 result in a higher expression rate than a reference value obtained with an identical con trol reporter vector lacking the candidate intron under otherwise unchanged conditions. The reporter gene may express visible markers. Reporter gene systems which express visible markers include p-glucuronidase and its substrate (X-Gluc), luciferase and its 15 substrate (luciferin), and [p-galactosidase and its substrate (X-Gal) which are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes (1995) Meth ods Mol Biol 55:121-131). The assay with p glucuronidase (GUS) being very especially preferred (Jefferson et a., GUS fusions: beta-glucuronidase as a sensitive and versa 20 tile gene fusion marker in higher plants. EMBO J. (1987) Dec 206(13):3901-3907). p glucuronidase (GUS) expression is detected by a blue color on incubation of the tissue with 5-bromo-4-chloro-3-indolyl-p-D-glucuronic acid. The selectable marker gene may confer antibiotic or herbicide resistance. Examples of reporter genes include, but are not limited to, the dhfr gene, which confers resistance to methotrexate (Wigler (1980) 25 Proc Natl Acad Sci 77:3567-3570); npt, which confers resistance to the aminoglyco sides neomycin and G-418 (Colbere-Garapin (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyl transferase, respectively. 30 Expect value when used in the context of DNA sequence alignments or DNA sequence database searches refers to the number of times a certain match or a better one would be expected to occur purely by chance in a search of the entire database. Thus, the lower the Expect value, the greater the similarity between the input sequence and the match. The Expect value (E) is a parameter that describes the number of hits one can 35 "expect" to see just by chance when searching a database of a particular size, It de creases exponentially with the Similarity Score (S) that is assigned to a match between two sequences. The higher the score, the lower the E value. Essentially, the E value describes the random background noise that exists for matches between sequences. The Expect value is used as a convenient way to create a significance threshold for 40 reporting results. An E value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size you might expect to see 1 match with a similar score simply by chance. The E-value is influenced by: a) length of sequence (the longer the query the lower the probability that it will find a sequence in the database by chance), b) size of database (the larger the database the higher the probability that the query will 45 find a match by chance), c) the scoring matrix (the less stringent the scoring matrix the higher the probability that the query will find a sequence in the database by chance). 12 Expressed sequence tag (EST): refers to a cDNA sequence that has been obtained from a single pass terminal DNA sequencing. An EST sequence denotes a sequence that is derived from a transcript, and hence from a gene that is transcribed. 5 Expressible nucleic acid sequence: as used in the context of this invention is any nu cleic acid sequence that is capable of being transcribed into RNA (e.g. mRNA, an tisense RNA, double strand forming RNA etc.) or translated Into a particular protein. Expression: refers to the biosynthesis of a gene product. For example, in the case of a 10 structural gene, expression involves transcription of the structural gene into mRNA and - optionally - the subsequent translation of mRNA into one or more polypeptides. Functional equivalents: with regard to the inventive introns has to be understood as natural or artificial mutations of said introns described in any of the SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. Mutations can be 15 insertions, deletions or substitutions of one or more nucleic acids that do not diminish the expression enhancing properties of said introns, These functional equivalents hav ing a identity of at least 80%, preferably 85%, more preferably 90%, most preferably more than 95%, very especially preferably at least 98% identity but less then 100% identity to the intron sequences as described by any of the SEQ ID NOs: 1, 2, 3, 5, 6, 20 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, wherein said identity is deter mined over a sequence of at least 95 consecutive base pairs, preferably at least 150 consecutive base pairs, more preferably at least 200 consecutive base pairs of the se quence as described by any of the SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,; 20, 21 or 22 and having essentially the same IME effect characteristics 25 as the intron sequences as shown in any of the SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. Functional equivalents are in particular homologs of said introns derived from other plant species. Homologs when used in reference to introns refers to introns with ex 30 pression enhancing properties isolated from a genomic nucleic acid sequence that en codes for a protein (i) sharing more than 60%, preferably 65%, 70%, 75%, 80%, more preferably 85%, 90%, 951% or most preferably more than 95% sequence identity on amino acid level with proteins that are encoded by genes from which the inventive introns with 35 the SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1 or 22 have been isolated, or (ii) catalyzing the same enzymatic reaction as the proteins encoded by genes from which the inventive introns SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 have been isolated, or 40 (iii) showing comparable spatial and temporal expression pattern as the proteins en coded by genes from which the inventive introns SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12,13,14,15,16, 17, 18,19, 20, 21 or 22 have been isolated. "Functional equivalents' as described above might have, compared with the inventive 45 introns a reduced or increased gene expression enhancing effect. In this context, the gene expression enhancing effect of the functional equivalent Intron is at least 50% higher, preferably at least 100% higher, especially preferably at least 300% higher, 13 very especially preferably at least 500% higher than a reference value obtained with any of the introns shown in SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21 or 22 under otherwise unchanged conditions. Functionally linked or operably linked: is to be understood as meaning, for example, the 5 sequential arrangement of a regulatory element (e.g. a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (such as e.g., a terminator) in such a way that each of the regulatory elements can fulfill its in tended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. The expression may result depending on the arrangement of 10 the nucleic acid sequences In relation to sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily required. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions that are further away, or indeed from other DNA molecules. The terms "functionally linked', "operably linked," "in operable combination," and "in 15 operable order" as used herein with reference to an inventive intron with gene expres sion enhancing properties refers to the linkage of at least one of said introns to a nu cleic acid sequences in a way that the expression enhancing effect is realized and, if functional splice sites have been included, that the intron can be spliced out by the cell factors responsible for the splicing procedure. In a preferred embodiment of the present 20 invention, the intron is introduced into the 5' non coding region of a nucleic acid se quence. Inventive expression constructs, wherein an inventive intron is functionally linked to an nucleic acid sequence are shown in the examples. More preferred ar rangements are those in which an intron functioning in intron mediated expression en hancement Is inserted between a promoter and a nucleic acid sequence, preferably 25 into the transcribed nucleic acid sequence, or in case of a nucleic acid sequence en coding for a protein, into the 5' untranslated region of a nucleic acid sequence. The distance between the promoter sequence and the nucleic acid sequence to be ex pressed recombinantly Is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs. Operable link 30 age, and an expression cassette, can be generated by means of customary recombina tion and cloning techniques as are described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring 35 Harbor (NY), in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and in Gelvin et al. (1990) In: Plant Molecular Biology Manual. However, further sequences which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences. The insertion of sequences may also 40 lead to the expression of fusion proteins. Preferably, the expression construct, consist ing of a linkage of promoter, intron and nucleic acid sequence to be expressed, can exist in a vector-integrated form and be inserted into a plant genome, for example by transformation. Gene: refers to a coding region operably linked to appropriate regulatory sequences 45 capable of regulating the expression of the polypeptide in some manner. A gene in cludes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, 14 etc.) preceding (upstream) and following (downstream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) be tween individual coding regions (Le., exons). Genes may also include sequences lo cated on both the 5'- and 3'-end of the sequences, which are present on the RNA tran 5 script. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5'-flanking region may contain regulatory sequences such as promoters and ienhancers, which control or influence the transcription of the gene. The 3'-flanking region may contain sequences, which direct the termination of transcription, 10 posttranscriptional cleavage and polyadenylation. Gene expression enhancing properties, gene expression enhancing effect or intron mediated gene expression enhancement (IME): when made in reference to an intron sequence refers to the ability of the intron to enhance quantitatively the expression 15 level of a nucleic acid sequence (e.g. a gene) that is part of an recombinant/transgenic DNA expression cassette (as defined herein), measured on the basis of the transcribed RNA, mRNA, protein amount or protein activity compared to the otherwise identical expression construct lacking the intron under otherwise unchanged conditions. Gene expression enhancing properties in plants: refers to an intron that is able to enhance 20 quantitatively the expression level of a plant derived nucleic acid sequence in a plant or plant cell and the enhancement of gene expression rate of a non-plant derived nucleic acid in a plant or a plant cell compared to the otherwise identical expression construct lacking the intron under otherwise unchanged conditions. In a preferred embodiment of the invention, the expression enhancing effect is understood as an increase in the RNA 25 steady state level, the protein steady state level or the protein activity of a nucleic acid sequence or the corresponding protein (e.g. a reporter gene or protein) of at least 50%, or at least 100%, or at least 200%, 300%, 400% or at least 500%, 600%, 700%, 800%, 900% or at least 1,000%, or more than 1,000% compared to the otherwise identical expression construct lacking the intron under otherwise unchanged conditions. Fur 30 therrnore expression enhancing effect or intron mediated enhancement has to be un derstood as the ability of an intron to change the tissue, organ or cell specific expres sion pattern of a nucleic acid sequence (e.g. a gene) that is part of an inventive ex pression cassette. Changing the tissue, organ or cell specific expression pattern of a nucleic acid sequence that is part of an inventive expression cassette refers to the fact 35 that due to the presence of an inventive intron, the expression level (mRNA or encoded protein steady state level, or the activity of a protein) of the respective gene is in creased above the detection threshold of the used detection method. Gene silencing: can be realized by antisense or double-stranded RNA or by co 40 suppression (sense-suppression). The skilled worker knows that he can use alternative cDNA or the corresponding gene as starting template for suitable antisense constructs. The 'antisense' nucleic acid is preferably complementary to the coding region of the target protein or part thereof. However, the 'antisense' nucleic acid may also be com plementary to the non-coding region or part thereof. Starting from the sequence infor 45 mation on a target protein, an antisense nucleic acid can be designed in the manner with which the skilled worker is familiar, taking into consideration Watson s and Crick s rules of base pairing. An antisense nucleic acid can be complementary to the entire or part of the nucleic acid sequence of a target protein. 15 Likewise encompassed is the use of the above-described sequences in sense orienta tion, which, as is known to the skilled worker, can lead to co-suppression (sense suppression). It has been demonstrated that expression of sense nucleic acid se quences can reduce or switch off expression of the corresponding gene, analogously to 5 what has been described for antisense approaches (Goring (1991) Proc. Nati Acad. Sci, USA 88:1770-1774; Smith (1990) Mol. Gen. Genet. 224:447-481; Napoli (1990) Plant Cell 2:279-289;Van der Krol (1990) Plant Cell 2:291-299). In this context, the construct introduced may represent the gene to be reduced fully or only in part. The possibility of translation is not necessary. Especially preferred is the use of gene regu 10 lation methods by means of double-stranded RNAi ('double-stranded RNA interfer ence'). Such methods are known to the person skilled in the art (e.g., Matzke 2000; Fire 1998; WO 99/32619; WO 99153050; WO 00/68374; WO 00/44914; WO 00144895; WO 00/49035; WO 00/63364). The processes and methods described in the refer ences stated are expressly referred to. 15 Genome and genomic DNA of an organism as used herein is the whole hereditary in formation of an organism that is encoded in the DNA (or, for some viruses, RNA). This includes both the genes and the non-coding sequences. Said genomic DNA comprises the DNA of the nucleus (also referred to as chromosomal DNA) but also the DNA of the 20 plastids (e.g., chloroplasts) and other cellular organelles (e.g., mitochondria). Prefera bly the terms genome or genomic DNA is referring to the chromosomal DNA of the nucleus. The term "chromosomal DNA' or "chromosomal DNA-sequence" is to be un derstood as the genomic DNA of the cellular nucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes or chromat 25 ids, they might be condensed or uncoiled. An insertion into the chromosomal DNA can be demonstrated and analyzed by various methods known in the art like e.g., poly merase chain reaction (PCR) analysis, Southem blot analysis, fluorescence in situ hy bridization (FISH), and in situ PCR. Heterologous: with respect to a nucleic acid sequence refers to a nucleotide sequence, 30 which is ligated to a nucleic acid sequence to which it is not ligated in nature, or to which It is ligated at a different location in nature. Hybridizing: as used herein includes "any process by which a strand of nucleic acid joins with a complementary strand through base pairing." (Coombs 1994, Dictionary of 35 Biotechnology, Stockton Press, New York N.Y.). Hybridization and the strength of hy bridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, strin gency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids. As used herein, the term "Tm" is used in reference to the "melting 40 temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indi cated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 45 M NaCl [see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)]. Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calcula 16 tion of Tm. The person skilled in the art knows well that numerous hybridization condi tions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) 5 and the concentration of the salts and other components (e.g., the presence or ab sence of formamide, dextran sulfate, polyethylene glycol) are considered and the hy bridization solution may be varied to generate conditions of either low or high hybridiza tion stringency Those skilled in the art know that higher stringencies are preferred to reduce or eliminate non-specific binding between the nucleotide sequence of an inven 10 tive intron and other nucleic add sequences, whereas lower stringencies are preferred to detect a larger number of nucleic add sequences having different homologies to the inventive nucleotide sequences. Such conditions are described by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) or in Current Protocols in Molecular Biology, 15 John Wiley & Sons, N. Y. (1989) 6.3.1-6.3.6. Preferred hybridization condition are dis close in the detailed description. Identity: when used in relation to nucleic acids refers to a degree of complementarity. Identity between two nucleic acids is understood as meaning the identity of the nucleic acid sequence over in each case the entire length of the sequence, which is calculated 20 by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the parameters being set as follows: Gap Weight: 12 Length Weight: 4 Average Match: 2,912 Average Mismatch:-2,003 25 For example, a sequence with at least 95% identity to the sequence SEQ ID NO. 1 at the nucleic acid level is understood as meaning the sequence that, upon comparison with the sequence SEQ ID NO. 1 by the above program algorithm with the above pa rameter set has at least 95% identity. There may be partial Identity (i.e., partial identity 30 of less then IO%) or complete identity (i.e., complete identity of 100%). Introducing a recombinant DNA expression construct: in plant cells refers to a recombi nant DNA expression construct that will be introduced into the genome of a plant by transformation and is stably maintained. The term "introducing' encompasses for ex 35 ample methods such as transfection, transduction or transformation. Identification, "Identifying' or "selecting': with regard to transformation of plants has to be understood as a screening procedure to identify and select those plant cells in which the recombinant expression construct has been introduced stably into the ge 40 nome. "Identifying' with regard to an intron with gene expression enhancing properties refers to a process for the selection of said intron out of a population of introns. Pref erably, "identifying' refers to an in si/lco selection process, more preferably to an auto mated in silico selection process, using the selection criteria of the inventive methods. Such an in silico identification process can comprise for instance the steps of 45 (1) generating an intron sequence database on the basis of DNA sequences present in a DNA sequence database (e.g. genomic DNA databases publicly available via the internet), 17 (2) screening of the generated intron DNA sequence database -or other genomic DNA sequences containing databases - for introns with gene expression enhancing properties using the criteria according to the inventive method, wherein the steps for retrieving or generating the DNA sequences; the generation of an 5 intron specific DNA sequence database and the screening of these DNA sequences using the criteria according to the inventive method - will be performed with the aid of appropriate computer algorithms and computer devices. Intron: refers to sections of DNA (intervening sequences) within aigene that do not en 10 code part of the protein that the gene produces, and that is spliced out of the mRNA that is transcribed from the gene before it is exported from the cell nucleus. Intron se quence refers to the nucleic acid sequence of an intron. Thus, introns are those regions of DNA sequences that are transcribed along with the coding sequence (exons) but are removed during the formation of mature mRNA. Introns can be positioned within the 15 actual coding region or in either the 5 or 3 untranslated leaders of the pre-mRNA (unspliced mRNA). Introns in the primary transcript are excised and the coding se quences are simultaneously and precisely ligated to form the mature mRNA. The junc tions of introns and exons form the splice site. The sequence of an intron begins with GU and ends with AG. Furthermore, in plants, two examples of' AU-AC introns have 20 been described: the fourteenth intron of the RecA-lke protein gene and the seventh intron of the G5 gene from Arabidopsis thaliana are AT-AC introns. Pre-mRNAs con taining introns have three short sequences that are beside other rsequences- essential for the intron to be accurately spliced. These sequences are the 5' splice-site, the 3 splice-site, and the branchpoint. mRNA splicing is the removal of intervening se 25 quences intronss) present in primary mRNA transcripts and joinirsg or ligation of exon sequences. This is also known as cis-splicing which joins two exons on the same RNA with the removal of the intervening sequence (intron). The functional elements of an intron comprising sequences that are recognized and bound by the specific protein components of the spliceosome (e.g. splicing consensus sequences at the ends of 30 introns). The interaction of the functional elements with the spliceosome results in the removal of the intron sequence from the premature mRNA and the rejoining of the exon sequences. Introns have three short sequences that are essential -although not suffi cient- for the Intron to be accurately spliced. These sequences are the 5' splice site, the 3' splice site and the branch point. The branchpoint sequence is important in splic 35 ing and splice-site selection in plants. The branchpoint sequence is usually located 10 60 nucleotides upstream of the 3' splice site. Plant sequences exhibit sequence devia tions in the branchpoint, the consensus sequences being 5-CURAY-3 (SEQ ID NO:75) or 5 -YURAY-3 (SEQ ID NO: 76). 40 "IME-intron' or intron mediated enhancement (IME)-intron: when made in reference to an intron sequence refers to an intron with gene expression enhancing properties in plants as defined herein (see gene expression enhancing properties, gene expression enhancing effect or intron mediated gene expression enhancement). 45 Isolation or isolated: when used in relation to an intron or gene, as in "isolation of an intron sequence' or "isolation of a gene" refers to a nucleic acid sequence that is identi fied within and isolated/separated from its chromosomal nucleic acid sequence context within the respective source organism. Isolated nucleic acid is nucleic acid present in a 18 form or setting that is different from that in which it is found in nature, In contrast, non isolated nucleic acids are nucleic acids such as DNA and RNA, which are found in the state they exist in nature. For example, a given DNA sequence (e.g. a gene) is found on the host cell chromosome in proximity to neighboring genes; intron sequences, are 5 imbedded into the nucleic acid sequence of a gene in an alternating sequence of in trons and exons. The isolated nucleic acid sequence may be present in single-stranded or double-stranded form. When an isolated nucleic acid sequence is to be utilized to express a protein, the nucleic acid sequence will contain at a minimum at least a por tion of the sense or coding strand (i.e., the nucleic acid sequence may be single 10 stranded). Alternatively, it may contain both the sense and anti-sense strands (Lo., the nucleic acid sequence may be double-stranded). Nucleic acid: refers to deoxyribonucleotides, ribonucleotides or polymers or hybrids thereof in single-or double-stranded, sense or antisense form. Unless otherwise indi cated, a particular nucleic acid sequence also implicitly encompasses conservatively 15 modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly Indicated. The term "nucleic acid" can be used to describe a "gene", "cDNA','DNA' "mRNA", "oligonudeotide," and "polynucleo tide". Nucleic acid sequence: as used herein refers to the consecutive sequence of deoxyri 20 bonucleotides or ribonucleotides (nucleotides) of a DNA fragment (oligonucleotide, polynudeotide, genomic DNA, cDNA etc.) as it can made be available by DNA se quencing techniques as a list of abbreviations, letters, characters or words, which rep resent nucleotides. 25 Organ: with respect to a plant (or "plant organ') means parts of a plant and may include (but shall not limited to) for example roots, fruits, shoots, stem, leaves, anthers, sepals, petals, pollen, seeds, etc. Otherwise unchanged conditions: means for temple - that the expression which is 30 initiated by one of the expression constructs to be compared is not modified by combi nation with additional genetic control sequences, for example enhancer sequences and is done in the same environment (e.g., the same plant species) at the same develop mental stage and under the same growing conditions. 35 Plant: is generally understood as meaning any single-or multi-celled organism or a cell, tissue, organ, part or propagation material (such as seeds or fruit) of same which is capable of photosynthesis. Included for the purpose of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Annual, perennial, monocoty ledonous and 'dicotyledonous plants are preferred. The term includes the mature 40 plants, seed, shoots and seedlings and their derived parts, propagation material (such as seeds or ricrospores), plant organs, tissue, protoplasts, callus and other cultures, for example celi cultures, and any other type of plant cell grouping to give functional or structural units. Mature plants refer to plants at any desired developmental stage be yond that of the seedling. Seedling refers to a young immature plant at an early devel 45 opmental stage. Annual, biennial, monocotyledonous and dicotyledonous plants are preferred host organisms for the generation of transgenic plants. The expression of 19 genes is furthermore advantageous in all ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawns. Plants which may be mentioned by way of example but not by limitation are angiosperms, bryophytes such as, for example, Hepaticae (liverworts) and Musci (mosses); Pteridophytes such as ferns, horsetail and 5 club mosses; gymnosperms such as conifers, cycads, ginkgo and Gnetatae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophy ceae, Bacillariophyceae (diatoms), and Eugtenophyceae. Preferred are plants which are used for food or feed purpose such as the families of the Leguminosae such as pea, alfalfa and soya; Gramineae such as rice, maize, wheat, barley, sorghum, millet, 10 rye, triticale, or oats; the family of the Umbeliferae, especially the genus Daucus, very especially the species carote (carrot) and Aplum, very especially the species Graveolens dulce (celery) and many others; the family of the Solanaceae, especially the genus Lycopersicon, very especially the species esculentum (tomato) and the ge nus Solanum, very especially the species tuberosum (potato) and melongena (egg 15 plant), and many others (such as tobacco); and the genus Capsicum, very especially the species annuum (peppers) and many others; the family of the Leguminosae, espe cially the genus Glycine, very especially the species max (soybean), alfalfa, pea, lu ceme, beans or peanut and many others; and the family of the Cruciferae (Brassica cae), especially the genus Brassica, very especially the species napus (oil seed rape), 20 campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and o/eracea cv Emperor (broccoli); and of the genus Arabldopsis, very especially the species thaliana and many others; the family of the Compositae, especially the genus Lactuca, very especially the species sativa (lettuce) and many others; the family of the Asteraceae such as sunflower, Tagetes, lettuce or Calendula and many other; the fam 25 ily of the Cucurbitaceae such as melon, pumpkin/squash or zucchini, and linseed. Fur ther preferred are cotton, sugar cane, hemp, flax, chillies, and the various tree, nut and wine species. Providing: when used in relation to an intron as in "physically providing an intron' refers 30 to the cloning of the DNA sequence representing said intron from a plant of interest and the provision of such an intron physically in an appropriate vector or plasmid for further cloning work and the subsequent application of said intron according to the invention. Producing: when used in relation to an intron as in "producing an intron' refers to the 35 synthesis of DNA molecules on the basis of DNA sequence information of an inventive intron. Promoter, promoter element or promoter sequence: as used herein, refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of control 40 ling the transcription of the nucleotide sequence of interest into mRNA. Thus, a pro moter is a recognition site on a DNA sequence that provide an expression control ele ment for a gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene. A promoter is typically, though not necessarily, located 5' (i.e., upstream) of a nucleotide sequence of interest (e.g., proximal to the 45 transcriptional start site of a structural gene). Promoters may be tissue specific or cell specific. The term "tissue specific" as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., petals) in the relative absence of expression of the same 20 nucleotide sequence of interest in a different type of tissue (e.g., roots). Promoters may be constitutive or regulatable. The term "constitutive" when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, 5 light, etc.). Typically, constitutive promoters are capable of directing expression of a transgene in substantially any cell and any tissue. In contrast, a "regulatable" promoter is one which Is capable of directing a level of transcription of an operably linked nuclei acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.) which is different from the level of transcription of the operably linked nucleic acid se 10 quence in the absence of the stimulus. A promoter sequence functioning in plants is understood as meaning, in principle, any promoter which is capable of governing the expression of genes, in particular foreign genes, in plants or plant parts, plant cells, plant tissues or plant cultures. In this context, expression can be, for example, constitu tive, inducible or development-dependent. A constitutive promoter is a promoter where 15 the rate of RNA polymerase binding and initiation is approximately constant and rela tively independent of extemal stimuli. Usable promoters are constitutive promoters (Benfey et al. (1989) EMBO J. 8:2195-2202), such as those which originate from plant viruses, such as 35S CAMV (Franck et al. (1980) Cell 21:285-294), 19S CaMV (see also US 5352605 and WO 84/02913), 34S FMV (Sanger et al (1990) Plant. Mol. Biol., 20 14:433-443), the parsley ubiquitin promoter, or plant promoters such as the Rubisco small subunit promoter described in US 4,962,028 or the plant promoters PRP1 [Ward et al. (1993) Plant. Mol. Biol. 22: 361-6], SSU, PGEL1, OCS [Leisner (1988) Proc Nat Acad Sci USA 85(5):2553-2557], lib4, usp, mas {Comai (1990) Plant Mol Biol 15(3):373-381]:, STLS1, ScBV (Schenk (1999) Plant Mol Biol 39(6):1221-1230), B33, 25 SADI or SAD2 (flax promoters, Jain et aL. (1999) Crop Science 39(6) :1696-1701) or nos [Shaw et al. (1984) Nucleic Acids Res. 12(20):7831-7846]. An inducible promoter is a promoter where the rate of RNA polymerase binding and initiation is modulated by external stimuli. Such stimuli include light heat, anaerobic stress, alteration in nutrient conditions, presence or absence of a metabolite, presence of a ligand, microbial attack, 30 wounding and the like (for a review, see Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). Chemically inducible promoters are particularly suitable when it is desired to express the gene in a time-specific manner. Examples of such promoters are a salicylic acid inducible promoter (WO 95/19443), and abscisic acid-inducible promoter (EP 335 528), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 35 2:397-404), a cyclohexanol- or ethanol-inducible promoter (WO 93/21334) or others as described herein. A viral promoter is a promoter with a DNA sequence substantially similar to the promoter found at the 5' end of a viral gene. A typical viral promoter is found at the 5' end of the gene coding for the p21 protein of MMTV described by Huang et al. ((1981) Cell 27:245). A synthetic promoter is a promoter that was chemi 40 cally synthesized rather than biologically derived. Usually synthetic promoters incorpo rate sequence! changes that optimize the efficiency of RNA polymerase initiation. A temporally regulated promoter is a promoter where the rate of RNA polymerase binding and initiation is modulated at a specific time during development. Examples of tempo rally regulated promoters are given In Chua et al. [(1989) Science 244:174-181]. A spa 45 tially regulated promoter is a promoter where the rate of RNA polymerase binding and initiation is modulated in a specific structure of the organism such as the leaf, stem or root. Examples of spatially regulated promoters are given in Chua et al. [(1989) Sci ence 244:174-181]. A spatiotemporally regulated promoter is a promoter where the rate 21 of RNA polymerase binding and initiation is modulated in a specific structure of the organism at a specific time during development. A typical spatiotemporally regulated promoter is the EPSP synthase-35S promoter described by Chua et al. [(1989) Science 244:174-181]. Suitable promoters are furthermore the oilseed rape napin gene pro 5 moter (US 5,608,152), the Vicia faba USP promoter (Bumlein dt al. (1991) Mol Gen Genet 225(3):459-67), the Arabidopsis oleosin promoter (WO 98/45461), the Phaseo lus vulgaris phaseolin promoter (US 5,504,200), the Brassica Bce4 promoter (WO 91/13980), the bean arc5 promoter, the carrot DcG3 promoter, or the Legumin B4 pro moter (LeB4; Bumlein at al. (1992) Plant Journal 2(2):233-9), and promoters which 10 bring about the seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like. Advantageous seed-specific promoters are the sucrose binding protein promoter (WO 00/26388), the phaseolin promoter and the napin promoter, Suitable promoters which must be considered are the barley lpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230), and the promoters described in 15 WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene and the rye se calin gene). Further suitable promoters are Amy32b, Amy 6-6 and Aleurain [US 5,677,474], Bce4 (oilseed rape) [US 5,530,149], glycinin (soya) [EP 571 741], phos 20 phoenolpyruvate carboxylase (soya) [JP 06/62870], ADR12-2 (soya) [WO 98/08962], isocitrate lyase (oilseed rape) [US 5,689,040] or a-amylase (barley) [EP 781 849]. Other promoters which are available for the expression of genes in plants are leaf specific promoters such as those described in DE-A 19644478 or light-regulated pro moters such as, for example, the pea petE promoter. Further suitable plant promoters 25 are the cytosolic FBPase promoter or the potato ST-LSI promoter (Stockhaus at al. (1989) EMBO J. 8:2445), the Glycine max phosphoribosylpyrophosphate amidotrans ferase promoter (GenBank Accession No. U87999) or the node-specific promoter de scribed in EP A 0 249 676. Other suitable promoters are those which react to biotic or abiotic stress conditions, for example the pathogen-induced PRPI gene promoter 30 (Ward et aL. (1993) Plant. Mol. Biol. 22:361-366), the tomato heat-inducible hsp8o promoter (US 5,187,267), the potato chill-inducible alpha-amylase promoter (WO 96/12814) or the wound-inducible pinli promoter (EP-A-0 375 091) or others as de scribed herein. Other promoters, which are particularly suitable, are those that bring about plastid-specific expression. Suitable promoters such as 'the viral RNA poly 35 merase promoter are described in WO 95/16783 and WO 97/06250, and the Arabidop sis cipP promoter, which is described in WO 99/46394. Other promoters, which are used for the strong expression of heterologous sequences in as many tissues as pos sible, in particular also in leaves, are, in addition to several of the abovementioned viral and bacterial promoters, preferably, plant promoters of actin or ubiquitin genes such 40 as, for example, the rice acting promoter. Further examples of constitutive plant pro moters are the sugarbeet V-ATPase promoters (WO 01/14572). Examples of synthetic constitutive promoters are the Super promoter (WO 95/14098) and promoters derived from G-boxes (WO 94/12015). If appropriate, chemical inducible promoters may fur thermore also be used, compare EP-A 388186, EP-A 335528, WO 97/06268. The 45 above listed promoters can be comprise other regulatory elements that affect gene expression in response to plant hormones (Xu et al., 1994, Plant Cell 6(8):1077-1085) biotic or abiotic environmental stimuli, such as stress conditions, as exemplified by 22 drought (Tran et aL (2004) Plant Cell 16(9):2481-2498), heat, chilling, freezing, salt stress, oxidative stress (US 5,290,924) or biotic stressors like bacteria, fungi or viruses. Polypeptide, peptide, oligopeptide, gene product, expression product and protein: are 5 used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues. Recombinant or transgenic DNA expression construct: with respect to, for example, a nucleic acid sequence (expression construct, expression cassette or vector comprising 10 said nucleic acid sequence) refers to all those constructs originating by experimental manipulations in which either a) said nucleic acid sequence, or b) a genetic control sequence linked operably to said nucleic acid sequence (a), for example a promoter, or 15 c) (a) and (b) is not located in its natural genetic environment or has been modified by experimental manipulations, an example of a modification being a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment refers to the natural chromosomal locus in the organism of origin, or to the presence in 20 a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1,000 bp, very espe cially preferably at least 5,000 bp, in length. A naturally occurring expression construct 25 - for example the naturally occurring combination of a promoter with the corresponding gene - becomes a transgenic expression construct when It is modified by non-natural, synthetic "artificial' methods such as, for example, mutagenesis. Such methods have been described (US 5,565,350; WO 00/15815). Recombinant polypeptides or proteins: refer to polypeptides or proteins produced by recombinant DNA techniques, Le., pro 30 duced from cells transformed by an exogenous recombinant DNA construct encoding the desired polypeptide or protein. Recombinant nucleic acids and polypeptide may also comprise molecules which as such does not exist in nature but are modified, changed, mutated or otherwise manipulated by man, An important use of the intron sequences of the invention will be the enhancement of the expression of a nucleic acid 35 sequence, which encodes a particular protein, a polypeptide or DNA sequences that interfere with normal transcription or translation, e.g. interference- or antisense-RNA. In one embodiment of the present invention, the recombinant DNA expression construct confers expression of one or more nucleic acid molecules. Said recombinant DNA ex pression construct according to the invention advantageously encompasses a promoter 40 functioning in plants, additional regulatory or control elements or sequences functioning in plants, an intron sequence with expression enhancing properties in plants and a ter minator functioning in plants. Additionally, the recombinant expression construct might contain additional functional elements such as expression cassettes conferring expres sion of e.g. positive and negative selection markers, reporter genes, recombinases or 45 endonucleases effecting the production, amplification or function of the expression cassettes, vectors or recombinant organisms according to the invention. Further more, the recombinant expression construct can comprise nucleic acid sequences 23 homologous to a plant gene of interest having a sufficient length in order to induce a homologous recombination (HR) event at the locus of the gene of interest after intro duction in the plant. A recombinant transgenic expression cassette of the invention (or a transgenic vector comprising said transgenic expression cassette) can be produced 5 by means of customary recombination and cloning techniques as are described (for example, in Maniatis 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Silhavy 1984, ) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and in Ausubel 1987, Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley 10 Interscience). The introduction of an expression cassette according to the invention into an organism or cells, tissues, organs, parts or seeds thereof (preferably into plants or plant cells, tissue, organs, parts or seeds) can be effected advantageously using vec tors, which comprise the above described nucleic acids, promoters, introns, termina tors, regulatory or control elements and functional elements. 15 Regeneration: as used herein, means growing a whole plant from a plant cell, a group of plant coils, a plant part or a plant piece (e.g., from a protoplast, callus, protocorm-like body, or tissue part). Regulatory sequence: refers to promoters, enhancer or other segments of DNA where 20 regulatory proteins such as transcription factors bind and thereby influencing the tran scription rate of a given gene. Substantially all introns of a plant genome represented in a genomic DNA sequence database or genomic DNA library: refers to more than 80%, preferably to more than 25 90%, more preferably to .more than 95%, still more preferably more than 98% of all introns present in the genome of the plant used as a source for the preparation of the genomic DNA sequence database or genomic DNA library. The construction of ge nomic libraries and the subsequent sequencing of the genomic DNA and the construc tion of a genomic or genome DNA sequence database using the obtained sequence 30 information is well established in the art (Mozo at al. (1998) Mol. Gen. Genet. 258:562 570; Choi et al. (1995) Weeds World 2 :17-20; Lui et al. (1999) Proc. Nati. Acad. Sci. USA 96:6535-6540; The Arabidopsis Genome initiative, Nature 402:761-777 (1999); The Arabidopsis Genome Initiative, Nature 408:796-826 (2000). 35 Structural gene: as used herein is intended to mean a DNA sequence that is tran scribed into mRNA which is then translated into a sequence of amino acids characteris tic of a specific polypeptide. Sufficient length: with respect to a homology sequence comprised in a DNA-construct 40 (e.g., the homology sequence A or B) is to be understood to comprise sequences of a length of at least 100 base pair, preferably at least 250 base pair, more preferably at least 500 base pair, especially preferably at least 1,000 base pair, most preferably at least 2,500 base pair. The term "sufficient homology' with respect to a homology se quence comprised in a DNA-construct (e.g., the homology sequence A or B) is to be 45 understood to comprise sequences having a homology to the corresponding target sequence comprised in the chromosomal DNA (e.g., the target sequence A or B ) of at least 70 %, preferably at least 80 %, more preferably at least 90 %, especially prefera 24 bly at least 95 %, more especially preferably at least 99%, most preferably 100 %, wherein said homology extends over a length of at least 50 base pair, preferably at least 100 base pair, more preferably at least 250 base pair, most preferably at least 500 base pair. 5 Target region/sequence: of a nucleic acid sequence is a portion of a nucleic acid se quence that is identified to be of Interest. A "coding region" of a nucleic acid sequence is the portion of the nucleic acid sequence, which is transcribed and translated in a sequence-specific manner to produce into a particular polypeptide or protein when 10 placed under the control of appropriate regulatory sequences. The coding region is said to encode such a polypeptide or protein. Tissue: with respect to a plant (or "plant tissue') means arrangement of multiple plant cells including differentiated and undifferentiated tissues of plants. Plant tissues may 15 constitute part of a plant organ (e.g., the epidermis of a plant leaf) but may also consti tute tumor tissues and various types of cells In culture (e.g., single cells, protoplasts, embryos, calli, iprotocorm-like bodies, etc.). Plant tissue may be in plant, in organ cul ture, tissue culture, or cell culture. 20 Transforming or transformation: as used herein refers to the introduction of genetic material (e.g., a transgene) into a cell. Transformation of a cell may be stable or transient. The term "transient transformation" or "transiently transformed" refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome. Transient transformation may be detected by, 25 for example, enzyme-linked immunosorbent assay (ELISA) which detects the presence of a polypeptide encoded by one or more of the transgenes. Alternatively, transient transformation may be detected by detecting the activity of the protein (e-g., p glucuronidase) encoded by the transgene (e.g., the uldA gene) as demonstrated herein [e.g., examples 1.6 and 2.4, histochemical assay of GUS enzyme activity by staining 30 with X-gluc which gives a blue precipitate in the presence of the GUS enzyme; and a chemiluminescent assay of GUS enzyme activity using the GUS-Light kit (Tropix)]. The term "transient transformant" refers to a cell which has transiently incorporated one or more transgenes. In contrast the term "stable transformation" or stablyy transformed" refers to the introduction and integration of one or more transgenes into the genome of 35 a cell, preferably resulting in chromosomal integration and stable heritability through meiosis. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences, which are capable of binding to one or more of the transgenes. Alternatively, stable transformation of a cell may also be detected by the polymerase chain reaction of genomic DNA of the cell to amplify 40 transgene sequences. The term "stable transformant" refers to a cell that has stably integrated one or more transgenes Into the genomic DNA. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene. Transformation also includes introduction 45 of genetic material into plant cells In the form of plant viral vectors involving extrachromosomal replication and gene expression, which may exhibit variable properties with respect to meiotic stability. 25 Transgenic or recombinant when used in reference to a cell refers to a cell which con tains a transgene, or whose genome has been altered by the introduction of a trans gene. The term "transgenic" when used in reference to a tissue or to a plant refers to a tissue or plant, respectively, which comprises one or more cells that contain a trans 5 gene, or whose genome has been altered by the introduction of a transgene. Trans genic cells, tissues and plants may be produced by several methods including the in troduction of a "transgene" comprising nucleic acid (usually DNA) into a target cell or integration of the transgene into a chromosome of a target cell by way of human inter vention, such as by the methods described herein, 10 Wild-type, natural or of natural origin: means with respect to an organism, polypeptide, or nucleic acid sequence, that said organism polypeptide, or nucleic acid sequence is naturally occurring or available in at least one naturally occurring organism polypeptide, or nucleic acid sequence which is not changed, mutated, or otherwise manipulated by 15 man. Vector: is a DNA molecule capable of replication in a host cell. Plasmids and cosmids are exemplary vectors. Furthermore, the terms "vector and "vehicle" are used inter changeably in reference to nucileic acid molecules that transfer DNA segment(s) from 20 one cell to another, whereby the cells not necessarily belonging to the same organism (e.g. transfer of a DNA segment form an Agrobecterium cell to a plant cell). The term "expression vector" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences neces 25 sary for the expression of the operably linked coding sequence in a particular host or ganism. DETAILED DESCRIPTION OF THE INVENTION The teaching of the present invention enables the identification of introns causing intron mediated enhancement (IME) of gene expression. Furthermore, the present invention 30 provides isolated plant introns that, if functionally combined with a promoter functioning in plants and a nucleic acid fragment, can enhance the expression rate of said nucleic acid in a plant or a plant cell. A first embodiment of the present invention relates to a method for identifying an in 35 tron with plant gene expression enhancing properties comprising selecting an intron from a plant genome, wherein said intron is characterized by at least the following fea tures 1) an intron length shorter than 1,000 base pairs, and II) presence of a 5' splice site comprising the dinucleotide sequence 5'-GT-3' (SEQ 40 ID NO: 78), and Ill) presence of a 3' splice site comprising the trinucleotide sequence 5'-CAG-3' (SEQ ID NO: 79), and IV) presence of a branch point resembling the consensus sequence 5'-CURAY-3' (SEQ ID NO:75) upstream of the 3'splice site, and 45 V) an adenine plus thymine content of at least 40% over 100 nucleotides down stream from the 5' splice site, and 26 VI) an adenine plus thymine content of at least 50% over 100 nucleotides upstream from the 3' splice site, and VII) an adenine plus thymine content of at least 50%, and a thymine content of at least 30% over the entire intron. 5 In another embodiment, the invention relates to a method for enriching the number of introns with expression enhancing properties In plants in a population of plant introns to a percentage of at least 50% of said population, said method comprising selecting in trons from said population, said introns are characterized by at least the following fea 10 tures I) an intron length shorter than 1,000 base pairs, and II) presence of a 5' splice site comprising the dinucleotide sequence 5'-GT-3' (SEQ ID NO: 78), and 111) presence of a 3' splice site comprising the trinucleotide sequence 5'-CAG-3' 15 (SEQ ID NO: 79), and IV) presence of a branch point resembling the consensus sequence 5-CURAY-3' (SEQ ID NO:75) upstream of the 3'splice site, and V) an adenine plus thymine content of at least 40% over 100 nucleotides down stream from the 5' splice site, and 20 VI) an adenine plus thymine content of at least 50% over 100 nucleotides upstream from the 3' splice site, and VII) an adenine plus thymine content of at least 50%, and a thymine content of at least 30% overthe entire intron. 25 The inclusion of any of the inventive introns described by SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12,13,14, 15,16,17,18, 19, 20, 21 or 22 into the 5' untranslated region (UTR) of the p-glucuronidase gene (GUS) driven by the Zea mays Ubiquitin promoter has led to strong expression enhancement of the reporter gene in maize protoplasts (Black Mexican Sweet) suspension cells and stable transformed plants (see examples). Fur 30 thermore, it could be shown that the gene expression enhancement properties of said introns are comparable to those known from the literature (e.g. the first intron of the Zea mays Ubiquitin gene, used as positive control in the expression assays). In a preferred embodiment, the number of introns - with gene expression enhancing 35 properties identified within a population of introns by applying the method of the in vention for enrichment is enriched to a percentage of at least 50%, preferably at least 55%, more preferably at least 60%, especially preferably at least 65%, or very espe cially preferably at least 70% (i.e., a given population of 100 introns pre-selected by using the inventive method will comprise at least 50, preferably at least 55, more pref 40 erably at least 60, especially preferably at least 65 or 70 introns with gene expression enhancing properties). More preferably, the number of introns - with gene expression enhancing properties identified within a population of introns by applying the method of the invention for enrichment is enriched to a percentage of at least 50%, wherein the selected introns, if part of an recombinant DNA expression construct leads to an in 45 crease in the gene expression of a given gene of at least 300% compared to the oth erwise identical expression construct lacking the intron under otherwise unchanged conditions. Most preferably, the enrichment is at least 60% percent, wherein the se lected introns, increasing the transcription of a gene driven by a given promoter of at 27 least 200%.Especially preferably, the enrichment is at least 70%, wherein the selected introns, increasing the transcription of a gene driven by a given promoter of at least 50%. 5 Preferably, the length of an inventive IME-intron is preferably shorter than 1,000 base pairs, more preferably shorter than 900 bp, most preferably shorter than 800 bp. In a preferred embodiment, the branchpoint sequence of the Intron identified by a method of the invention is described by the nucleotide sequences 5'-CURAY-3' (SEQ ID NO. 75) or 5'-YURAY-3' (SEQ ID NO. 76), wherein the U and A are essential nucleotides, and 10 purines and pyrimidines are preferred nucleotides at positions 3 and 5 respectively. In position 1, pyrimidines are preferred but also C is preferred to U. The sequence context of the 5 splice-site surrounding the GT dinucleotide may vary. Preferred are 5 splice sites of the sequence 5'-RR/GT(RTXRT)(GY)-3' (SEQ ID NO. 77), wherein R stands for the nucleotides G or A, Y stands for the nucleotides C or T. The nucleotides given in 15 brackets describing alternative nucleotides at the respective position. In a preferred embodiment of the invention, the adenine/thymine (AT) content of an inventive intron over the entire sequence is at least 50%, more preferably at least 55%, even more preferably at least 60%. 20 In a preferred embodiment of the invention the populations of plant introns to which the inventive methods will be applied comprises a) substantially all introns of a plant ge nome represented in a DNA sequence database or b) a plant genomic DNA library. In an additional embodiment of the invention, the population of introns to which the inven 25 five methods will be applied to is selected from the group consisting of a) introns lo cated between two protein encoding exons, and b) introns located within the 5' un translated region of the corresponding gene. In order to identify an intron with expres sion enhancing properties in plants or plant cells located within a coding region (be tween two protein encoding exons) or in the 5'untranslated region of a given gene, the 30 coding regions and the 5' untranslated regions from a set of genes (e.g., present in a sequence database) can be screened for the presence of intros located in said re gions and the identified introns are subsequently screened using: one of the inventive methods. Such an in silicon identification process using bioinformatics tools known to the persons skilled in the art can be performed by screening a) specific DNA sequence 35 databases (e.g., containing solely coding regions or the 5' untranslated regions), or b) other publicly accessible genomic DNA sequences containing databases. In a pre ferred embodiment of the invention, the introns with expression enhancing properties located in the 5'untranslated regions are identified by a method comprising the steps of: 40 a. identifying a coding sequences within a set of genes present in a sequence data base, and b. identifying EST sequences corresponding to the genes identified under (a), and c. comparing said coding sequences and EST sequences with the genomic sequence of the respective genes, and 45 d. selecting EST sequences comprising the 5' untranslated region, and e. Identifying introns located in said 5' untranslated regions. 28 Preferably, the steps of retrieving or generating DNA sequences or the generation of specific DNA sequence database and screening the same (e.g. using the criteria ac cording to the inventive methods) can be performed with the aid of appropriate bioin formatic computer algorithms and appropriate computer devices known to a skilled 5 person. In a preferred embodiment, the introns where selected from a population of introns derived from monocotyledonous plants, especially preferred are monocotyle donous plants selected from the group consisting of the genera Hordeum, Avena, So cale, Triticum, Sorghum, Zea, Saccharum and Oryza. 10 In a furthermore preferred embodiment of the invention, the population of introns to which the inventive methods will be applied are selected from a population of plant genes representing the 10% fraction (9* decile) of genes with the highest expression rate in a gene expression analysis experiment performed using a plant cell, plant tissue or a whole plant. 15 To allow the determination of gene expression levels, a number of different techniques have been proposed (Milosavljevic, A. at al. (1996) Genome Res. 6:132 141; Shoe maker, D. et al. (1996) Nature Genet. 14:450 456; Sikela,J.M. and Auffray,C. (1993) Nature Genet. 3:189 191; Meier-Ewert S. et al. (1998) Nucleic Acids Research 20 26(9):2216-2223). Therefore, a number of different gene expression analysis systems could be employed in accordance with the instant invention, including, but not limited to microarray analysis, "digital northern', clone distribution analysis of cDNA libraries us ing the "DNA sequencing by hybridization method' (Strezoska, Z. at al. (1991) Proc. Nati. Acad. Sci USA 88:10089-10093) and Serial Analysis of Gene Expression (SAGE, 25 Velculescu, V. E. at al. (1995) Science 270:484-487). By using the cDNA microarray hybridization technology the expression profiles of thou sands of genes can be monitored at once. The DNA array analysis has become a standard technique in the molecular biology laboratory for monitoring gene expression. 30 Arrays can bemade either by the mechanical spotting of pre-synthesized DNA prod ucts or by the de novo synthesis of oligonucleotides on a solid substrate, usually a de rivatized glass slide. Typically arrays are used to detect the presence of mRNAs that may have been transcribed from different genes and which encode different proteins. The RNA is extracted from many cells, or from a single cell type, then converted to 35 cDNA or cRNA. The copies may be "amplified" by (RT-) PCR. Fluorescent tags are enzymatically incorporated into the newly synthesized strands or can be chemically attached to the new strands of DNA or RNA. A CDNA or cRNA molecule that contains a sequence complementary to one of the single-stranded probe sequences will hybridize, or stick, via base pairing to the spot at which the complementary probes are affixed. 40 The spot will then fluoresce when examined using a microarray scanner. Increased or decreased fluorescence intensity indicates that cells in the sample have recently tran scribed, or ceased transcription, of a gene that contains the probed sequence. The intensity of the fluorescence is proportional to the number of copies of a particular mRNA that were present and thus roughly indicates the activity or expression level of 45 that gene. Microarrys (and the respective equipment needed to perform the expression analysis experiments) that can be employed in accordance with the present invention are commercially available. The GeneChip Arabidopsis ATHI Genome Array, pro duced from Affimetrix (Santa Clara, CA), contains more than 22,500 probe sets repre 29 senting approximately 24,000 genes. The array is based on information from the inter national Arabidopsis sequencing project that was formally completed in December 2000 (http://www.affymetrix. com). Thus, the expression rate of the analyzed genes can be ranked (according to the intensity of the fluorescence of the respective genes 5 after the hybridization process) and the genes belonging to the 10% of genes showing the highest gene expression rate can be identified by using microarray analysis. Databases containing microarray expression profiling results are publicly available via the internet e.g. the Nottingham Arabidopsis Stock Center s microarray database or the 10 OSMID (osmotic stress microarray information) database. The Nottingham Arabidopsis Stock Center s microarray database containing a wide selection of microarray data from Affimetrix gene chips (http://affymetrix.arabidopsis. info). The OSMID database (http:/www.osmid.org) contains the results of approximately 100 microarray experi ments performed at the University of Arizona. This includes analysis of NaCI, cold, and 15 drought treatments of Arabidopsis thaliana, rice (Oyza sativa), barley, (Hordeum vul garis), ice plant (Mesembryanthemum crystallinum), and com (Zea mays). Thus, by using the expression profiles present in sequence/expression databases the expres sion rate of genes can be ranked (according to the clone distribution of the respective cDNA in the library) and genes belonging to the 10% of genes showing the highest 20 (abundance) gene expression rate can be identified. "Digital Northern are generated by padly sequencing thousands of randomly se lected clones from relevant cDNA libraries. Differentially expressed genes can then be detected from variations in the counts of their cognate sequence tags. The sequence 25 tag-based method consists of generating a large number (thousands) of expressed sequence tags (ESTs) from 3'-directed regional non-normalized cDNA libraries. The concept of a "digital Northern comparison is the following: a number of tags is re ported to be proportional to the abundance of cognate transcripts in the tissue or cell type used to make the cDNA library. The variation in the relative frequency of those 30 tags, stored in computer databases, is then used to point out the differential expression of the corresponding genes (Okubo et a!. 1992; Matsubara and Okubo 1994). The SAGE method is a further development of this technique, which requires only nine nu cleotides as a tag, therefore allowing a larger throughput. Thus, the expression rate of the analyzed genes by using the "digital Northem' method can be ranked (according to 35 the abundance of the tags of the respective gene in the cDNA library) and the genes belonging to the 10% of genes showing the highest (abundance) gene expression rate can be Identified. Using the "sequencing by hybridization method' described in the US patents US 40 5,667,972, US 5,492,806, US 5,695,940, US 5,972,619, US 6,018,041, US 6,451,996, US 6,309,824 it is possible to perform in sllico clone distribution analysis of complete cDNA libraries. The entire content of said US patents is incorporated by reference. This technology is commercially available and customized experiments can be con ducted in collaboration with the company HySeq Inc.. To determine clone distribution 45 by using the "sequencing by hybridization method', or "HySeq-technology' plants are grown under a variety of conditions and treatments, and then tissues at different devel opmental stages are collected. This is done in a strategic manner so the probability of harvesting all expressible genes in at least one or more of the libraries is maximized. 30 mRNA is then extracted from each of the collected samples and used for the library production. The libraries can be generated from mRNA purified on oligo dT columns. Colonies from transformation of the cDNA library into E.coll are randomly picked and placed into microtiter plates and subsequently spotted DNA onto a surface. The cDNA 5 inserts from each clone from the microtiter plates are PCR amplified and spotted onto a nylon membrane. A battery of 288 33 -P radiolabeled seven-mer oligonucleotides are then sequentially hybridized to the membranes. After each hybridization a blot image is captured during a phosphorimage scan to generate a profile for each single oligonu cieotide. Absolute identity is maintained by barcoding for image cassette, filter and ori 10 entation within the cassette. The filters are then treated using relatively mild conditions to strip the bound probes and then returned to the hybridization chambers for another round. The hybridization and imaging cycle is repeated until the set of 288 oligomers is completed. After completion of hybridizations, each spot (representing a cDNA insert) will have recorded the amount of radio signal generated from each of the 288 seven 15 mer oligonucleotides. The profile of which oligomers bound, and to what degree, to each single cDNA insert (a spot on the membrane) is defined as the signature gener ated from that clone. Each clone's signature is compared with all other signatures gen erated from the same organism to identify clusters of related signatures. This process "sorts' all of the clones from an organism into so called "clusters' before sequencing. 20 In the clustering process, complex or tissue specific cDNA libraries are "mined' using a series of 288 seven base-pair oligonucleotides. By collecting data on the hybridization signature of these oligos, the random set of clones in a lbrary can be sorted into "clus ters'. A cluster is indicative for the abundance of each gene in a particular library and is therefore a measure of the gene expression rate of an individual gene. Thus, the 25 expression rate of genes can be ranked using the 'HySeq' technology and the genes belonging to the 10% of genes showing the highest (abundance) gene expression rate can be identified. The genes, cDNAs or expressed sequence tags chosen for the identification of the 30 inventive introns, belonging to the 10%, preferably 5%, more preferably 3% most pref erably 1% of genes showing the highest gene expression rate in a gene expression analysis experiment, wherein the gene expression rate can be calculated indirectly by using the above described methods. In a preferred embodiment of the invention, the nucleic acid sequences of the genes belonging to the 10% of genes showing the high 35 est gene expression rate where used to isolate the complete genomic DNA sequence including the intron sequences- of the respective genes by screening of e.g. appropri ate DNA sequences containing databases, or genomic DNA or genomic DNA libraries using hybridization methods or RACE cloning techniques (rapid amplification of cDNA ends), or chromosome walking techniques. After sequence determination of the iso 40 lated complete genomic DNA of the respective candidate gene, the intron sequences present in said genes were screened using the above described criteria to identify those introns, having expression enhancing properties. The described in silco methods for the selection of introns with expression enhancing properties have a high probability of success, but the efficiency of the described methods may be further increased by 45 combination with other methods. Therefore, in one preferred embodiment of the inven tion independent validation of the genes representing the 10% of genes showing the highest gene expression rate in a gene expression analysis experiment is done using 31 alternative gene expression analysis tools, like Northern analysis, or real time PCR analysis (see examples). In a preferred embodiment of the invention the method for the identification or enrich 5 merit of introns with gene expression enhancing properties in plants is applied to DNA sequence databases using an automated process, more preferably using a computer device and an algorithm that defines the instructions needed for accomplishing the se lection steps for identifying or enriching introns with gene expression enhancing proper ties in plants within the screened population of DNA sequences. A further embodiment 10 of the invention is a computer algorithm that defines the instructions needed for ac complishing the selection steps for identifying or enriching Introns with plant gene ex pression enhancing properties as described above. Useful computer algorithms are well known in the art of bioinformatics or computational biology. Bloinformatics or com putational biology is the use of mathematical and informational techniques to analyze 15 sequence data (e.g. generation of sequence data, sequence alignments, screening of sequence data) usually by creating or using computer programs, mathematical models or both. One of the main areas of bloinformatics is the data mining and analysis of data gathered from different sources. Other areas are sequence alignment, protein structure prediction. Another aspect of bioinformatics in sequence analysis is the automatic 20 search for genes or regulatory sequences within a genome (e.g. intron sequences within a stretch of genomic DNA), Sequence databases can be searched using a vari ety of methods. The most common is probably searching for a sequence similar to a certain target gene whose sequence is already known to the user. A useful program is the BLAST (Basic Local Alignment Search Tool) program a method of this type. 25 BLAST is an algorithm for comparing biological sequences, such as DNA sequences of different genes. Given a library or database of sequences, a BLAST search enables a researcher to look for specific sequences. The BLAST algorithm and a computer pro gram that implements it were developed by Stephen Altschul at the U.S. National Cen ter for Biotechnology Information (NCBI) and is available on the web at 30 http://www.ncbi.nlm.nih.gov/BLAST. The BLAST program can either be downloaded and run as a command-line utility "blastall" or accessed for free over the web. The BLAST web server, hosted by the NCBI, allows anyone with a web browser to perform similarity searches against constantly updated databases of proteins and DNA that include most of the newly sequenced organisms. BLAST is actually a family of pro 35 grams (all included in the blastall executable) including beside others the Nucleotide nucleotide BLAST (BLASTN). This program, given a DNA query, returns the most simi lar DNA sequences from the DNA database that the user specifies. A person skilled in the art knows how to produce or retrieve sequence Data from e.g. public sequence database and to design algorithms to screen the set of sequences in a customized way 40 (see examples). Additionally, the invention relates to computer algorithm that defines the instructions needed for accomplishing the selection steps for identifying or enriching introns with gene expression enhancing properties in plants from a plant genome or a population of 45 introns selected from the group consisting of introns located between two protein en coding exons, and/or introns located within the 5' untranslated region of the corre sponding gene and/or introns located in the DNA sequences of genes representing the 10% fraction of genes with the highest expression rate in a gene expression analysis 32 experiment performed using a plant cell, plant tissue and/or a whole plant. Another embodiment of the invention is a computer device or data storage device comprising the algorithm. A storage device can be a hard disc" (or "hard drive") or an optical data storage medium like a CD-ROM ("Compact Disc Read-Only Memory" (ROM) or DVD 5 (digital versatile disc) or any other mechanically, magnetically, or optically data storage medium. Another embodiment of the invention relates to a method for isolating, providing or pro ducing an intron with gene expression enhancing properties in plants comprising the 10 steps of a) performing an identification or enrichment of introns with gene expression enhancing properties in plants as described above and providing the sequence information of said identified or enriched introns, and b) providing the physical nucleotide sequence of said introns identified or enriched un 15 dera)and c) evaluating the gene expression enhancing properties of the intron sequence pro vided under b) in an in vivo or in vitro expression experiment, and d) isolating introns from said expression experiment c), which demonstrate expression enhancing properties. 20 Preferably, evaluation of the gene expression enhancing properties of the isolated in trons comprises, ci) providing a recombinant expression constructs by functionally linking an individual nucleotide sequence from step b) with at least one promoter sequence functioning 25 in plants or plant cells, and at least one readily quantifiable nucleic acid sequence, and c2) introducing said recombinant DNA expression construct in plant cells and evaluat ing the gene expression enhancing properties of the isolated intron. 30 Preferably, the evaluation of the gene expression enhancing properties is done in a plant cell or stable transformed plants and wherein said isolated intron enhances ex pression of a given gene at least twofold (see examples). An additional subject matter of the invention relates to a recombinant DNA expression 35 construct comprising at least one promoter sequence functioning in plants cells, at least one nucleic acid sequence and at least one intron selected from the group con sisting of the sequences described by SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22, and functional equivalents thereof, wherein said promoter sequence and at least one of said intron sequences are functionally linked to 40 said nucleic acid sequence and wherein said intron is heterologous to said nucleic acid sequence or to said promoter sequence. Furthermore, the invention relates to recom binant expression constructs comprising at least one promoter sequence functioning in plants cells, at least one nucleic acid sequence and at least one functional equivalents of an intron described by any of sequences SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 45 14,15,16,17,18,19, 20, 21 and 22. 33 Preferably, said functional equivalents comprising the functional elements of an intron, wherein said promoter sequence and at least one of said intron sequences are func tionally linked to said nucleic acid sequence and wherein said intron is heterologous to said nucleic acid sequence or to said promoter sequence. More 'preferably, the func 5 tional equivalent is further characterized by i) having at least 50 consecutive base pairs of the intron sequence described by any of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, or ii) having an identity of at least 80% over a sequence of at least 95 consecutive nu 10 cleic acid base pairs to a sequences described by any of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 or iii) hybridizing under high stringent conditions with a nucleic acid! fragment of at least 50 consecutive base pairs of a nucleic acid molecule described by any of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, 15 In a preferred embodiment of the invention, the introns comprising at least 50 bases pairs, more preferably at least 40 bases pairs, most preferably 30 bases pairs of the sequences/exons 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respec tively. In another embodiment of the in, the recombinant DNA expression construct of 20 the invention further comprises one or more additional regulatory sequences function ally linked to a promoter. Those regulatory sequences can be selected from the group consisting of heat shock-, anaerobic responsive-, pathogen responsive-, drought re sponsive-, low temperature responsive-, ABA responsive-elements, 5 -untranslated gene region, 3 -untranslated gene region, transcription terminators, polyadenylation 25 signals and enhancers. Cis- and trans-acting factors Involved in ABA-induced gene expression have been reviewed by Bray (1997) Trends Plant Sci. 2:48 54; Busk at at (1998) Plant Mol. Biol. 37:425 435 and Shinozaki and Yamaguchi-Shinozaki (2000) Curr. Opin. Plant Biol. 3:217 223). Many ABA-inducible genes contain a conserved, ABA-responsive, cis-acting element named ABRE (ABA-responsive element; 30 PyACGTGGC) in their promoter regions (Guiltinan et al. (1990) Science 250 :267 271; Mundy et at (1990) Proc. Natt Acad. ScL USA 87:406 410). The promoter region of the rd29A gene was analyzed, and a novel cis-acting element responsible for dehydra tion- and cold-induced expression was identified at the nucleotide sequence (Yamagu chi-Shinozaki and Shinozaki (1994) Plant Cell 6:251 264.). A 9-bp conserved se 35 quence, TACCGACAT, termed the dehydration-responsive element (DRE), is essential for the regulation of dehydration responsive gene expression. DRE-related motifs have been reported in the promoter regions of cold- and drought-inducible genes such as kin1, cor6.6, and rIT17 (Wang et al. (1995) Plant Mol. Biol. 28:60|5 617; Iwasaki et at (1997) Plant Physiol. 115:1287). The thermoinducibility of the heat shock genes is at 40 tributed to activation of heat shock factors (HSF). HSF act through a highly conserved heat shock promoter element (HSE) that has been defined as adjacent and inverse repeats of the motif 5'-nGAAn-3' (Amin et al (1988) Mol Cell Biol 8:3761-3769). Exam ples for defense or pathogen response elements are the W-box (TTGACY) and W-box like elements, representing binding sites for plant-specific WRKY transcription factors 45 involved in plant development and plant responses to environmental stresses (Eulgem et al. (2000) Trends Plant Sci 5:199 206; Robatzek Set aL (2001) Plant J 28:123 133), and the Myc-element (CACATG) (Rushton PJ et at (1998) Curr Opin Plant Biol 1:311 315). Such regulatory sequences or elements that can be employed in con 34 junction with a described promoter, encompass the 5 -untranslated regions, enhan cer sequences and plant polyadenylation signals. Examples of translation enhan cers, which may be mentioned, are the tobacco mosaic virus 5 leader sequence (Gal lie et al (1987) Nucl Acids Res 15:8693-8711), the enhancer from the octopine syn 5 thase gene and the like. Furthermore, they may promote tissue specificity (Rouster J et al. (1998) Plant J 15:435-440). The recombinant DNA expression construct will typically include the gene of interest along with a 3' end nucleic acid sequence that acts as a signal to terminate transcription and subsequent polyadenylation of the RNA. Preferred plant polyadenylation signals are those, which essentially correspond to T-DNA 10 polyadenylation signals from Agrobacterum tumefaciens, in particular gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACHS (Gielen et al (1984) EMBO J 3:835-46) or functional equivalents thereof. Examples of terminator sequences, which are especially suitable, are the OCS (octopin synthase) terminator and the NOS (nopaline synthase) terminator. An expression cassette and the vectors de 15 rived from it may comprise further functional elements. The term functional element is to be understood in the broad sense and refers to all those elements, which have an effect on the generation, amplification or function of the expression cassettes, vectors or recombinant organisms according to the invention. The following may be mentioned by way of example, but not by limitation: 20 1. Selection markers Selection markers are useful to select and separate successfully transformed or homologous recombined cells. To select cells which have successfully undergone homologous recombination, or else to select transformed cells, It Is, also typically nec 25 essary to introduce a selectable marker, which confers resistance to a biocide (for ex ample herbicide), a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic to the cells which have successfully undergone recombina tion. The selection marker permits the selection of the transformed cells from untrans formed ones (McCormick et al. (1986) Plant Cell Reports 5:81-84). 30 1.1 Negative selection markers Selection markers confer a resistance to a biocidal compound such as a metabolic inhibitor (e.g, 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin 35 or glyphosate). Especially preferred negative selection markers are those which confer resistance to herbicides. Examples which may be mentioned are: - Phosphinothricin acetyltransferases (PAT; also named Bialophos resistance; bar; de Block et al. (1987) EMBO J 6:2513-2518) - 5-enolpyruvyishikimate-3-phosphate synthase (EPSPS) confer- ring resistance to 40 Glyphosate (N-(phosphonomethyl)glycine), - Glyphosate degrading enzymes (Glyphosate oxidoreductase; gox), - Dalapon inactivating dehalogenases (deh) - sulfonylurea- and imidazolinone-inactivating acetolactate syntheses (for example mutated ALS variants with, for example, the S4 and/or Hra mutation) 45 - Bromoxynil degrading nitrilases (bxn) - Kanamycin- or G418- resistance genes (NPTIl; NPTI) coding e.g., for neomycin phosphotransferases, 35

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2 -Desoxyglucose-6-phosphate phosphatase (DOGRI-Gene product; WO 98/45456; EP 0 807 836) conferring resistance against 2 -desokyglucose (Randez Gil et aL, 1995 Yeast 11:1233-1240). Additional suitable negative selection marker are the aadA gene, which confers 5 resistance to the antibiotic spectinomycin, the streptomycin :phosphotransferase (SPT) gene, which allows resistance to streptomycin and the hygromycin phos photransferase (HPT) gene, which mediates resistance to hygromycin. Especially preferred are negative selection markers that confer resistance against the toxic effects imposed by D-amino acids like e.g., D-alanine and D-serine (WO 03/060133; Erikson 10 2004). Especially preferred as negative selection marker in this contest are the daol gene (EC: 1.4. 3.3 : GenBank Acc.-No.: U60066) from the yeast Rhodotorua gracilis (Rhodosporidium toruloides) and the E. coli gene dsdA (D-serine dehydratase (D serine deaminase) [EC: 4.3.1.18; GenBank Acc.-No.: J01603). 15 1.2) Counter selection marker Counter selection markers are especially suitable to select organisms with defined deleted sequences comprising said marker (Koprek T et al. (1999) Plant J 19(6): 719-726). Examples for counter selection marker comprise thymidin kinases (TK), cytosine deaminases (Gleave AP et aL. (1999) Plant Mol Biol. 40(2):223-35; Perera RJ 20 pt al. (1993) Plant Mol. Biol 23(4): 793-799; Stougaard J. (1993) Plant J 3:755-761), cytochrom P450 proteins (Koprek et al. (1999) Plant J 16:719-726), haloalkandehalo genases (Naested H (1999) Plant J 18:571-576), iaaH gene products (Sundaresan V at al. (1995) Genes & Development 9:1797-1810), cytosine deaminase codA (Schlaman HRM and Hooykaas PJJ (1997) Plant J 11:1377-1385), or tms2 gene products (Fe 25 doroff NV & Smith DL, 1993, Plant J 3:273- 289). 1.3 Positive selection marker Furthermore, positive selection marker can be employed. Genes like isopentenyltrans ferase from Agrobacterium tumefaciens (strain:P022; Genbank Acc.-No.: AB025109) 30 may as a key enzyme of the cytokinin biosynthesis facilitate regeneration of trans formed plants (e.g., by selection on cytokinin-free medium). Corresponding selection methods are described (Ebinuma 2000a,b). Additional positive selection markers, which confer a growth advantage to a transfon-ned plant in comparison with a non transformed one, are described e.g., in EP-A 0 601 092. Growth stimulation selection 35 markers may include (but shall not be limited to) p-Glucuronidase (in combination with e.g., a cytokinin glucuronide), mannose-6-phosphate isomerase (in combination with mannose), UDP-galactose-4-epimerase (in combination with e.g., galactose), wherein mannose-6-phosphate isomerase in combination with mannose is especially preferred. 40 2) Reporter genes Reporter genes encode readily quantifiable proteins and, via their color or enzyme activity, make possible an assessment of the transformation effi cacy, the site of expression or the time of expression. Very especially preferred in this context are genes encoding reporter proteins (Schenbom E and Groskreutz D. (1999) 45 Mol Biotechnol. 13(1):29-44) such as the green fluorescent protein (GFP) (Sheen et al. (1995) Plant Joumal 8(5):777-784; Haseloff et al. (1997) Proc Natl Acad Sci USA 94(6):2122-2127; Reichel et al. (1996) Proc Natl Acad Sci USA 93(12):5888-5893; Tian et al. (1997) Plant Cell Rep 16:267-271;WO 97/41228; Chui WL et al. (1996) Curr Biol 36 6:325-330; Leffel SM et al. (1997) Biotechniques. 23(5):912-8), chloramphenicol transferase, a luciferase (Ow et al. (1986) Science 234:856-859; Millar et al. (1992) Plant Mol Biol Rep 10:324-414), the aequorin gene (Prasher et a. (1985) Biochem Biophys Res Commun 126(3):1259-1268), l1 galactosidase, R locus gene (encoding a 5 protein which regulates the production of anthocyanin pigments (red coloring) in plant tissue and thus makes possible the direct analysis of the promoter activity without addi tion of further auxiliary substances or chromogenic substrates; Dellaporta et al. (1988) in: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genet ics Symposium 11:263-282), with B glucuronidase being very especially preferred (Jef 10 ferson et al. (1987) EMBO J. 6:3901-3907). 3) Origins of replication, which ensure amplification of the expression cassettes or vec tors according. to the invention in, for example, E. col. Examples which may be men tioned are ORI (origin of DNA replication), the pBR322 or or the P15A ori (Sambrook 15 et al.: Molecular Cloning. A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). 4) Elements which are necessary for Agmbacterium-mediated plant transformation, such as, for example, the right or left border of the T-DNA or the vir region. 20 The inventive recombinant expression construct contains expressible nucleic acid se quences in addition to, or other than, nucleic acid sequences encoding for marker pro teins. In a preferred embodiment of the invention the recombinant DNA expression construct comprises an nucleic acid sequence encodes for i) a protein or ii) a sense, 25 antisense, or double-stranded RNA sequence. In a further preferred embodiment of the present invention, the recombinant DNA expression construct contains a -nucleic add sequence encoding a protein. In yet another embodiment of the invention the recombi nant DNA expression construct may contain a DNA for the purpose of expressing RNA transcripts that function to affect plant phenotype without being translated into a pro 30 tein. Such non protein expressing sequences comprising antisense RNA molecules, sense RNA molecules, RNA molecules with ribozyme activity, double strand forming RNA molecules (RNAi). The transgenic expression constructs of the invention can be employed for suppressing or reducing expression of endogenous target genes by "gene silencing'. The skilled worker knows preferred genes or proteins whose suppres 35 sion brings about an advantageous phenotype. Examples may include but are not lim ited to down-regulation of the p-subunit of Arabidopsis G protein for increasing root mass (Ullah et aL (2003) Plant Cell 15:393-409), inactivating cyclic nucleotide-gated ion channel (CNGC) for improving disease resistance (WO 2001007596), and down regulation of 4-coumarate-CoA ligase (4CL) gene for altering lignin and cellulose con 40 tents (US 2002138870). In yet another preferred embodiment of the invention, the transgenic expression constructs of the invention contain nucleic acids, which when transcribed, produce RNA enzymes (Ribozymes) which can act as endonucleases and catalyze the cleavage of RNA molecules with selected sequences. The cleavage of the selected RNA can result in the reduced production of their encoded polypeptide prod 45 ucts. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Ceck 1987, Proc. Nati. Acad. Sci. USA, 84:8788-8792; Gerlach et al, 1987, Nature, 328:802-805; Forster and Symons, 1987, Cell, 49:211-220). Several different ribozyme motifs have been described with RNA cleavage activity (Symons, 1992, 37 Annu. Rev. Biochem., 61: 641-671). Examples include sequences from group I self splicing introns including Tobacco Ringspot Virus (Prody ot al., 1986, Science, 231:1577-1580). Other suitable ribozymes include sequences from RNaseP with cleavage activity (Yan et al. (1992) Proc. Natg. Acad. Sci. USA 87:4144-4148), hairpin 5 ribozyme structures (Berzal-Herranz et al. (1992) Genes and Devel. 98:1207-1210) and Hepatitis Delta virus based ribozyme (U.S. Pat, No. 5,625,047). The general de sign and optimization of ribozymes directed RNA cleavage activity has been discussed on detail (Haseloff and Gerlach (1988) Nature 224:585-591; Symons (1992) Annu. Rev. Biochem. 61: 641-671). The choice of a particular nucleic acid sequence to be 10 delivered to a host cell or plant depends on the aim of the transformation. In general, the main goal of producing transgenic plants is to add some beneficial traits to the plant. In another embodiment of the invention, the recombinant expression construct com 15 prises a nucleic acid sequence encoding for a selectable marker protein, a screenable marker protein, a anabolic active protein, a catabolic active protein, a biotic or abiotic stress resistance protein, a male sterility protein or a protein affecting plant agronomic characteristics. Such traits include, but are not limited to, herbicide resistance or toler ance, insect resistance or tolerance, disease resistance or tolerance (viral, bacterial, 20 fungal, nematode); stress tolerance, as exemplified by tolerance to drought, heat chill ing, freezing, salt stress, oxidative stress; increased yield, food content, male sterility, starch quantity and quality, oil content and quality, vitamin content and quality (e.g. vitamin E) and the like. One may desire to incorporate one or more nucleic acid se quences conferring any of such desirable traits. Furthermore, the recombinant expres 25 sion constructs of the invention can comprise artificial transcription factors (e.g. of the zinc finger protein type; Beerli (2000) Proc Natl Aced Sci USA 97(4):1495-500). These factors attach to the regulatory regions of the endogenous genes to be expressed or to be repressed and, depending on the design of the factor, bring about expression or repression of the endogenous gene. The following may be mentioned by way of exam 30 ple but not by way of limitation as nucleic acid sequences or polypeptides which can be used for these applications: Improved protection of the plant embryo against abiotic stresses such as drought, high or low temperatures, for example by overexpressing the antifreeze polypeptides from 35 Myoxocephalus scorpius (WO 00/00512), Myoxocephalus octodecemspinosus, the Arabidopsis thailana transcription activator CBF1, glutamate dehydrogenases (WO 97/12983, WO 98/11240), a late embryogenesis gene (LEA), for example from barley (WO 97/13843), calcium-dependent protein kinase genes (WO 98/26045), calcineurins (WO 99/05902), famesyl transferases (WO 99/06580, Pei 1998), ferritin (Deak 1999), 40 oxalate oxidase (WO 99/04013; Dunwell 1998), DREBIA factor (dehydration response element B IA; Kasuga 1999), mannitol or trehalose synthesis genes, such as treha lose-phosphate synthase or trehalose-phosphate phosphatase (WO 97/42326), or by inhibiting genes such as the trehalase gene (WO 97/50561). Especially preferred nu cleic acids are those which encode the transcriptional activator CBF1 from Arabidopsis 45 thaliana (GenBank Acc. No.: U77378) or the Myoxocephalus octodeoemspinosus anti freeze protein (GenBank Acc. No.: AF306348), or functional equivalents of these. For expression in plants, the nucleic acid molecule must be linked operably to a suitable promoter. The plant specific promoter, regulatory element and the terminator of the 38 inventive recombinant expression construct needs not be of plant origin, and may origi nate from viruses or microorganisms, in particular for example from viruses which at tack plant cells. 5 An additional subject matter of the invention is the introduction of an inventive intron sequence into a target nucleic acid sequence via homologous recombination (HR). As a prerequisite for the HR between the recombinant expression construct and the ge nomic target nucleic acid sequence, the recombinant expression construct must con tain fragments of the target nucleic acid sequence of sufficient length and homology. In 10 a preferred embodiment of the invention, the intron sequences that has to be inserted into the gene of interest via HR is (within the recombinant expression construct) placed between a pair of DNA sequences identical to the region 5'and 3' to the preferred place of insertion. In this case, the recombinant expression construct can comprises only the intron sequence and the nucleic acid sequences needed to in 15 duce the HR event. In a preferred embodiment of the invention, the intron sequence that is flanked by the nucleic acid sequence of the target DNA, contains an expression cassette that enables the expression of an selectable marker protein which allows the selection of transgenic plants in which a homologues or illegitimate recombination had occurred subsequent to the transformation. The expression cassette driving the ex 20 pression of the selection marker protein can be flanked by HR control sequences that are recognized by specific endonucleases or recombinases, facilitating the removal of the expression cassette from the genome. Such so called marker exci sion methods e.g. the cre/lox technology permit a tissue-specific, if appropriate induc ible, removal of the expression cassette from the genome of the host organism (Sauer 25 B (1998) Methods. 14(4):381-92). In this method, specific flanking sequences (lox se quences), which later allow removal by means of cre recombinase, are attached to the target gene. Specifically, the present invention relates to transgenic expression cassettes compris 30 ing the following introns with gene expression enhancing properties in plants: 1) The sequence of the first iniron (BPSLI, SEQ ID NO: 1) isolated from the Oryza saiva metallothioneine-like gene (Gene Bank accession No. AP002540, Oryza sativa (Japonica cultivar group) genomic DNA, Chromosome 1, PAC clone: P0434B04, gene-id = "P0434B04.31, protein id ="BAB4401 0.1 ", complement joined sequences: 35 142304..142409, 143021..143098, 143683..143747; Hsieh, H.M. et al., RNA expres sion patterns of a type 2 metallothioneine-like gene from rice. Plant Mol. Biol. 32 (3), 525-529 (1996)). The gene comprises two introns and three exons. The first intron of the Oryza sathra metallothioneine-like gene (BPSI.1, SEQ ID NO:1) is flanked by the 5' (5'-GU-3', base pair (bp) 1-2 in SEQ ID NC:1) and 3' (5'-CAG-3',bp 582-584 in SEQ 40 ID NO:1) splice sites. In a preferred embodiment of the invention, the first intron of the Oryza sativa metallothioneine-like gene (BPSI.1, SEQ ID NC:1) comprises at least 28 bases pairs, more preferably at least 40 bases pairs, most preferably at least 50 base pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively (SEQ ID NO: 82). On nucleotide level, the Oryza sativa metallothionein 45 like gene shares high homology or identity with the coding region of orthologous genes from other monocotyledonous or dicotyledonous plants e.g. 89% identity to the Zea mays CL1155_3 mRNA sequence (acc. No. AY109343), 88% identity to the Poa secunda metallothionein-like protein type 2 mRNA (acc. No. AF246982.1), 93% identity 39 to the Triticum aestivum metallothioneine mRNA, partial coding sequence (acc No.AF470355.1), 89% identity to the Nicotiana plumbaginifolia metallothionein-like pro tein mRNA (acc. No. NPU35225), 86% identity to the Brassica oleracea cultivar Green King metallothioneine-like protein 2 (acc. No. AF200712), 95% and 88% identity to the 5 Hotreum vulgare subsp. vulgare partial mRNA for metallothioneine type2 mt2b (acc. No. HVU511346) and mtb2a (acc. No. HVU511345) genes, respectively (identities have been calculated using the BLASTN 2.2.9 algorithm [May-01-2004] Altschul, Stephen F. et a/., (1997), Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25:3389-3402). 10 2) The sequence of the first intron (BPSI.2, SEQ ID NO:2) isolated from the Oryza sa tiva Sucrose UDP Glucosyltransferase-2 gene (Gene Bank accession No. AC084380, Oryza sativa (Japonica cultivar group) genomic DNA, chromosome 3, BAC OS JNBa0090P23, gene ID ="OSJNBaO09OP23.15", Protein ID=AAK5219.1, complement 15 join (nucleotide 62884 to. 65255, 65350..65594, 65693-66011, 66098..66322, 66427..66593, 66677..66793, 66881..67054, 67136..67231, 67316..67532, 67652..67770, 67896..68088, 68209..68360, 68456..68585, ' 69314..69453 and 70899..72082). The gene comprises 13 introns and 14 exons. The first intron of the Oryza sativa Sucrose UDP Glucosyltransferase-2 gene (BPSI.2, SEQ ID NO: 2) is 20 flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:2) and 3' (5'-CAG-3',bp 726-728 in SEQ ID NO: 2) splice sites. In a preferred embodiment of the invention, the first intron of the Oryza safiva Sucrose UDP Glucosyltransferase-2 gene (SEQ ID NO:2) com prises at least 19 bases pairs of the sequence 5' to the 5'-splice site and 23 bases pairs of the sequences/exons 3' to the 3'-splice site of the intron (SEQ ID NO: 83). In a 25 particularly preferred embodiment the intron BPSI.2 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and13' adjacent to the 5' and 3' splice sites of the intron, respectively 3) The sequence of the second intron isolated from the Oryza sativa Sucrose UDP 30 Glucosyltransferase-2 gene (BPSI.3, SEQ ID NO:3). Said the second intron is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:3) and 3' (5'-CAG-3',bp 93-95 in SEQ ID NO: 3) splice sites. In a preferred embodiment of the invention, the second intron of the Oryza saliva Su 35 crose UDP Glucosyltransferase-2 gene (SEQ ID NO:3) comprises at least 25 bases pairs of the sequence 5' to the 5'-splice site and 30 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 84). In a particularly preferred embodiment the intron BPSI.3 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, 40 respectively. On nucleotide level, the Oryza sativa Sucrose UDP Glucosyltransferase-2 gene shares high homology or identity with the coding region of orthologous genes from other monocotyledonous or dicotyledonous plants e.g. 88% identity to the Zee mays sucrose synthase (Sus1) mRNA (acc. No. L22296.1), 85% identity to the Triticum aestivum mRNA for sucrose synthase type 2 (acc. No. AJ000153), 85% identity to the 45 H. vulgare mRNA for sucrose synthase (acc No. X69931), 80% identity to the Saccha rum officinarum sucrose synthase-2 mRNA (acc No. AF263384,), 95% identity to the Rice mRNA for sucrose synthase (8464 gene), partial sequence (acc. No. D10418), 79% identity to the Glycine max sucrose synthase mRNA (acc. No. AF03231). Identi 40 ties have been calculated using the BLASTN 2.2.9 algorithm [May-01-2004] Altschul, Stephen F. et al., (1997), Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25:3389-3402). 5 4) The sequence of the eighth intron (BPSI.5, SEQ ID NO:5) isolated from the Oryza sativa gene encoding for the Sucrose transporter (Gene Bank accession No. AF 280050). Said the eighth intron (SEQ ID NO:5) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:5) and 3' (5'-CAG-3',bp 223-225 In SEQ ID NO: 5) splice sites. In a pre ferred embodiment of the invention, the eighth intron of the Oryza saeva gene encoding 10 for the Sucrose transporter (SEQ ID NO:5) comprises at least 35 bases pairs of the sequence 5' to the 5'-splice site and 30 bases pairs of the sequences 3' to the 3' splice site of the intron (SEQ ID NO: 86). In a particularly preferred embodiment the intron BPSI.5comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respec 15 tively. In a more preferred embodiment, the 5' and 3' splice sites of the eighth intron (BPSI.5, SEQ ID NO:5) are modified in order to match the plant consensus sequences for 5' splice sites 5'-AG::GTAAGT-3' (SEQ ID NO: 80) and 3' splice sites 5'-CAG::GT 3' (SEQ ID NO: 81) using a PCR mutagenesis approach (SEQ ID NO:87). 20 5) The sequence of the fourth intron (BPSI.6, SEQ ID NO:6) isolated from the Oryza sativa gene (Gene Bank accession No. BAA94221) encoding for an unknown protein with homology to the A. theliana chromosome Il sequence from clones T22013, F12K2 encoding for a putative lipase (AC006233). Said the fourth intron (SEQ ID NO:6 ) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:6) and 3' (5'-CAG-3', bp 768-770 in 25 SEQ ID NO:6):splice sites, In a preferred embodiment of the invention, the fourth intron of the Oryza sativa gene (accession No. BAA94221) (SEQ ID NO:6) comprises at least 34 bases pairs of the sequence 5' to the 5'-splice site and 34 bases pairs of the se quences 3' to the 3'-splice site of the intron (SEQ ID NO: 88). In a particularly preferred embodiment the intron BPSI.6 comprises at least 40 bases pairs, more preferably at 30 least 50 bases, pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively. In a more preferred embodiment, the 5' and 3' splice sites of fourth intron (BPSI.6, SEQ ID NO:6) are modified in order to match the plant consen sus sequences for 5' splice sites 5'-AG::GTAAGT-3' (SEQ ID NO: 80) and 3' splice sites 5'-CAG::GT-3' (SEQ ID NO: 81) using a PCR mutagenesis approach (SEQ ID 35 NO:89). 6) The sequence of the fourth intron (BPSI.7, SEQ ID NO:7) Isolated from the Oryza sativa gene (accession No. BAB90130) encoding for a putative cinnamyl-alcohol dehy drogenase. Said the fourth intron (SEQ ID NO:7 ) is flanked by the 5' (5'-GU-3', bp 1-2 40 in SEQ ID NO:7) and 3' (5'-CAG-3', 713-715 bp in SEQ ID NO: 7) splice sites. In a preferred embodiment of the invention, the fourth intron of the Oryze sativa gene (ac cession No. BAB90130) (SEQ ID NO:7) comprises at least 34 bases pairs of the se quence-.5.' to the 5'-splice site and 26 bases pairs of the sequences 3' to the 3tsplice site of the intron (SEQ ID NO: 90). In a particularly preferred embodiment the intron 45 BPSI.7 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respec tively. In a more preferred embodiment, the 5' and 3' splice sites of the fourth intron (BPSI.7, SEQ ID NO:7) are modified in order to match the plant consensus sequences 41 for 5' splice sites 5'-AG::GTAAGT-3' (SEQ ID NO: 80) and 3' splice sites 5'-CAG::GT 3' (SEQ ID NO: 81) using a PCR mutagenesis approach (SEQ ID NO:91). 7) The sequence of the third intron (BPSI.10, SEQ ID NO:10) isolated from the Oryza 5 sativa gene (accession No. AP003300) encoding for a putative protein kinase. Said the third intron (SEQ ID NO:10) is flanked by the 5' (5'-GU-3', bp 112 in SEQ ID NO:10) and 3' (5'-CAG-3', 536-538 bp in SEQ ID NO: 10) splice sites. In a preferred embodi ment of the invention, the third intron of the Oryza sativa gene (accession No. AP003300) (SEQ ID NO:10) comprises at least 31 bases pairs of the sequence 5' to the 10 5'-splice site and 31 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 94). In a particularly preferred embodiment the intron BPSL10 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively. In a more pre ferred embodiment, the 5' and 3' splice sites of the third intron (BPSI.10, SEQ ID 15 NO:10) are modified In order to match the plant consensus sequences for 5' splice sites 5'-AG::GTAAGT-3' (SEQ ID NO: 80) and 3' splice sites 5'-CAG::GT-3' (SEQ ID NO: 81) using a PCR mutagenesis approach (SEQ ID NO:95). 8) The sequence of the first intron (BPS.11, SEQ ID NO:11) isolated from the Oryza 20 sativa gene (accession No. L37528) encoding for a MADS3 box protein. Said the first intron (SEQ ID NO:11) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:11) and 3' (5'-CAG-3', bp 329-331 in SEQ ID NO: 11) splice sites. In a preferred embodiment of the invention, the first intron of the Oryza safiva gene (accession No. L37528) (SEQ ID NO:1 1) comprises at least 35 bases pairs of the sequence 5' to the 5'-splice site and 25 34 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 96). In a particularly preferred embodiment the intron BPSl1 1 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively, In a more preferred embodiment, the 5' and 3' splice sites of the first intron (BPSI.11, SEQ ID NO:11) are modified in 30 order to match the plant consensus sequences for 5' splice sites 5'-AG::GTAAGT-3' (SEQ ID NO: 80) and 3' splice sites 5'-CAG::GT-3' (SEQ ID NO: 81) using a PCR mutagenesis approach (SEQ ID NO:97). 9) The sequence of the first intron (BPSI.12, SEQ ID NO:12) isolated from the Oryza 35 sativa gene (accession No. CB625805) encoding for a putative Adenosylmethionine decarboxylase. Said the first intron (SEQ ID NO:12) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:12) and 3' (5'-CAG-3', bp 959-961 in SEQ ID NO: 12) splice sites. In a preferred embodiment of the invention, the first intron of the Oryza sativa gene (accession No. CB625805) (SEQ ID NO:12) comprises at least 26 bases pairs of the 40 sequence 5' to the 5'-splice site and 26 bases pairs of the sequences 3' to the 3' splice site of the intron (SEQ ID NO: 98). In a particularly preferred embodiment the intron BPSI.12 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively. 45 10) The sequence of the first intron (BPSI.13, SEQ ID NO:13) isolated from the Oryza saliva gene (accession No. CF297669) encoding for an Aspartio proteinase. Said the first intron (SEQ ID NO:13) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:13) 42 and 3' (5'-CAG-3', bp 593-595 in SEQ ID NO: 13) splice sites. In a preferred embodi ment of the invention, the first intron of the Oryza sativa gene (accession No. CF297669 ) (SEQ ID NO:13) comprises at least 26 bases pairs of the sequence 5' to the 5'-splice site and 24 bases pairs of the sequences 3' to the 3-splice site of the 5 intron (SEQ ID NO: 99). In a particularly preferred embodiment the intron BPSI.13 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the se quences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively. 11) The sequence of the first intron (BPS].14, SEQ ID NO:14) isolated from the Oryza 10 sativa gene (accession No. CB674940) encoding for a Lecl4b protein. Said the first intron (SEQ ID NO:14) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:14) and 3' (5'-CAG-3', bp 143-145 in SEQ ID NO: 14) splice sites. In a preferred embodiment of the invention, the first intron of the Oryza sativa gene (accession No. CB674940) (SEQ ID NO:14) comprises at least 26 bases pairs of the sequence 5' to the 5'-splice site 15 and 25 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 100). In a particularly preferred embodiment the intron BPSI.14 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adja cent to the 5' and 3' splice sites of the intron, respectively. 20 12) The sequence of the first intron (BPSI.15, SEQ ID NO:15) isolated from the 5 UTR of the Oryza saiva gene (accession No. BAD37295.1) encoding for a putative SalT protein precursor. Said the first intron (SEQ ID NO:15) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:15) and 3' (5'-CAG-3', bp 312-314 in SEQ ID NO: 15) splice sites. In a preferred embodiment of the invention, the first intron of the Oryza saiva 25 gene (accession No.BAD37295.1) (SEQ ID NO:15) comprises at least 26 bases pairs of the sequence 5' to the 5'-splice site and 25 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 101). In a particularly preferred embodiment the intron BPSI.15 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, 30 respectively. 13) The sequence of the first intron (BPSI.16, SEQ ID NO:16) isolated from the Oryza sativa gene (accession No. BX928664) encoding for a putative reticulon. Said the first intron (SEQ ID NO:16) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:16) and 3' 35 (5'-CAG-3', bp 650-652 in SEQ ID NO: 16) splice sites. In a preferred embodiment of the invention, the first intron of the Oryza satfva gene (accession No. BX928664) (SEQ ID NO:16) comprises at least 26 bases pairs of the sequence 5' to the 5'-splice site and 23 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 102). In a particularly preferred embodiment the intron BPSI.16 comprises at least 40 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adja cent to the 5' and 3' splice sites of the intron, respectively. 14) The sequence of the first intron (BPSL17, SEQ ID NO:17) isolated from the Oryza sativa gene (accession No. AA752970) encoding for a glycolate oxidase. Said the first 45 intron (SEQ ID NO:17) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:17) and 3' (5'-CAG-3', bp 266-268 in SEQ ID NO:17) splice sites. In a preferred embodiment of the invention, the first intron of the Oryza saiva gene (accession No. AA752970 ) (SEQ ID NO:17) comprises at least 26 bases pairs of the sequence 5' to the 5'-splice site 43 and 35 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 103). In a particularly preferred embodiment the intron BPSI.17 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adja cent to the 5' and 3' splice sites of the intron, respectively. 5 15) The sequence of the first intron (BPSI.18, SEQ ID NO:18) isolated from the Oryza sativa clone GI 40253643 (accession No. AK064428) Is similar to AT4g33690. Said the first intron (SEQ ID NO:18) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:18) and 3' (5'-CAG-3', bp 544-546 In SEQ ID NO:18) splice sites. In a preferred embodi 10 ment of the invention, the first intron of the Oryza sativa gene (accession No. AK064428) (SEQ ID NO:18) comprises at least 26 bases pairs of the sequence 5' to the 5'-splice site and 21 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 104). In a particularly preferred embodiment the intron BPSI.1 8 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the se 15 quences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively. 16) The sequence of the first intron (BPSI.19, SEQ ID NO:19) isolated from the Oryza sativa clone GI 51091887 (accession No. AK062197)). Said the first intron (SEQ ID NO:19) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:19) and 3' (5'-CAG-3', bp 20 810-812 in SEQ ID NO:19) splice sites. In a preferred embodiment of the invention, the first intron of the Oryza sativa gene (accession No. AK0621197) (SEQ ID NO:19) com prises at least 26 bases pairs of the sequence 5' to the 5'-splice site and 26 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 105). In a par ticularly preferred embodiment the intron BPSI.19 comprises at feast 40 bases pairs, 25 more preferably at least 50 bases pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively. 17) The sequence of the first intron (BPSI.20, SEQ ID NO:20) isolated from the Oryza saiva gene (accession No. CF279761) encoding for a hypothetical protein done (GI 30 33657147). Said the first intron (SEQ ID NO:20) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:20) and 3' (5'-CAG-3', bp 369-371 in SEQ ID NO:20) splice sites, In a preferred embodiment of the invention, the first intron of the Oryza saliva gene (acces sion No. CF279761) (SEQ ID NO:20) comprises at least 26 bases pairs of the se quence 5' to the 5'-splice site and 27 bases pairs of the sequences 3' to the 3'-splice 35 site of the intron (SEQ ID NO: 106). In a particularly preferred embodiment the intron BPSI.20 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respec tively. 40 18) The sequence of the first intron (BPSI.21, SEQ ID NO:21) isolated from the Oryza sativa gene (accession No. CF326058) encoding for a putative membrane transporter. Said the first intron (SEQ ID NO:21) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ ID NO:21) and 3' (5'-CAG-3', bp 720-722 in SEQ ID NO:21) splice sites. In a preferred embodiment of the invention, the first intron of the Oyza sativa :gene (accession No. 45 CF326058) (SEQ ID NO:21) comprises at least 26 bases pairs of the sequence 5' to the 5'-splice site and 25 bases pairs of the sequences 3' to the 3'-spice site of the intron (SEQ ID NO: 107). In a particularly preferred embodiment the intron BPSI.21 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the se quences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively. 44 19) The sequence of the first intron (BPSI.22, SEQ ID NO:22) isolated from the Oryza sativa gene (accession No. C26044) encoding for a putative ACT domain repeat pro tein. Said the first intron (SEQ ID NO:22) is flanked by the 5' (5'-GU-3', bp 1-2 in SEQ 5 ID NO:22) and 3' (5'-CAG-3', bp 386-388 in SEQ ID NO:22) splice sites. In a pre ferred embodiment of the invention, the first intron of the Oryza saliva gene (accession No. C26044) (SEQ ID NO:22) comprises at least 26 bases pairs of the sequence 5' to the 5-spice site and 28 bases pairs of the sequences 3' to the 3'-splice site of the intron (SEQ ID NO: 108). In a particularly preferred embodiment the intron BPSI.22 10 comprises at least 40 bases pairs, more preferably at least 50 bases pairs of the se quences 5' and 3' adjacent to the 5' and 3' splice sites of the intron, respectively. Table 1: Genes from which the introns of the invention are preferably isolated, putative function of said genes, cDNA and the protein encoded by said genes. 15 Intron Rice Gi Accesion SEQ Sequence homology nuMber No. ID NO.Seunehmlg BPSL1 AP002540 1 metallothioneine-like gene BPSIL2 AC084380 2 Sucrose UDP Glucosyttransferase-2 gene, first Intron BPSI.3 AC084380 3 Sucrose UDP Gluoosyltransferase-2 gene, second Intron BPSI4 AC084380 4 Sucrose UDP Glucosyltransferase-2 gene, third Intron BPSL.5 9624451 AF280050 5 Sucrose transporter BPSL6 7523493 BAA94221 6 Similar to A. thaliana chromosome Il sequence from clones T22013, F12K2; putative lipase (AC006233) BPSL7 20161203 BAB90130 7 putative cinnamyi-alcohol dehydrogenase BPSI10 20160990 AP003300 10 Putative protein kinase BPS1.11 886404 L37528 11 MADS3 box protein BPSL12 29620794 CB625805 12 putative Adenosylmethionine decarboxylase BPSL13 33666702 CF297669 13 Aspartic proteinase BPSL14 29678665 CB674940 14 Lec14b protein BPSI.15 51535011 BAD37295 15 putative SalT protein precursor BPSI.16 41883853 BX928664 16 Putative Reticulon BPSI.17 2799981 AA752970 17 Glycolate oxidase BPSI.18 40253643 AK06442 18 Putative non-coding (Similar to AT4g33690) BPSI.19 51091887 AK062197 19 Putative non-coding BPSI.20 33657147 CF279761 20 Hypothetical protein BPSI.21 3380379 CF326058 21 Putative membrane transporter BPSL22 2309889 C26044 22 Putative ACT domain repeat protein It is disclosed by the examples of this invention, that the inventive introns with the SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10 and 11 have an impact on the expression rate of the GUS gene in transient expression assays and stable transformed plants, respectively. It 20 could be shown that the inclusion of said Introns into the 5' UTR of the GUS gene has 45 led to a strong enhancement in the expression rate of this gene in transiently and sta ble transformed cell, respectively, compared to a control construct that lacks the first intron (see examples 1.6.1 (table 7), 1.6.2 (table 8), 2.4 (table 15). The expression enhancing properties of the introns with the SEQ ID NOs: 12, 13, 14, 5 15, 16, 17, 18, 19, 20, 21 or 22 can be demonstrated by performing the above de scribed transient or stable expression assays. Functional equivalents of the inventive introns can be identified via homology searches in nucleic acid databases or via DNA hybridization (screening of genomic DNA librar 10 ies) using a fragment of at least 50 consecutive base pairs of the nucleic acid molecule described by any of the SEQ ID NOs: 1, 2, 3; 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 and stringent hybridization conditions. In a preferred embodiment of the present invention the stringent hybridizing conditions can be chosen as follows: The hybridization puffer contains Formamide, NaCI and PEG 6000 (Polyethyleneglykol 15 MW 6000). Formamide has a destabilizing effect on double strand nucleic acid mole cules, thereby, when used in hybridization buffer, allowing the reduction of the hybridi zation temperature to 42*C without reducing the hybridization stringency. NaCl has a positive impact on the renaturation-rate of a DNA duplex and the hybridization effi ciency of a DNA probe with its complementary DNA target. PEG increases the viscosity 20 of the hybridization buffer, which has in principle a negative impact on the hybridization efficiency. The composition of the hybridization buffer is as follows: 250 mM Sodium phosphate-buffer pH 7,2 1 mM EDTA (ethylenediaminetetraacetic acid) 7 % SDS (glv) (sodium dodecyl sulfate) 250 mM NaCI (Sodiumchloride) 10 pg/ml single stranded DNA 5 % Polyethylengiykol (PEG) 6000 40 % Formamide The hybridization is preferably performed over night at 42'C. In the morning, the hy 25 bridized filter will be washed 3 x for 10 minutes with 2xSSC + 0,1 % SDS. Hybridization should advantageously be carried out with fragments of at least 50, 60, 70 or 80 bp, preferably at least 90 bp. In an especially preferred embodiment the hybridization should be carried out with the entire nucleic acid sequence with conditions described above. 30 Combination of the introns of the invention with different plant promoters has clearly demonstrated their expression enhancing and/or modulating properties. In a preferred embodiment of the invention the recombinant DNA expression construct comprises (functionally linked to an intron of the invention) a promoter sequence functioning in 35 plants or plant cells selected from the group consisting of a) the rice chloroplast protein 12 (Os.CP12) promoter as described by nucleotide 1 to 854 of SEQ ID NO: 113 (the 'fragment'), or a sequence having at least 60% (pref erably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) identity to said fragment, or a sequence hybridizing under 40 stringent conditions (preferably under conditions equivalent to the high stringency conditions defined in the paragraph above) to said fragment, or a sequence com 46 prising at least 50 (preferably at least 100, more preferably at least 200 or 300, most preferably at least 400 or 500) consecutive nucleotides of said fragment, and b) the maize hydroxyproline-rich glycoprotein (Zm.HRGP) promoter as described by nucleotide 1 to 1184 of SEQ ID NO: 114, or a sequence having at least 60% (pref 5 erably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) Identity to said fragment, or a sequence hybridizing under stringent conditions (preferably under conditions equivalent to the high stringency conditions defined in the paragraph above) to said fragment or a sequence com prising at least 50 (preferably at least 100, more preferably at least 200 or 300, 10 most preferably at least 400 or 500) consecutive nucleotides of said fragment, and c) the rice p-caffeoyl-CoA 3-0-methyltransferase (Os.CCoAMT1) promoter as de scribed by nucleotide 1 to 1034 of SEQ ID NO: 115, or a sequence having at least 60% (preferably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) identity to said fragment, or a sequence hybridiz 15 ing under stringent conditions (preferably under conditions equivalent to the high stringency conditions defined in the paragraph above) to said fragment, or a se quence comprising at least 50 (preferably at least 100, more preferably at least 200 or 300, most preferably at least 400 or 500) consecutive nucleotides of said fragment, and 20 d) the maize Globulin-1 (Zm.Gibi) promoter (W64A) as described by nucleotide I to 1440 of SEQ ID NO: 116, or a sequence having at least 60% (preferably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) identity to said fragment, or a sequence hybridizing under stringent condi tions (preferably under conditions equivalent to the high stringency conditions de 25 fined in the paragraph above) to said fragment, or a sequence comprising at least 50 (preferably at least 100, more preferably at least 200 or 300, most preferably at least 400 or 500) consecutive nucleotides of said fragment, and e) the putative Rice H+-transporting ATP synthase (Os.V-ATPase) promoter as de scribed by nucleotide I to 1589 of SEQ ID NO: 117, or a sequence having at least 30 60% (preferably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) identity to said fragment, or a sequence hybridiz ing under stringent conditions (preferably under conditions equivalent to the high stringency conditions defined in the paragraph above) to said fragment, or a se quence comprising at least 50 (preferably at least 100, more preferably at least 35 200 or 300, most preferably at least 400 or 500) consecutive nucleotides of said fragment, and f) the putative rice C-8,7 sterol isomerase (Os.C8,7 SI) promoter as described by nucleotide 1 to 796 of SEQ ID NO: 118, or a sequence having at least 60% (pref erably at least 70% or 80%, more preferably at least 90% or 95%, most preferably 40 at least 98% or 99%) identity to said fragment or a sequence hybridizing under stringent conditions (preferably under conditions equivalent to the high stringency conditions defined in the paragraph above) to said fragment or a sequence com prising at least 50 (preferably at least 100, more preferably at least 200 or 300, most preferably at least 400 or 500) consecutive nucleotides of said fragment, and 45 g) the maize lactate dehydrogenase (Zm.LDH) promoter as described by nucleotide 1 to 1062 of; SEQ ID NO: 119, or a sequence having at least 60% (preferably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) identity to said fragment, or a sequence hybridizing under stringent condi 47 tions (preferably under conditions equivalent to the high stringency conditions de fined in the paragraph above) to said fragment, or a sequence comprising at least 50 (preferably at least 100, more preferably at least 200 or 300, most preferably at least 400 or 500) consecutive nucleotides of said fragment, and 5 h) the rice Late Embryogenesis Abundant (Os.Lea) promoter as described by nucleo tide 1 to 1386 of SEQ ID NO: 121, or a sequence having at least 60% (preferably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) identity to said fragment, or a sequence hybridizing under stringent conditions (preferably under conditions equivalent to the high stringency conditions 10 defined in the paragraph above) to said fragment, or a sequence comprising at least 50 (preferably at least 100, more preferably at least 200 or 300, most pref erably at least 400 or 500) consecutive nucleotides of said fragment. Preferably said expression construct is comprising a combination of one of the above 15 defined promoters with at least one intron selected from the group consisting of i) the BPSL1 intron as described by nucleotide 888 to 1470 of SEQ ID NO: 113 or a sequence having at least 60% {preferably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) identity to said fragment, or a sequence hybridizing under stringent conditions (preferably under conditions 20 equivalent to the high stringency conditions defined above) to said fragment, or a sequence comprising at least 50 (preferably at least 100, more preferably at least 200 or 300, most preferably at least 400 or 500) consecutive nucleotides of said fragment and ii) the BPSI.5 intron as described by nucleotide 1068 to 1318 of SEQ ID NO: 120, or a 25 sequence having at least 60% (preferably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) identity to said fragment, or a sequence hybridizing under stringent conditions (preferably under conditions equivalent to the high stringency conditions defined above) to said fragment, or a sequence comprising at least 50 (preferably at least 100, more preferably at least 30 200 or 300, most preferably at least 400 or 500) consecutive nucleotides of said fragment. More preferably expression construct is comprising a combination of promoter and in tron selected from the group consisting of 35 i) sequences as described by any of SEQ ID NO: 113, 114, 115; 116, 117, 118, 119, 120, or 121, and ii) sequences having at least 50 (preferably at least 100, more preferably at least 200 or 300, most preferably at least 400 or 500) consecutive nucleotides of a sequence described by any of SEQ ID NOs: 113, 114, 115, 116, 117, 118, 119, 120, or 121, 40 and iii) sequences having an identity of at least 60% (preferably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 98% or 99%) to a sequence described by any of SEQ ID NOs: 113, 114, 115, 116, 117, 118, 119, 120, or 121, and 45 iv) sequences hybridizing under stringent conditions (preferably under conditions equivalent to the high stringency conditions defined above) with sequence described by any of SEQ ID NOs: 113, 114, 115, 116, 117, 118, 119, 120; or 121. 48 A preferred subject matter of the invention, is a vector, preferably a plant transforma tion vector, containing an inventive recombinant expression construct. The expression cassette can be introduced into the vector via a suitable restriction cleavage site. The plasmid formed is first introduced into Ecol. Correctly transformed E.coli are selected, 5 grown, and the recombinant plasmid is obtained by the methods familiar to the skilled worker. Restriction analysis and sequencing may serve to verify the cloning step. Pre ferred vectors are those, which make possible stable integration of the expression cassette into the host genome. An expression construct according to the invention can advantageously be introduced into cells, preferably into plant cells, using vectors. 10 In one embodiment, the methods of the invention involve transformation of organism or cells (e.g. plants or plant cells) with a transgenic expression vector comprising at least a transgenic expression cassette of the invention. The methods of the invention are not limited to the expression vectors disclosed herein. Any expression vector which is ca pable of introducing a nucleic acid sequence of interest into a plant cell is contemplated 15 to be within the scope of this invention. Typically, expression vectors comprise the transgenic expression cassette of the invention in combination with elements which allow cloning of the vector into a bacterial or phage host. The vector preferably, though not necessarily, contains an origin of replication which is functional in a broad range of prokaryotic hosts. A selectable marker is generally, but not necessarily, included to 20 allow selection: of cells bearing the desired vector. Preferred are those vectors that al lowing a stable integration of the expression construct into the host genome. In the case of injection or electroporation of DNA into plant cells, the plasmid used need not meet any particular requirements. Simple plasmids such as those of the pUC series can be used. if intact plants are to be regenerated from the transformed cells, it is nec 25 essary for an additional selectable marker gene to be present on the plasmid. A variety of possible plasmid vectors are available for the introduction of foreign genes into plants, and these plasmid vectors contain, as a rule, a replication origin for multiplica tion in E.coli and a marker gene for the selection of transformed bacteria. Examples are pBR322, pUC series, M13mp series, pACYC1 84 and the like. The expression construct 30 can be introduced into the vector via a suitable restriction cleavage site. The plasmid formed is first introduced into E.coli. Correctly transformed Ecol are selected and grown, and the recombinant plasmid is obtained by methods known to the skilled worker. Restriction analysis and sequencing can be used for verifyingthe cloning step. 35 Depending on the method by which DNA is introduced, further genes may be neces sary on the vector plasmid. Agrobacterium tumefaclens and A. rhizogenes are plant-pathogenic soil bacteria, which genetically transform plant cells. The 1 and Ri plasmids of A. tumefaciens and A. 40 rhizogenes, respectively, carry genes responsible for genetic transformation of the plant (Kado (1991) Cdt Rev Plant Sci 10:1). Vectors of the invention may be based on the Agrobactelrium Ti- or Ri-plasmid and may thereby utilize a natural system of DNA transfer into the plant genome. As part of this highly developed parasitism Agrobacte rium transfers a defined part of its genomic information (the T-DNA; flanked by about 45 25 bp repeats,,named left and right border) into the chromosomal DNA of the plant cell (Zupan (2000) Plant J 23(1): 11-28). By combined action of the so-called vir genes (part of the original Ti-plasmids) said DNA-transfer is mediated. For utilization of this natural system, Ti-plasmids were developed which lack the original tumor inducing genes 49 ("disarmed vectors"). In a further improvement, the so called "binary vector systems", the T-DNA was physically separated from the other functional elements of the Ti plasmid (e.g., the vir genes), by being incorporated into a shuttle vector, which allowed easier handling (EP-A 120 516; US 4.940,838). These binary vectors comprise (beside 5 the disarmed T-DNA with its border sequences), prokaryotic sequences for replication both in Agrobacterium and E. coiL. It is an advantage of Agrobacterium-rnmediated trans formation that in general only the DNA flanked by the borders is transferred into the genome and that preferentially only one copy is inserted. Descriptions of Agrobacte rium vector systems and methods for Agrobacterlum-mediated gene transfer are 10 known in the art (Miki 1993, "Procedures for Introducing Foreign DNA into Plants" in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY; pp.67-88; Gruber 1993, "Vectors for Plant Transformation," in METHODS IN PLANT MOLECU LAR BIOLOGY AND BIOTECHNOLOGY; pp.89-119; Moloney (1989) Plant Cell Re ports 8: 238-242). The use of T-DNA for the transformation of plant cells has been 15 studied and described intensively (EP 120516; Hoekema 1985, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; Fraley (1985) CRC Crit. Rev. Plant. Sci. 4:1-45; and An (1985) EMBO J. 4:277-287). Various binary vectors are known, some of which are commercially available such as, for example, pBIN19 (Clontech Laboratories, Inc. U.S.A). Hence, for Agrobacterium-mediated trans 20 formation the transgenic expression construct of the invention is integrated into specific plasmids, either into a shuttle or intermediate vector, or into a binary vector. If a Ti or Ri plasmid is to be used for the transformation, at least the right border, but in most cases the right and left border, -of the Ti or Ri plasmid T-DNA is linked to the transgenic ex pression construct to be introduced in the form of a flanking region. Binary vectors are 25 preferably used. Binary vectors are capable of replication both in E.coli and in Agrobac terium. They may comprise a selection marker gene and a linker or polylinker (for in sertion of e.g. the expression construct to be transferred) flanked by the right and left T DNA border sequence. They can be transferred directly into Agrobacterlum (Holsters (1978) Mol Gen Genet 163:181-187). The selection marker gene permits the selection 30 of transformed agrobacteria and is, for example, the nptll gene, which confers resis tance to kanamycin. The Agrobacterium which acts as host organism in this case should already contain a plasmid with the vir region. The latter is required for transfer ring the T-DNA to the plant cell. An Agrobacterium transformed in this way can be used for transforming plant cells. The use of T-DNA for transforming plant cells has been 35 studied and described intensively (EP 120 516; Hoekema (1985) Nature 303:179-181; An (1985) EMBO J. 4:277-287; see also below). Common binary vectors are based on "broad host range"-plasmids like pRK252 (Bevan (1984) NucI Acid Res 12:8711-8720) or pTJS75 (Watson (1985) EMBO J 4(2):277-284) derived from the P-type plasmid RK2. Most of these vectors are derivatives of pBIN19 (Bevan 1984, Nucl Acid Res 40 12:8711-8720). Various binary vectors are known, some of which are commercially available such as, for example, pB101.2 or pBIN19 (Clontech Laboratories, Inc. USA). Additional vectors were improved with regard to size and handling (e.g. pPZP; Hajduk iewicz (1994) Plant Mol Biol 25:989-994). Improved vector systems are described also in WO 02100900. In a preferred embodiment Agrobacterium strains for use in the prac 45 tice of the invention Include octopine strains, e.g., LBA4404 or agropine strains, e.g., EHAI 01 or EHA1 05. Suitable strains of A. tumefaciens for DNA transfer are for exam ple EHA101-pEHA101 (Hood (1986) J Bacteriol 168:1291-1301), EHA105{pEHA105] (Li (1992) Plant Mol Biol 20:1037-1048), LBA4404[pAL4404] (Hoekema (1983) Nature 50 303:179-181), :058C1[pMP90] (Koncz (1986) Mol Gen Genet 204:383-396), and C58C1[pGV2260] (Deblaere (1985) Nucl Acids Res 13:4777-4788. Other suitable strains are Agrobacterium tumefaciens C58, a nopaline strain. Other suitable strains are A. tumefaciens C58CI (Van Larebeke (1974) Nature 252:169-170, A136 (Watson 5 (1975) J. Bacteriol 123:255-264) or LBA4011 (Klapwijk (1980) J. Bacteriol. 141:128 136 In a preferred embodiment, the Agrobacterium strain used to transform the plant tissue pre-cultured with the plant phenolic compound contains a L,L-succinamoplne type Ti-plasmid, preferably disarmed, such as pEHA101. In another preferred embodi ment the Agrobacterium strain used to transform the plant tissue pre-cuitured with the 10 plant phenolic compound contains an octopine-type Ti-plasmid, preferably disarmed, such as pAL4404. Generally, when using octopine-type TI-plasmids or helper plasmids, it is preferred that the vid~ gene be deleted or inactivated (Jarchow (1991) Proc. Nati. Acad. Sci. USA 88:10426-10430). in a preferred embodiment the Agrobactedum strain used to transform the plant tissue pre-cultured with the plant phenolic compound such 15 as acetosyringone. The method of the invention can also be used in combination with particular Agrobacterium strains, to further increase the transformation efficiency, such as Agrobacterium strains wherein the vir gene expression and/or induction thereof is altered due to the presence of mutant or chimeric virA or virG genes (e.g. Hansen (1994) Proc. Nat. Acad. Sci. USA 91:7603-7607;Chen 1991 J. Bacteriol. 173:1139 20 1144; Scheeren-Groot (1994) J. Bacteriol 176:6418-6426). A binary vector or any other vector can be modified by common DNA recombination techniques, multiplied in E coil, and introduced into Agrobacterium by e.g., electroporation or other transformation techniques (Mozo (1991) Plant Mol. Biol. 16:917-918). Agrobacterium is grown and used as described in the art. The vector comprising Agrobacterium strain may, for ex 25 ample, be grown for 3 days on YP medium (5 g/L yeast extract 10 g/L peptone, 5 g/L Nail, 15 g/L agar, pH 6.8) supplemented with the appropriate antibiotic (e.g., 50 mg/L spectinomycin). Bacteria are collected with a loop from the solid medium and resus pended. 30 An additional subject matter of the invention relates to transgenic non-human organ isms transformed with at least one vector containing a transgenic expression construct of the invention. In a preferred embodiment the invention relates to bacteria, fungi, yeasts, more preferably to plants or plant cell. in a preferred embodiment of the inven tion, the transgenic organism is a monocotyledonous plant. In a yet more preferred 35 embodiment, the monocotyledonous plant is selected from the group consisting of the genera Hordeum, Avena, Secale, Triticum, Sorghum, Zee, Saccharum and Oryza, very especially preferred are plants selected from the group consisting of Hordeum vulgare, Triticum aestivum, Triticum aestivum subsp.spelta, Triticale, Avene sativa, Secale ce reale, Sorghum bicolor, Saccharum officinarum, Zea mays and Oryza salive trans 40 formed with the inventive vectors or containing the inventive recombinant expression constructs. Preferred bacteria are bacteria of the genus Escherichla, Erwinia, Agrobac terium, Flavobacterium, A/calgenes or cyanobacteria, for example of the genus Synechocystis: Especially preferred are microorganisms which are capable of infecting plants and thus of transferring the constructs according to the invention. Preferred mi 45 croorganisms are those from the genus Agrobacterium and, in particular, the species Agrobacterium tumefaciens. Preferred yeasts are Candida, Saccharomyces, Han senula or Pichia. Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria or other fungi. Plant organisms are furthermore, for the purposes 51 of the invention, other organisms which are capable of photosynthetic activity such as, for example, algae or cyanobacteria, and also mosses. Preferred algae are green al gae such as, for example, algae of the genus Haematococcus, Phaedactylum tricor natum, Volvox or Dunaliella, Furthermore the invention relates cell cultures, tissues, 5 organs (e.g., leaves, roots and the like in the case of plant organisms), or propagation material derived from transgenic non-human organisms like bacteria, fungi, yeasts, plants or plant cells transformed with at least one vector containing a transgenic ex pression construct of the invention. 10 An additional subject matter of the invention relates to a method for providing an ex pression cassette for enhanced expression of a nucleic acid in al plant or a plant cell, comprising the step of functionally linking the inventive introns tb a plant expression cassette not comprising said intron. In a yet another preferred embodiment, the inven tion relates to a method for enhancing the expression of a nucleic acid sequence in a 15 plant or a plant cell, comprising functionally linking the inventive introns to said nucleic acid sequence. Preferably, the method for providing an expression cassette for en hanced expression of a nucleic acid in a plant or a plant cell and the method for en hancing the expression of a nucleic acid sequence in a plant or a plant cell further comprises the steps of 20 I) providing an recombinant expression cassette, wherein the nucleic acid sequence is functionally linked with a promoter sequence functional in plants and with an intron sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 6, 7, 10, 11, 12, 13,14, 15, 16,17,18,19, 20, 21 and 22, ii) introducing said recombinant expression into a plant cell or a plant, 25 iii) identifying or selecting the transgenic plant cell comprising said transgenic expres sion construct. In another preferred embodiment, the above-described method fur ther comprises the steps of iv) regenerating transgenic plant tissue from the transgenic plant cell. In an alternative preferred embodiment, the method further comprises 30 v) regenerating a transgenic plant from the transgenic plant cell. The generation of a transformed organism or a transformed cell requires introducing the DNA in question into the host cell in question. A multiplicity of methods is available for this procedure, which is termed transformation (see also Keown (1990) Methods in 35 Enzymology 185:527-537). For example, the DNA can be introduced directly by micro injection or by bombardment via DNA-coated microparticles. Also, the cell can be per meabilized chemically, for example using polyethylene glycol, so that the DNA can en ter the cell by diffusion. The DNA can also be introduced by protoplast fusion with other DNA-containing units such as minicells, cells, lysosomes or liposomes. Another suit 40 able method of introducing DNA is electroporation, where the cells are permeabilized reversibly by an electrical pulse. Methods for introduction of a transgenic expression construct or vector into plant tissue may include but are not limited to, e.g., electroinjec Von (Nan (1995) In "Biotechnology in Agriculture and Forest" Ed. Y. P. S. Bajaj, Springer-Verlag Berlin Heidelberg 34:145-155; Griesbach (1992) Hort Science 27:620); 45 fusion with liposomes, lysosomes, cells, minicells or other fusible lipid-surfaced bodies (Fraley (1982) Proc. Nati. Acad. Sci. USA 79:1859-1863); polyethylene glycol (Krens (1982) Nature 296:72-74); chemicals that increase free DNA uptake; transformation using virus, and the like. Furthermore, the biolistic method with the gene gun, electro 52 poration, incubation of dry embryos in DNA-containing solution, and microinjection may be employed. Protoplast based methods can be employed (e.g., for rice), where DNA is delivered to the protoplasts through liposomes, PEG, or electroporation (Shimamoto (1989) Nature 338:274-276; Datta (1990) Bio/Technology 8:736-740). Transformation 5 by electroporation involves the application of short, high-voltage electric fields to create "pores" in the cell membrane through which DNA is taken-up. These methods are for example - used to produce stably transformed monocotyledonous plants (Paszkowski (1984) EMBO J 3:2717-2722; Shillito (1985) Bio/Technology, 3:1099-1103; Fromm (1986) Nature 319:791-793) especially from rice (Shimamoto (1989) Nature 338:274 10 276; Datta (1990) Bio/Technology 8:736-740; Hayakawa (1992) Proc Natl Acad Sci USA 89:9865-9869). Particle bombardment or "biolistics" is a widely used method for the transformation of plants, especially monocotyledonous plants. In the "biolistics" (microprojectile-mediated DNA delivery) method microprojectile particles are coated with DNA and accelerated by a mechanical device to a speed high enough to penetrate 15 the plant cell wall and nucleus (WO 91/02071). The foreign DNA gets incorporated into the host DNA and results in a transformed cell. There are many variations on the "bio listics" method (Sanford (1990) Physiologia Plantarium 79:206-209; Fromm (1990) Bio/rechnology 8:833-839; Christou (1988) Plant Physiol 87:671-674; Sautter (1991) Bio/Technology 9:1080-1085). The method has been used to produce stably trans 20 formed monocotyledonous plants including rice, maize, wheat, barley, and oats (Chris tou (1991) BlotTechnology 9:957-962; Gordon-Kamm (1990) Plant Cell 2:603-618; Va sil (1992) Bio/Technology 10:667-674, (1993) Bio/Technology 11:1153-1158; Wan (1994) Plant Physiol. 104:3748; Somers (1992) Bio/Technology 10:1589-1594). In ad dition to these 'direct' transformation techniques, transformation can also be effected 25 by bacterial infection by means of Agrobacterium tumefaciens or Agrobacterum rhizogenes. These strains contain a plasmid (Ti or Ri plasmid) which is transferred to the plant following Agrobacterium infection. Part of this plasmid, termed T-DNA (trans ferred DNA), is integrated into the genome of the plant cell (see above for description of vectors). To transfer the DNA to the plant cell, plant explants are cocultured with a 30 transgenic Agrobacterium tumefaciens or Agrobacterium rhizogenes. Starting from infected plant material (for example leaf, root or stem sections, but also protoplasts or suspensions of plant cells), intact plants can be generated using a suitable medium which may contain, for example, antibiotics or biocides for selecting transformed cells. The plants obtained can then be screened for the presence of the DNA introduced, in 35 this case the expression construct according to the invention. As soon as the DNA has integrated into the host genome, the genotype in question is, as a rule, stable and the insertion in question is also found in the subsequent generations. The plants obtained can be cultured and hybridized in the customary fashion. Two or more generations should be grown in order to ensure that the genomic integration is stable and heredi 40 tary. The abovementioned methods are described (for example, in Jenes (1983) Tech niques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by Kung & Wu, Academic Press 128-143; and in Potrykus (1991) Ann Rev Plant Physiol Plant Mol Biol 42:205-225). One of skill in the art knows that the efficiency of transformation by ,Agrobacteriumrn may be enhanced by using a number of methods 45 known in the art. For example, the inclusion of a natural wound response molecule such as acetosyringone (AS) to the Agrobacterium culture has been shown to enhance transformation efficiency with Agrobacterium tumefaciens (Shahla (1987) Plant Mot. Biol. 8:291-298). Alternatively, transformation efficiency may be enhanced by wounding 53 the target tissue to be transformed. Wounding of plant tissue may be achieved, for ex ample, by punching, maceration, bombardment with microprojedtiles, etc. (see, e.g., Bidney (1992) Plant Molec. Biol. 18:301-313). A number of other methods have been reported for the transformation of plants (especially monocotyledbnous plants) includ 5 ing, for example, the "pollen tube method" (WO 93/18168; Luo (1988) Plant Mol. Biol. Rep. 6:165-174), macro-injection of DNA into floral tillers (Du (1989) Genet Manip Plants 5:8-12), injection of Agrobacterium into developing caryopses (WO 00/63398), and tissue incubation of seeds in DNA solutions (T6pfer (1989) Plant Cell 1:133-139). Direct injection of exogenous DNA into the fertilized plant ovule at the onset of em 10 bryogenesis was disclosed in WO 94/00583. WO 97/48814 disclosed a process for producing stably transformed fertile wheat and a system of transforming wheat via Agrobacterium based on freshly isolated or pre-cultured immature embryos, embryo genic callus and suspension cells. 15 As a rule, the expression construct integrated contains a selection marker, which im parts a resistance to a biocide (for example a herbicide) or an antibiotic such as kana mycin, G 418, bleomycin, hygromycin or phosphinotricin and the like to the transformed plant. The selection marker permits the selection of transformed cells from untrans formed cells (McCormick 1986) Plant Cell Reports 5:81-84). The plants obtained can 20 be cultured and hybridized in the customary fashion. Two or more generations should be grown in order to ensure that the genomic integration is stable and hereditary. The abovementioned methods are described (for example, in Jenes 11983; and in Potrykus 1991). As soon as a transformed plant cell has been generated, an intact plant can be obtained using methods known to the skilled worker. Accordingly, the present invention 25 provides transgenic plants. The transgenic plants of the invention are not limited to plants in which each and every cell expresses the nucleic acid sequence of interest under the control of the promoter sequences provided herein. Included within the scope of this invention is any plant which contains at least one cell which expresses the nu cleic acid sequence of interest (e.g., chimeric plants). It is preferred, though not neces 30 sary, that the transgenic plant comprises the nucleic acid sequence of interest in more than one cell, and more preferably in one or more tissue. Once transgenic plant tissue, which contains an expression vector, has been obtained, transgenic plants may be regenerated from this transgenic plant tissue using methods known in the art. Species from the following examples of genera of plants may be regenerated from transformed 35 protoplasts: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonela, Vigna, Citrus, Unum, Geranium, Manihot Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Hyoscyamus, Lycopersicon, Nicotiana, Soianum, Petunia, Digitalis, Majo rana, Ciohorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocalis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, 40 Cucumis, Browaalia, Glycine, Pisum, Lolium, Zea, Triticum, Sorghum, and Datura. For regeneration of transgenic plants from transgenic protoplasts, a suspension of trans formed protoplasts or a Petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, somatic embryo formation can be induced in the callus tissue. 45 These somatic embryos germinate as natural embryos to form plants. The culture me dia will generally contain various amino acids and plant hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on 54 the medium, on the genotype, and on the history of the culture. These three variables may be empirically controlled to result in reproducible regeneration. Plants may also be regenerated from cultured cells or tissues. Dicotyledonous plants which have been shown capable: of regeneration from transformed individual cells to obtain transgenic 5 whole plants include, for example, apple (Malus pumfia), blackberry (Rubus), Black berrylraspberry hybrid (Rubus), red raspberry (Rubus), carrot (Daucus carota), cauli flower (Brassica oleracea), celery (Apium graveolens), cucumber (Cucumis sativus), eggplant (Solanum melongena), lettuce (Lactuca sativa), potato (Solanum tuberosum), rape (Brassica; napus), wild soybean (Giycine canescens), soybean (Glycine max), 10 strawberry (Fragaria ananassa), tomato (Lycopersicon esculentum), walnut (Juglans regia), melon (Cucumis mleo), grape (Vitis vinifera), and mango (Manglfera indica). Monocotyledonous plants which have been shown capable of regeneration from trans formed individual cells to obtain transgenic whole plants include, for example, rice (Oryza sativa), rye (Secale camale), and maize (Zea mays). 15 in addition, regeneration of whole plants from cells (not necessarily transformed) has also been observed in: apricot (Prunus arneniaca), asparagus (Asparagus officinalis), banana (hybrid Musa), bean (Phaseolus vulgaris), cherry (hybrid Prunus), grape (Vitis vinifera), mango (Mangifera indica), melon (Gucumis melo), ochra (Abelmoschus escu 20 lentus), onion (hybrid All/um), orange (Cfitms sinensis), papaya (Carrca papaya), peach (Prunus persica), plum (Prunus domestica), pear (Pyrus communes), pineapple (Ananas comosus), watermelon (Citrullus vulgar/s), and wheat (Triticum aestivum). The regenerated plants are transferred to standard soil conditions and cultivated in a con ventional manner. After the expression vector is stably incorporated into regenerated 25 transgenic plants, it can be transferred to other plants by vegetative propagation or by sexual crossing. For example, in vegetatively propagated crops, the mature transgenic plants are propagated by the taking of cuttings or by tissue culture techniques to pro duce multiple identical plants. In seed propagated crops, the mature transgenic plants are self crossed to produce a homozygous inbred plant which is capable of passing the 30 transgene to its progeny by Mendetian inheritance. The inbred plant produces seed containing the nucleic acid sequence of interest These seeds can be grown to produce plants that would produce the selected phenotype. The inbred plants can also be used to develop new hybrids by crossing the inbred plant with another inbred plant to pro duce a hybrid. 35 Confirmation of the transgenic nature of the cells, tissues, and plants may be per formed by PCR analysis, antibiotic or herbicide resistance, enzymatic analysis and/or Southern blots to verify transformation. Progeny of the regenerated plants may be ob tained and analyzed to verify whether the transgenes are heritable. Heritability of the 40 transgene is further confirmation of the stable transformation of the transgene in the plant. The resulting plants can be bred in the customary fashion. Two or more genera tions should be grown in order to ensure that the genomic integration is stable and he reditary. Corresponding methods are described, (Jenes 1993; Potrykus 1991). 45 Also in accordance with the invention are cells, cell cultures, tissues, parts, organs, such as, for example, roots, leaves and the like in the case of transgenic plant organ isms derived from the above-described transgenic organisms, and transgenic propa gation material such as seeds or fruits. 55 Preferably, the method for enhancing the expression of a nucleic acid sequence in a plant or a plant cell further comprises, linking the introns with expression enhancing properties to the expression cassette by insertion via homologous recombination comprising the following steps: 5 a) providing in vivo or in vitro a DNA construct comprising said introns flanked by se quences allowing homologous recombination into a pre-existing expression cassette between the promoter and the nucleic acid of said expression cassette, b) transforming a recipient plant cell comprising said cassette, c) regenerating a transgenic plant where said intron has been inserted into the 10 genomic DNA of said promoter nucleic acid construct via homologous recombina ton. Two different ways for the integration of DNA molecules into genomes are possible: Either regions of sequence identity between the partners are used (homologous re 15 combination (HR), "gene targeting') or no sequence-specific requirements have to be fulfilled (illegitimate recombination also referred to as non-homologous end joining (NHEJ)). Gene targeting (GT) is the generation of specific mutations in a genome by homologous recombination-mediated integration of foreign DNA sequences. In contrast to natural recombination processes, one of the recombination partners is artificial and 20 introduced by transformation In gene targeting. The integration 'of transformed DNA follows pre-existing recombination pathways. Homologous recombination is a reaction between any pair of DNA sequences having a similar sequence of nucleotides, where the two sequences interact (recombine) to form a new recombinant DNA species. The frequency of homologous recombination Increases as the length of the shared nucleo 25 tide DNA sequences increases, and is higher with linearized plasmid molecules than with circularized plasmid molecules. Homologous recombination can occur between two DNA sequences that are less than identical, but the recombination frequency de clines as the divergence between the two sequences increases. Introduced DNA se quences can be targeted via homologous recombination by linking a DNA molecule of 30 interest to sequences sharing homology with endogenous sequences of the host cell. Once the DNA enters the cell, the two homologous sequences can interact to insert the introduced DNA at the site where the homologous genomic DNA sequences were lo cated. Therefore, the choice of homologous sequences contained on the introduced DNA will determine the site where the introduced DNA is integrated via homologous 35 recombination. For example, if the DNA sequence of interest is linked to DNA se quences sharing homology to a single copy gene of a host plant cell, the DNA se quence of interest will be inserted via homologous recombination at only that single specific site. However, if the DNA sequence of interest is linked to DNA sequences sharing homology to a multicopy gene of the host eucaryotic cell, then the DNA se 40 quence of interest can be inserted via homologous recombination at each of the spe cific sites where a copy of the gene is located. For example, if one wishes to insert a foreign gene into the genomic site where a selected gene is located, the introduced DNA should contain sequences homologous to the selected gene. A double recombi nation event can be achieved by flanking each end of the DNA sequence of interest 45 (the sequence intended to be inserted into the genome) with DNA sequences homolo gous to the selected gone. A homologous recombination event involving each of the homologous flanking regions will result in the insertion of the foreign DNA. Thus only 56 those DNA sequences located between the two regions sharing genomic homology become integrated into the genome. In the case of gene targeting via homologous recombination, the inventive intron that 5 has to be introduced in the chromosome, preferably in the 5'UTR of a gene (a pre existing expression cassette), is (for example) located on a DNA construct and is 5' and 3' flanked by nucleic acid sequences of sufficient homology to the target DNA (such an construct is called "gene targeting substrate') In which the intron should be integrated. Said flanking regions must be sufficient in length for making possible re 10 combination. They are, as a rule, in the range of several hundred bases to several kilo bases in length (Thomas KR and Capecchi MR (1987) Cell 51:503; Strepp et al. (1998) Proc Natl Acad Sci USA 95(8):4368-4373). In a preferred embodiment of the invention, the gene targeting substrate comprises an selection marker that is co-integrated with the intron into the genomic region of interest, allowing the selection of recombination 15 events. Preferably, the gene targeting substrate is integrated via a double cross over event between pairs of homologous DNA sequences of sufficient length and homology resulting in the insertion of the intron sequence (and if desired additional nucleic acid sequences e.g. selection marker). Using homologous recombination, a intron of the invention can be placed in the 5' non coding region of the target gene (e.g., an en 20 dogenous plant gene) to be transgenically expressed, by linking said intron to DNA sequences which are homologous to, for example, endogenous sequences upstream and/or downstream of the reading frame of the target gene. After a cell has been trans formed with the DNA construct in question, the homologous sequences can interact and thus place the intron of the invention at the desired site so that the intron sequence 25 of the invention becomes operably linked to the target gene and constitutes an expres sion construct of the invention. For homologous combination or gene targeting, the host organism - for example a plant - is transformed with the recombination construct using the methods described herein, and clones, which have successfully undergone recombination, are selected, for example using a resistance to antibiotics or herbicides. 30 If desirable to target the nucleic acid sequence of interest to a particular locus on the plant genome, site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, fbr example, homologous recombination using Agrobacterlum-derived sequences. Generally, plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homolo 35 gous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer DNA (T-DNA) sequences, as previously described (US 5,501,967, the entire contents of which are herein incorporated by reference). One of skill in the art knows that homologous recombination may be achieved using 40 targeting vectors which contain sequences that are homologous to any part of the tar geted plant gene, whether belonging to the regulatory elements of the gene, or the cod ing regions of the gene. Homologous combination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known. Gene targeting is a relatively rare event in higher eucaryotes, espe 45 cially in plants. Random integrations into the host genome predominate. One possibility of eliminating the randomly integrated sequences and thus increasing the number of cell clones with a correct homologous recombination is the use of a sequence-specific recombination system as described in US 6,110,736, by which unspecifically integrated 57 sequences can be deleted again, which simplifies the selection of events which have integrated successfully via homologous recombination. An efficient variant of gene targeting has been reported for Drosophila melanogaster 5 (Rong and Golic 2000 Science. 2000 Jun 16;288(5473):2013-8). In this method the construct for targeting is integrated into the host genome flanked by two recognition sites of a site-specific recombinase and Includes a site for a rare cutting restriction en donuclease. By induced expression of the site-specific recombinase a DNA circle is excised from the genome. This circle is then linearized after the restriction enzyme (in 10 this case I-Scel) has been expressed resulting in an "activated' DNA molecule with both ends homologous to the target sequence. In the female germ line of Drosophila, gene targeting occurred in about one out of 500 cells. Selection of gene targeting events from events of illegitimate recombination can be facilitated by certain combina tions of positive and negative selection techniques (WO 99120780). 15 Counter selection is a powerful approach in mammalian and plant systems to enrich for gene targeting events. In plants the bacterial codA gene as a cell autonomous negative selection marker can be used for selection in tissue culture (Schilman and Hooykaas Plant J 11:1377-1385, 1997; Thylaer et aL, Plant Mol Biol. 1997 Nov;35(4):523-30.). 20 Negative selection in plants allowed a more than a thousand-fold suppression of ran dom integration (Risseeuw et at, Plant J. 1997 Apr1 1(4):717-28..; Gallego et at, Plant Mol Biol. 1999 Jan;39(1):83-93; Terada et at, Nat Biotechnol. 2002 Oct;20(10):1030-4. Epub 2002 Sep 09. ). Exploratory approaches to increase gene targeting in plants comprise expression of proteins like RecA (WO 97/08331) or RecA-homologues de 25 rived from other species like e.g., Rad52 (WO 01/68882) or RecAlirE2 fusion-proteins (WO 01/38504). Use of poly(ADPribose)polyrnerase inhibitors has demonstrated an increased HR in plants (Puchta H et al. (1995) Plant J 7:203-210). Initiation of se quence-unspecific DNA double-strand breaks was also found to increase efficiency of HR in plants (Puchta H et al. (1995) Plant J 7(2),203-210; Lebel EG et al. (1993) Proc 30 Natl Acad Sci USA 90(2):422-426). However, sequence-unspecific induction of DNA strand breaks is disadvantageous because of the potential mutagenic effect. Se quence-specific induction of DNA strand-breaks may also increase efficiency of HR but is limited to artificial scenarios (Siebert R and Puchta H (2002) Plant Cell 14(5):1121 1131). 35 It is specifically contemplated by the inventors that one could employ techniques for the site-specific integration or excision of transformation constructs prepared in accordance with the instant invention. An advantage of site- specific integration or excision is that it can be used to overcome problems associated with conventional, transformation tech 40 niques, in which transformation constructs typically randomly integrate into a host ge nome in multiple copies. This random insertion of introduced DNA into the genome of host cells can be lethal if the foreign DNA inserts into an essential gene. In addition, the expression of a transgene may be influenced by "position effects' caused by the sur rounding genomic DNA. Further, because of difficulties associated with plants possess 45 ing multiple transgene copies, including gene silencing, recombination and unpredict able inheritance, it is typically desirable to control the copy number of the inserted DNA, often only desiring the insertion of a single copy of the DNA sequence. Site specific integration or excision of transgenes or parts of transgenes can be achieved in 58 plants by means of homologous recombination (see, for example, U.S. 5,527, 695). The DNA-constructs utilized within the method of this invention may comprise addi tional nucleic acid sequences. Said sequences may be for example localized in different positions with respect to the homology sequences. Preferably, the additional 5 nucleic acid sequences are localized between two homology sequences and may be introduced via homologous recombination into the chromosomal DNA, thereby resem bling an insertion mutation of said chromosomal DNA. However, the additional se quences may also be localized outside of the homology sequences (e.g., at the 5 - or 3 -end of the DNA-construct). In cases where the additional sequence resembles a 10 counter selection marker this may allow a distinction of illegitimate insertion events from correct insertion events mediated by homologous recombination. Corresponding negative markers are described below and suitable methods are well known in the art (WO 99/20780). 15 In a preferred embodiment of the invention, efficiency of the method of the invention may be further increased by combination with other methods suitable for increasing homologous recombination. Said methods may include for example expression of HR enhancing proteins (like e.g., RecA; WO 97/08331; Reiss B at at. (1996) Proc Nat! Acad Sci USA 93(7):3094-3098; Reiss B et a]. (2000) Proc Nail Aced Sci USA 20 97(7):3358-3363) or treatment with PARP inhibitors (Puchta H ot al. (1995) Plant J. 7:203-210). Various PARP inhibitors suitable for use within this invention are known to the person skilled in the art and may include for example preferably 3-Aminobenzamid, 8-Hydroxy-2-methylquinazolin-4-on (NU1025), 1,11b-Dihydro-[2H]benzopyrano{4,3,2 de]isoquinolin-3-on (GPI 6150), 5-Aminoisoquinolinon, 3,4-Dihydro-5-[4-(1-piperidinyl) 25 butoxy]-1 (2H)-isoquinolinon or compounds described in WO 00/26192, WO 00/29384, WO 00/32579, WO 00/64878, WO 00/68206, WO 00/67734,WO 01123386 or WO 01/23390. Furthermore, the method may be combined with other methods facilitation homologous recombination and/or selection of the recombinants like e.g., posi tive/negative selection, excision of illegitimate recombination events or induction of 30 sequence-specific or unspecific DNA double-strand breaks. In a preferred embodiment, the method for enhancing the expression of a nucleic acid sequence in a plant or a plant cell further via linking the intron with expression enhancing properties to the ex pression cassette by insertion via homologous recombination is applied to monocotyle donous plants or plant cells, more preferably to plants selected from the group consist 35 ing of the genera Hordeum, Avena, Secale, Triticum, Sorghum, Zee, Saccharum, and Oryza, most preferably a maize plant. The nucleic acid sequence in which one of the inventive intron is inserted and function ally linked (via the inventive methods), encodes for a selectable marker protein, a 40 screenable marker protein, a anabolic active protein, a catabolic active protein, a biotic or abiotic stress resistance protein, a male sterility protein or a protein affecting plant agronomic characteristics as described above and/or a sense, antisense, or double stranded RNA as described above. In a preferred embodiment of the present invention, said nucleic acid sequence encodes a protein. In yet another embodiment of the inven 45 tion the method is applied to recombinant DNA expression construct that contain a DNA for the purpose of expressing RNA transcripts that function to affect plant pheno type without being translated into a protein. Such non protein expressing sequences 59 comprising antisense RNA molecules, sense RNA molecules, RNA molecules with ribozyme activity, double strand forming RNA molecules (RNAI) as described above. Additionally, a further subject matter of the invention relates to the use of the above 5 describes transgenic organism or of cell cultures, parts of transgenic propagation mate rial derived there from, produced with the inventive method, for the production of food stuffs, animal feeds, seeds, pharmaceuticals or fine chemicals. Preferred is furthermore the use of transgenic organisms for the production of pharmaceuticals or fine chemi cals, where a host organism is transformed with one of the above-described expression 10 constructs, and this expression construct contains one or more structural genes which encode the desired fine chemical or catalyze the biosynthesis of the desired fine chemical, the transformed host organism is cultured, and the desired fine chemical is isolated from the culture medium. This process can be used widely for fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic fla 15 vorings, aroma substances and colorants. Especially preferred is the production of to copherols and tocotrienols, carotenoids, oils, polyunsaturated fatty acids etc. Culturing the transformed host organisms, and isolation from the host organisms or the culture medium, is performed by methods known to the skilled worker. The production of pharmaceuticals such as, for example, antibodies, vaccines, enzymes or pharmaceut 20 cally active proteins is described (Hood (1999) Curr Opin Biotechnol. 10(4):382-6;Ma (1999) Curr Top Microbiol. Immunol. 236:275-92; Russel (1999) Current Topics in Mi crobiology and Immunology 240:119-138; Cramer et a/. (1999) Current Topics in Mi crobiology and Immunology 240:95-118; Gavilondo (2000) Biotechniques 29(1):128 138 ; Holliger (1999) Cancer & Metastasis Reviews 18(4):411419). 25 Furthermore the present invention relates to recombinant DNA expression construct comprising at least one promoter sequence functioning in plants or plant cells, at least one intron with expression enhancing properties In plants or plant cells characterized by 30 VIlI) an intron length shorter than 1,000 base pairs, and IX) presence of a 5' splice site comprising the dinucleotide sequence 5'-GT-3' (SEQ ID NO: 78), and X) presence of a 3' splice site comprising the trinucleotide sequence 5'-CAG-3' (SEQ ID NO: 79), and 35 XI) presence of a branch point resembling the consensus sequence 5'-CURAY 3' (SEQ ID NO: 75) upstream of the 3'splice site, and X1i) an adenine plus thymine content of at least 40% over 100 nucleotides down stream from the 5' splice site, and XIII) an adenine plus thymine content of at least 50% over 100 nucleotides up 40 stream from the 3' splice site, and XIV) an adenine plus thymine content of at least 55%, and a thymine content of at least 30% over the entire intron, and at least one nucleic acid sequence, wherein said promoter sequence and at least one of said intron sequences are func 45 tionally linked to said nucleic acid sequence and wherein said intron is heterologous to said nucleic acid sequence and/or to said promoter sequence. 60 Sequences 1. SEQ ID NO: 1 BPSI.1: Sequence of the first intron isolated from the Oryza sativa metallothioneine-lIke gene (accession No. AP002540) 5 2. SEQ ID NO: 2 BPSL.2: Sequence of the first Intron isolated from the Oryza sativa Sucrose UDP Glucosyltransferase-2 gene (accession No. AC084380) 3. SEQ ID NO: 3 BPSI.3: Sequence of the second intron isolated from the Oryza sativa Sucrose UDP Glucosyltransferase-2 gene (accession No. 10 AC084380) 4. SEQ ID NO: 4 BPS14: Sequence of the third intron isolated from the Oryza sativa Sucrose UDP Glucosyltransferase-2 gene (accession No. AC084380) 5. SEQ ID NO: 5 BPSI.5: Sequence of the eighth intron isolated from the 0. sa 15 tive gene encoding for the Sucrose transporter (accession No. AF 280050). 6. SEQ ID NO: 6 BPSI.6: Sequence of fourth intron isolated from the Oryza saiva gene (accession No. BAA94221) encoding for an un known protein with homology to the A. theliana chromosome I 20 sequence from clones T22013, F12K2 encoding for a putative lipase (AC006233). 7. SEQ ID NO: 7 BPS1.7: Sequence of the fourth intron isolated from the Oryza sativa gene (accession No. BAB90130) encoding for a putative cinnamyl-alcohol dehydrogenase. 25 8. SEQ ID NO: 8 BPSI.8: Sequence of the second intron isolated from the Oryza sativa gene (accession No. AC084766) encoding for a putative ribonucleoprotein. 9. SEQ ID NO: 9 BPSI.9: Sequence of the fifth intron isolated from the Oryza sativa clone GI 12061241. 30 10. SEQ ID NO: 10 BPSI.10: Sequence of the third intron isolated from the 0. sa tiva gene (accession No. AP003300) encoding for a putative protein kinase. 11. SEQ ID NO: 11 BPS. 11: Sequence of the first intron Isolated from the 0. sativa gene (accession No. L37528) encoding for a MADS3 box pro 35 tein. 12. SEQ ID NO: 12 BPSI.12: Sequence of the first intron isolated from the Oryza sativa gene (accession No. CB625805) encoding for a putative Adenosylmethionine decarboxylase. 61 13. SEQ ID NO: 13 BPSI.13: Sequence of the first intron isolated from the 0. sat/va gene (accession No. CF297669) encoding for an Aspartic pro teinase. 14. SEQ ID NO: 14 BPSI.14: Sequence of the first intron isolated from the 0. saitva 5 gene (accession No. CB674940) encoding for a Lec14b protein. 15. SEQ ID NO: 15 BPSI.15: Sequence of the first intron isolated from the Oryza sativa gene (accession No. BAD37295.1) encoding for a puta tive SalT protein precursor 16. SEQ ID NO: 16 BPSI.16: Sequence of the first intron isolated from the 0. safiva 10 gene (accession No. BX928664) encoding for a putative Reticu Ion. 17. SEQ ID NO: 17 BPSI.17: Sequence of the first intron isolated from the 0. satva gene (accession No. AA752970) encoding for a glycolate oxi dase. 15 18. SEQ ID NO: 18 BPSI.18: Sequence of the first intron isolated from the Oryza sativa clone (accession No. AK06442 encoding putative non coding 19. SEQ ID NO: 19 BPSL1.9: Sequence of the first intron isolated from the Oryza sativa clone (accession No. AK062197) encoding putative non 20 coding 20. SEQ ID NO: 20 BPSI.20 sequence of the first intron Isolated from the 0. sativa gene (accession No. CF279761) encoding for a hypothetical protein. 21. SEQ ID NO: 21 BPSI.21 Sequence of the first intron isolated from the Oryza 25 sativa gene (accession No. CF326058) encoding for a putative membrane transporter. 22. SEQ ID NO: 22 BPSL,22: Sequence of the firsit intron isolated from the Oryza sativa gene (accession No. C26044) encoding for a putative ACT domain repeat protein 30 23. SEQ ID NO: 23 Sucrose-UDP glucosyltransferase 2 forward (for) primer 24. SEQ ID NO: 24 Sucrose-UDP glucosyltransferase 2 reverse (rev) primer 25. SEQ 1D NO: 25 Putative Bowman-Kirk trypsin inhibitor (for) primer 26. SEQ ID NO: 26 Putative Bowman-Kirk trypsin inhibitor rev primer 27. SEQ ID NO: 27 Hypothetical protein Ace. No. CF279761 (for) primer 35 28. SEQ ID NO: 28 Hypothetical protein Acc. No. CF279761 rev primer 29. SEQ ID NO: 29 Phenylalanine ammonia-lyase (for) primer 62 30. SEQ ID NO: 30 Phenylalanine ammonia-lyase rev primer 31. SEQ ID NO: 31 Metallothioneine-like protein 1 (for) primer 32. SEQ ID NO: 32 Metallothioneine-like protein I rev primer 33. SEQ ID NO: 33 Catalase (for) primer 5 34. SEQ ID NO: 34 Catalase rev primer 35. SEQ ID NO: 35 Putative stress-related protein (for) primer 36. SEQ ID NO: 36 Putative stress-related protein rev primer 37. SEQ ID NO: 37 Putative translation Initiation factor SUl (for) primer 38. SEQ ID NO: 38 Putative translation initiation factor SUIli rev primer 10 39. SEQ ID NO: 39 Polyubiquitin (for) primer 40. SEQ ID NO: 40 Polyubiquitin rev primer 41. SEQ ID NO: 41 Glutathione S-transferase II (for) primer 42. SEQ ID NO: 42 Glutathione S-transferase 11 rev primer 43. SEQ ID NO: 43 Metallothioneine-like protein 2 (for) primer 15 44. SEQ ID NO: 44 Metallothioneine-like protein 2 rev primer 45. SEQ ID NO: 45 Translational initiation factor eiF1 (for) primer 46. SEQ ID NO: 46 Translational initiation factor elF1 rev primer 47. SEQ ID NO: 47 OSJNBaOO24F24.10 (unknown protein) (for) primer 48. SEQ ID NO: 48 OSJNBaOO24F24.10 (unknown protein) rev primer 20 49. SEQ ID NO: 49 Protein similar to Histone 3.2-614 (for) primer 50, SEQ ID NO: 50 Protein similar to Histone 3.2-614 rev primer 51. SEQ ID NO: 51 OSJNBaOO42L1 6.3 (for) primer 52. SEQ ID NO: 52 OSJNBaOO42LI 6.3 rev primer 53. SEQ ID NO: 53 BPSI.1-5' primer 25 54. SEQ ID NO: 54 BPSI.1-3' primer 55. SEQ ID NO: 55 BPSI.2-5' primer 56. SEQ ID NO: 56 BPSI.2-3' primer 63 57. SEQ ID NO: 57 BPSI.3-5' primer 58. SEQ ID NO: 58 BPSI.3-3' primer 59. SEQ ID NO: 59 BPSI.4-5' primer 60. SEQ ID NO: 60 BPSI.4-3' primer 5 61. SEQ ID NO: 61 BPSI.5-5'primer 62. SEQ ID NO: 62 BPSI.5-3' primer 63. SEQ ID NO: 63 BPSI.6-5' primer 64. SEQ ID NO: 64 BPSL 6-3' primer 65. SEQ ID NO: 65 BPSi.7-5' primer 10 66. SEQ ID NO: 66 BPSI.7-3' primer 67. SEQ ID NO: 67 BPSL8-5' primer 68. SEQ ID NO: 68 BPSI.8-3' primer 69. SEQ ID NO: 69 BPSI.9-5' primer 70. SEQ ID NO: 70 BPSI.9-3' primer 15 71. SEQ ID NO: 71 BPSI.10-5' primer 72. SEQ ID NO: 72 BPSI.10-3' primer 73. SEQ ID NO: 73 BPSI.11-5' primer 74. SEQ ID NO: 74 BPSI.11-3' primer 75. SEQ ID NO: 75 5'-CURAY-3' plant branchpoint consensus sequences 1 20 76. SEQ ID NO: 76 5'-YURAY-3' plant branchpoint consensus sequences 2 77. SEQ ID NO: 77 5'-(AG)(AG)/GT(AGT)(AGT)(GTC)-3' preferred 5 splice-site 78. SEQ ID NO: 78 5'splice site dinucleotide 5'-GT-3' 79. SEQ 1D NO: 79 3'splice site trinucleotide 5'-CAG-3' 80. SEQ ID NO: 80 5' splice site plant consensus sequence 5'-AG::GTAAGT-3' 25 81. SEQ ID NO: 81 3' splice site plant consensus sequence 5'-CAG::GT-3' 82. SEQ ID NO: 82 Sequence of the first intron isolated from the Oryza sativa met allothioneine-like gene (accession No. AP002540) including 64 sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.1 (SEQ ID NO:l) 83. SEQ ID NO: 83 Sequence of the first intron isolated from the 0. safiva Sucrose UDP Glucosyltransferase-2 gene (accession No. AC084380) 5 including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.2 (SEQ ID NO:2) 84. SEQ ID NO: 84 Sequence of the second intron isolated from the 0. saliva Su crose UDP Glucosyltransferase-2 gene (accession No. AC084380) including sequences 5' and 3' adjacent to the 5' 10 and 3' splice sites of the intron sequence BPSI.3 (SEQ ID NO:3) 85. SEQ ID NO: 85 Sequence of the third intron isolated from the 0. sativa Sucrose UDP Glucosyltransferase-2 gene (accession No. AC084380) including sequences 5' and 3' adjacent to the 5' and 3' splice 15 sites of the intron sequence BPSL4 (SEQ ID NO:4) 86. SEQ ID NO: 86 Sequence of the eighth intron isolated from the Oryza sativa gene encoding for the Sucrose transporter (GenBank acces sion No. AF 280050) including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.5 (SEQ 20 ID NO:5) 87. SEQ ID NO: 87 Sequence of the eighth intron isolated from the Oiyza sativa gene encoding for the Sucrose transporter (accession No. AF 280050) including modified 5' and 3' splice sites and se quences 5' and 3' adjacent to the 5' and 3' splice sites of the 25 intron sequence BPSI.5 (SEQ ID NO:5) 88. SEQ ID NO: 88 Sequence of the fourth intron isolated from the Oyza sativa gene encoding for an unknown protein with homology to the A.thaliana chromosome || sequence from clones T22013, F12K2 encoding for a putative lipase (AC006233) including se 30 quences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.6 (SEQ ID NO:6) 89. SEQ ID NO: 89 Sequence of the fourth intron isolated from the Oryza saiva gene encoding for an unknown protein with homology to the A.thaliana chromosome 11 sequence from clones T22013, 35 F121K2 encoding for a putative lipase (AC006233) including modified 5' and 3' splice sites and sequences 5' and 3' adja cent to the 5' and 3' splice sites of the intron sequence BPSI.6 (SEQ ID NO:6) 90. SEQ ID NO: 90 Sequence of the fourth intron isolated from the Oryza sativa 40 gene (accession No. BAB90130) encoding for a putative cin namyl-alcohol dehydrogenase including sequences 5' and 3' 65 adjacent to the 5' and 3' splice sites of the intron sequence BPSI.7 (SEQ ID NO:7) 91. SEQ iD NO: 91 Sequence of the fourth intron isolated from the Oryza sativa gene (accession No. BAB901 30) encoding for a putative cin 5 namyl-alcohol dehydrogenase including modified 5' and 3' splice sites and sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.7 (SEQ ID NO:7) 92. SEQ ID NO: 92 Sequence of the second intron isolated from the Oryza sativa gene (accession No. AC084766) encoding for a putative ribo 10 nucleoprotein including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSl.8 (SEQ ID NO:8) 93. SEQ ID NO: 93 Sequence of the second Intron isolated from the Oryza sativa gene (accession No, AC084766) encoding for a putative ribo 15 nucleoprotein including modified 5' and 3' splice sites and se quences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.8 (SEQ ID NO:8) 94. SEQ ID NO: 94 Sequence of the third intron isolated from the Oryza sativa gene (accession No. AP003300) encoding for a putative protein 20 including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.10 (SEQ ID NO:10) 95. SEQ ID NO: 95 Sequence of the third intron isolated from the Oryza sativa gene (accession No. AP003300) encoding for a putative protein including modified 5' and 3' splice sites and sequences 5' and 25 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSL10 (SEQ ID NO:10) 96. SEQ ID NO: 96 Sequence of the first intron isolated from the Oryza sativa gene (accession No. L37528) encoding for a MADS3 box protein in cluding sequences 5' and 3' adjacent to the 5' and 3' splice 30 sites of the intron sequence BPSIL1 (SEQ ID NO:i 1) 97. SEQ ID NO: 97 Sequence of the first intron isolated from the Oryza sativa gene (accession No. L37528) encoding for a MADS3 box protein in cluding modified 5' and 3' splice sites and sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence 35 BPSII 1 (SEQ ID NO:11) 98. SEQ ID NO: 98 Sequence of the first intron isolated from the Oryza sativa gene (accession No. CB625805) encoding for a putative Adenosyl methionine decarboxylase including sequences 5' and 3' adja cent to the 5' and 3' splice sites of the intron sequence 40 BPSI.12 (SEQ ID NO:12) 66 99. SEQ ID NO: 99 Sequence of the first intron isolated from the Oryza sativa gene (accession No. CF297669) encoding for a Aspartic proteinase including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.13 (SEQ ID NO:13) 5 100. SEQ ID NO: 100 Sequence of the first intron isolated from the Oryza sativa gene (accession No. CB674940) encoding for a Lecl4b protein in cluding sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI,14 (SEQ ID NO:14) 101. SEQ ID NO: 101 Sequence of the first intron isolated from the 0. safiva gene 10 (accession No. CA128696) encoding for a putative mannose binding rice lectin including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.15 (SEQ ID NO:15) 102. SEQ ID NO: 102 Sequence of the first intron isolated from the Oryza sativa gene 15 (accession No. BX928664) encoding for a putative Reticulon including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.16 (SEQ ID NO:16) 103. SEQ ID NO: 103 Sequence of the first intron isolated from the Oryza saiva gene (accession No. AA752970) encoding for a glycolate oxidase in 20 cluding sequences 5' and 3' adjacent to the -5' and 3' splice sites of the intron sequence BPSI.17 (SEQ ID NO:17) 104. SEQ ID NO: 104 Sequence of the first intron isolated from the Oryza sativa clone GI 34763855 including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.18 (SEQ ID 25 NO:18) 105. SEQ ID NO: 105 Sequence of the first Intron isolated from the Oryza sativa clone GI 32533738 including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.19 (SEQ ID NO:19) 30 106. SEQ ID NO: 106 Sequence of the first intron isolated from the Oryza sativa gene (accession No. CF279761) encoding for a hypothetical protein including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPS.20 (SEQ ID NO:20). 107. SEQ ID NO: 107 Sequence of the first intron isolated from the 0. sativa gene 35 (accession No. CF326058) encoding for a putative membrane transporter including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.21 (SEQ ID NO:21). 108. SEQ ID NO: 108 Sequence of the first intron isolated from the 0. saliva gene 40 (accession No. C26044) encoding for a putative ACT domain 67 repeat protein including sequences 5' and 3' adjacent to the 5' and 3' splice sites of the intron sequence BPSI.22 (SEQ ID NO:22). 109. SEQ ID NO: 109 Binary vector pBPSMM291 5 110. SEQ ID NO: 110 Binary vector pBPSMM305 111. SEQ ID NO: 111 Binary vector pBPSMM350 112. SEQ ID NO: 112 Binary vector pBPSLM139 113. SEQ ID NO: 113 Artificial sequence: cassette from vector pBPSMM355 (OsCP12::BPSI.1) comprising Os CP12 promoter (bp 1 - 854) 10 and BPSI.1 intron (bp 888 - 1470). 114. SEQ ID NO: 114 Artificial sequence: cassette from from vector pBPSMM355 (ZmHRGP::BPSI.1) comprising Maize [HRGP] hydroxyproline rich glycoprotein (extensin) 57UTR promoter (bp 1 - 1184) and oryza sativa BPSI.1 intron (bp 1217- 1799) 15 115. SEQ ID NO: 115 Artificial sequence: cassette from vector pBPSMM358 (OsC CoAMT1::BPSI.1)comprising p-caffeoyl-CoA 3-0 methyltransferase [CoA-O-Methyl] promoter (bp 1 - 1034)and BPSI.1 intron (1119 - 1701) 116. SEQ ID NO: 116 Artificial sequence: cassette from vector EXS1025 (ZmGlobu 20 lin1::BPSI.1) comprising Maize Globulin-1 [ZmGlbl] promoter (W64A) (bp 1 - 1440)and BPSI.1 intron (1443 - 1999) 117. SEQ ID NO: 117 Artificial sequence: cassette from vector pBPSMM369 (OsV ATPase::BPSI.L1)comprising putative Rice H+-transporting ATP synthase 57UTR promoter (1 - 1589) and BPSL.1 intron (1616 25 2198) 118. SEQ ID NO: 118 Artificial sequence: cassette from vector pBPSMM366 (OsC8,7SI::BPSL.1) comprising Putative Rice C-8,7 Sterol iso merase promoter (1 - 796) and BPSL.1 intron (827 - 1409) 119. SEQ ID NO: 119 Artificial sequence: cassette from vector pBPSMM357 30 (ZmLDH::BPSLI) comprising maize gene Lactate Dehydroge nase 57/UTR promoter (bp 1 - 1062) and BPSL.1 intron (bp 1095 - 1677). 120. SEQ ID NO: 120 Artificial sequence: cassette from vector pBPSLM229 (ZmLDH::BPSI.S) comprising maize gene Lactate Dehydroge 35 nase 5'7UTR promoter (bp 1 - 1062) and BPSI.5 intron (bp 1068 -1318) 121. SEQ ID NO: 121 Artificial sequence: cassette from vector pBPSMM371 (Os Lea::BPSl.1)comprising rice Lea (Late Ernbryogenesis Abun dant) promoter (bp 1 - 1386) and BPSI1 intron (bp 1387 40 2001) 68 EXAMPLES Chemicals Unless indicated otherwise, chemicals and reagents in the Examples were obtained 5 from Sigma Chemical Company (St. Louis, MO), restriction endonucleases were from New England Biolabs (Beverly, MA) or Roche (Indianapolis, IN), oligonucleotides were synthesized by MWG Biotech Inc. (High Point, NC), and other modifying enzymes or kits regarding biochemicals and molecular biological assays were from Clontech (Palo Alto, CA), Phermacia Biotech (Piscataway, NJ), Promega Corporation (Madison, WI), 10 or Stratagene (La Jolla, CA). Materials for cell culture media were obtained from Gibco/BRL (Gaithersburg, MD) or DIFCO (Detroit, MI). The cloning steps carried out for the purposes of the present invention, such as, for example, restriction cleavages, aga rose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to ni trocellulose and nylon membranes, linking DNA fragments, transformation of E, coli 15 cells, growing bacteria, multiplying phages and sequence analysis of recombinant DNA, are carried out as described by Sambrook (1989). The sequencing of recombi nant DNA molecules is carried out using ABI laser fluorescence DNA sequencer follow ing the method of Sanger (Sanger 1977). 20 Example 1: Identification and characterization of IME-Introns in highly express Ing genes 1.1 Identification of strongly and constitutively expressed Oryza sativa gene candidates. 25 Using the above described "sequencing by hybridization method' in silicon clone distri bution analysis of rice cDNA libraries have been performed. The rice cDNA clone distribution profiles were derived from about 7.6 million rice cDNA clones, which were generated over 23 rice cDNA libraries of different tissues at differ ent developmental stages and biotic/abiotic treatments. Method for the production of 30 cDNA libraries are well known in the art (e.g. Gubler U, and Hoffman BJ. (1983) A sim ple and very efficient method for generating cDNA libraries. Gene 25(2-3):263-269.; Jung-Hwa Oh et al. (2003) An improved method for constructing a full-length enriched cDNA library using small amounts of total RNA as a starting material. EXPERIMENTAL and MOLECULAR MEDICINE 35(6):586-590; Lanfranchi et al. (1996) Identification of 35 4370 expressed sequence tags from a 3'-end-specific cDNA library of human skeletal muscle by DNA sequencing and filter hybridization. Genome Res. 6(1):35-42). Fur thermore, a comprehensive description of cDNA library construction is provided in 1) Cowell and Austin. cDNA Library Protocols. In Methods in Molecular Biology, Volume 69, October 1996, Humana Press, Scientific and medical publishers, ISBN: 0-89603 40 383-X; and 2) Ying, Shao-Yao. Generation of cDNA Libraries, Methods and Protocols. In Methods in Molecular Biology, Volume 221, February 2003, Humana Press, Scien tific and medical publishers, ISBN: 1-588294)66-2. All of the clones were clustered into a total of 300,408 rice clusters using the above 45 described (see "sequencing by hybridization method', or "HySeq-technology') high throughput technology of 288 plant-specific 7 mer-oligonucleotide fingerprinting. For each generated cluster, clones have further been clustered into different variants using more stringent cutoff value of the hybridization patten similarity, leading to 335,073 69 rice done variants. Therefore, within each variant for given cluster, clones are more homogeneous. The distribution of rice cDNA clones over the 23 normalized cDNA li braries for given variants provides the rice variant expression profiles. The normalized cDNA library was produced by first adjusting the orignal library clone size to the aver 5 age clone size of all of the 23 libraries, then adjusting the number of clones per variant in that library based on the adjusted total number of clones in that library. Rice clones are selected from the rice clusters for sequencing to generate rice EST data. In using the clones distribution profiles of 335,073 rice variarts, 145 variants were selected based on the number of clones exceeding top 1% of the clone distribution 10 across the entire library for over each of 23 libraries, and genes were identified using the homologs to the EST sequences derived from the variants. These candidate genes showed strong, constitutive, and ubiquitous expression. The rice EST sequences ho molog to these candidate genes were mapped to the rice genomic DNA sequences. Top 15 candidates out of 145 were selected based on availability of genomic se 15 quences, annotation, and high level of expression (Table 2). Table 2. Gene candidates for potential IME-introns Candidate gene Annotation 1 sucrose-UDP glucosyltransferase 2 2 putative Bowman-Kirk trypsin inhibitor 3 Hypothetical Protein 4 phenylalanine ammonia-lyase 5 metallothioneine-like protein 6 Catalase 7 utative stress-related protein 8 putative translation initiation factor SUM1 9 Polyubiquitin 10 glutathione S-transferase 11 11 metallothioneine-like protein2 12 translational initiation factor elF1 13 OSJNBaOO24F24.10 (Unknown Protein) 14 Similar to Histone 3.2-614 15 OSJNBaOO42L16.3 1.2 Validation of highly expressing gene candidates using real time RT-PCR 20 Expression levels of the endogenous genes representing these 15 candidates were measured at the mRNA levels using LightCycler. Total RNA was extracted from rice plants at various developmental stages and tissues with and without drought stress (6, 12, 24, and 48 hr by withholding water) using Qiagen RNeasy Plant Mini Kit (Cat. No 74904). Quality and quantity of the RNA were determined using Molecular Probes Ri 25 boGreen Kit (Cat. No. R-11490) on the Spectra MAX Gemini. One lkg of RNA was used for RT-PCR (Roche RT-PCR AMV kit, Cat. No. 1483188) in the reaction solution I (1 pg RNA, 2 [iL 1ox Buffer, 4 4L 25 mM MgC 2 , 2 iL 1 mM dNTPs, 2 RL 3.2 Rg Ran dom Primers, 1 [LL 50 units RNase Inhibitor, 0.8 sxL 20 units AMV-RT polymerase, fill to 70 20 pAL with sterile water) under the optimized PCR program (25*C 10 min, 42*C 1hr, 990C 5 min, 40 stop reaction). The RT-PCR samples were used for the LightCycler reaction (11.6 gaL sterile water, 2.4 tL 25mM MgC 2 , 2 4L SYBR Green Polymerase mix, 2 [LL 10mM Specific Primer Mix, 5 2 RL RT-PCR reaction product) under the optimized program (950C 5 min, 95*C 30 sec, 614C 40 sec, 720C 40 sec and repeat steps 2-4 for 30 cycles, 720C 10 min, and 4*C stop reaction) provided by Roche (LightCycler FastStart DNA Master SYBR Green I, Cat. No.3003230). 10 Table 3. Primer sequences of the gne candidates Gene Primers SEQ ID NO. Sucrose-UDP glucosyltransferase 2Fwd: 5 -tttgtgcagcccgctttctacgag 23 Rev: 5 -acggccaacgggacggtgcta 24 Putative Bowman-Birk trypsin in- Fwd: 5 -gtcctcgccggcatcgtcac 25 hibitor Rev: 5 -cagaacggcgggttgatcc 26 Hypothetical protein Fwd: 5 -agctcgctcgcggtctt 27 Acc. No. CF279761 Rev: 5 -acagggcccaagtcgtgtgc 28 Phenylalanine ammonia-lyase Fwd: 5 -aggtctcgccatcgtcaatg 29 Rev: 5 -cgagacgggcgttgt 30 Methallothioneine-like protein 1 Fwd: 5 -ggtgcggaggatgcaagatg 31 Rev: 5 -ggggttgcaggtgcagttgtcg 32 Catalase Fwd: 5 -ggcgtcaacacctacacctt 33 R ev: 5 -tgcactg2cagcatctttgc 34 Putative stress-related protein Fwd: 5 -ggtggatgccacggtgcaagag 35 Rev: 5 -ggggaggtactgtgotc 36 Putative translation initiation factor Fwd: 5 -tgcggaagccaatgotga 37 SUl1 Rev: 5 -ccagcctaactaggaacgtc 38 Polyubiquitin Fwd: 5 -tcaggggaggcatgcaaa 39 Rev: 5 -tgcataccaccacggagacgaa 40 Glutathione S-transferase 11 Fwd: 5 -cgatttctccaaaggcgagcac 41 Rev: 5 -tgcgggtatgcgtccaaca 42 Metaliothioneine-like protein 2 Fwd: 5 -acagccaccaccaagacottog 43 Rev: 5 -ctgcagctggtgccacacttgc 44 Translational initiation factor eli Fw d:5 -tcccaactgccttOgatccctt 45 Rev: 5 -tggacagtggtcaggctcttacgg 46 OSJNBaOO24F24.1 0 (unknown Fwd: 5 -gagttctaccagttcagcgacc 47 protein) Rev: 5 -aacccaaggcgttgac 48 Similar to Histone 3.2-614 Fwd: 5 -agaccgcccgcaagtc 49 Rev: 5 -cttgggcatgatggtgacgc 50 OSJNBaOO42L1 6.3 Fwd: 5 ccaagagggagtgtgtatgcca1 51 Rev: 5 -acgaggaccaccacggtacccat 52 Standardizing the concentration of RNA (1 Rg) in each of the RT-PCR reactions was sufficient to directly compare the samples if the same primers were used for each Ughtcycler reaction. The output results were a number that corresponds to the cycle of 15 PCR at which the sample reaches the inflection point in the log curve generated. The lower the cycle numbers the higher the concentration of target RNA present in the 71 sample. Each sample was repeated in triplicate and an average was generated to pro duce the sample "crosspoint' value. The lower the crosspoint, the stronger the target gene was expressed in that sample. (Roche Molecular Biochemicals LightCycler Sys tem: Reference Guide May 1999 version) Based on the LightCycler results, 11 candi 5 dates were selected (Table 4). Table 4. LightCycler results representing expression of the rice gene candidates at the mRNA levels. Gene candidates Drought stressed rice root (R) and shoot (S) Well-watered conditions [strong & constitu- (hr withholding water) ive expression] R6 R12 R24 R48 S6 S12 S24 S48 seedling Panicle shoots flowers during flowering Unknown 21.1 21.6 N/A 20.3 0.5 1.7 N/A 21.0 23.3 22.7 21.4 23.7 Catalase 21.2 2.7 26.7 .0 21.9 21.7 N/A 27.8 22.8 31 20.6 23.5 GSTii 20.620.3 23.3 23.7 21.8 23.2 N/A 20.6 24.4 22.6 22.1 24.8 Hypothetical Pro- 31 31 31 31 31 31 31 31 31 31 27.4 27.0 tein Metallothioneine 1 20.1 21.5 16.5 16.3 18.3 19.6 A 19.2 21.0 22.5 20.6 20.6 Metallothioneine2 20.2120.8 23.8 24.8 16.5 18.7 N/A 18.7 19.9 17.8 21.2 19.2 PolyUbuiquitin 19.5 19.1 19.4 20.4 19.1 20.4 N/A 19.8 22.8 20.7 20.0 22.6 Stress Related 24.1 23.9 2.7 24.0 23.4 23.4 N/A 23.3 24.6 24.0 23.6 24.9 Protein Sucrose-UDP 21.3 21.9 26.6 26.7 20.7 20.9 27.2 22-6 20.9 19.1 20.7 26.0 glucoryltransferase 2 SUli 21.3 1.1 23.1 23.6 1.9 2.8 A 21.7 4.4 23.8 22.9 30.2 TLF 23.6 23.6 N/A 22.9 22.1 23.3 N/A 23.1 24.6 23.8 22.8 23.7 Trypsin Inhibitor 24.0 23.8 24.5 25.0 22.8 23.3 23.5 23.2 26.2 23.2 23.2 23.05 The numbers represent PCR cycle that reaches the start of the exponential curve of 10 the PCR product. Lower the number indicates that higher the expression of the en dogenous gene is. 1.3 Identification of IME-introns Candidate introns were isolated using the public available genomic DNA sequences 15 (e.g. http://www.ncbi.nim.nih.gov/genomes/PLANTS/PlantUst.htm), leading to a total of 20 introns, mostly first, second, and/or third introns from the targeted genes. These intron sequences were screened by the following IME criteria: - 5' splice site GT, 3' splice site CAG * At least 40% AT rich over 100 nucleotides downstream from the 5' splice 20 site GT - At least 50% AT rich over 100 nucleotides upstream from the 3' splice site CAG . At least 55% AT rich and 35% T rich over the entire intron - CURAY branch point 25 - Intron size less than 1kb 72 Selected intron candidates can retain up to 50 bp exon sequences upstream and downstream of the 5' and 3' splice sites, respectively. After screening the intron sequences against the IME criteria described above, four out 5 of the 20 candidates were chosen and named as follows. Table 5. The intron candidates Intron name Annotation BPSI.1 (SEQ ID No. 1) Metallothioneinel first intron BPSI.2 (SEQ ID No. 2) Sucrose-UDP glucosyltransferase2 first intron BPSI.3 (SEQ ID No. 3) Sucrose-UDP glucosyltransferase2 second intron BPSI.4 (SEQ ID No. 4) 1 Sucrose-UDP glucosyltransferase2 third intron 1.4 Isolation of the intron candidates 10 Genomic DNA from rice was extracted using the Qiagen DNAeasy Plant Mini Kit (Qiagen). Genomic DNA regions containing introns of interest were Isolated using con ventional PCR. Approximately 0.1 pg of digested genomic DNA was used for the regu lar PCR reaction (see below). The primers were designed based on the rice genomic sequences. One pL of the diluted digested genomic DNA was used as the DNA tem 15 plate in the primary PCR reaction. The reaction comprised six sets of primers (Table 6) in a mixture containing Buffer 3 following the protocol outlined by an Expand Long PCR kit (Cat #1681-842, Roche-Boehringer Mannheim). The isolated DNA was employed as template DNA in a PCR amplification reaction using the following primers: 20 Table 6. Primer sequences Primer name Sequence BPSI.1-5 (SEQ ID No. 53) 5 -ccogggcaccctgcggagggtaagatccgatcacc BPSI.1-3 (SEQ ID No. 54) 5 -cggaccggtacatcttgcatctgcatgtac BPSI.2-5 (SEQ iD No. 55) 5 -ccogggcacccttcaccaggttcgtgctgatttag BPSI.2-3 (SEQ ID No. 56) 5 -cggaccgaaccagcctgccaastaacag BPSI.3-5 (SEQ ID No. 57) 5 -cccgggcacctcctgagtgcacaggtttg BPSI.3-3 (SEQ ID No. 58) 5 -cggaccgggagataacaatcccctcctgcatg BPSI.4-5 (SEQ ID No. 59) 5 -cccgggcacccagcttgtggaagaagggtatg BPSI.4-3 (SEQ ID No. 60) 5 -cggaccggttgttggtgcgaaatatacatc Amplification was carried out in the PCR reaction (5 4L 1OX Advantage PCR Mix [Ep pendorf], 5 sLL genomic DNA [corresponds to approximately 80 ng], 2.5 mM of each dATP, dCTP, dGTP and dTTP [Invitrogen: dNTP mix], 1 FL of 20 pxM 5 -intron specific 25 primer 20pM, I L of 20 pM 3 intron specific primer, 14L TripleMaster DNA Poly merase mix [Eppendort], in a final volume of 50 sL) under the optimized PCR program (1 cycle with 15 sec at 94 0 C and 1 min at 80*C 35cycles with 15 sec at 94*C, I min at 58*C and I min at 72 0 C) provided by Thermocycler (T3 Thermocycler Biometra). 30 The PCR product was applied to an 1% (wlv) agarose gel and separated at 80V. The PCR products were excised from the gel and purified with the aid of the Qiagen Gel Extraction Kit (Qlagen, Hilden, Germany). The PCR product can be cloned directly into vector pCR4-TOPO (Invitrogen) following the manufacturer s instructionsj.e. the PCR 73 product obtained was inserted into a vector having T overhangs with its A overhangs and a topoisomerase. 1.5 Vector Construction 5 The base vector to which the intron candidates were clone in was pBPSMM267. This vector comprises the maize ubiquitin promoter with no intronic sequence, followed by multiple cloning sites (MCS) to be used for addition of introns of interest then the GUS int ORF (including the potato invertase [PN]2 intron to prevent bacterial expression), followed by nopaline synthase (NOS) terminator. The intron-containing expression vec 10 tors were generated by ligation of Xmal-Rsril digested intron PCR products into Xmal Rsril linearized pBPSMM267, thereby resulting in the following vectors (Table 7). Table 7. GUS chimeric constructs containing introns in the 5 UTR pUC-based Binary vector Composition of the expression cassette expression (promoter::intron::reporter gene::terminator) vector pBPSMM291 pBPSMM350 Zm.ubiquitin promoter::BPSI.1::GUS::NOS3 pBPSMM293 pBPSMM353 Zm.ubiquitin promoter: BPSI .2: -GUS:: NOS3 pBPSMM294 pBPSMM312 Zm.ubiquitin promoter-:BPSI.3: -GUS:: NOS3 pBPSMM295 pBPSMM310 Zm.ubiquitin promoter::BPSI.4: ;GU S:: NOS3 15 1.6 Plant analysis for Identifying IME-Introns These experiments were performed by bombardment of plant tissues or culture cells (Example 4.1), or by Agrobacterium-mediated transformation (Example 4.3). The target tissues for these experiments can be plant tissues (e.g. leaf or root), cultured cells (e.g. maize BMS), or plant tissues (e.g. immature embryos) for Agrobacterium protocols. 20 1.6.1 Transient assays To identify IME-introns, four introns (BPSL1, 2, 3, and 4) were tested using Micropro jectile bombardment. The maize ubiquitin promoter (Zm.ubiquitin) without any intronic sequence was used as basal expression (negative control). Introns of interest were 25 cloned into the 5 UTR region of Zm.ubiquitin promoter. Maize ubiquitin intron was used as a positive control to measure the relative levels of expression enhanced by introns of interest based on GUS expression. Strong enhancement with BPSI.1 and BPSI.2 introns was detected (Table 8). BPSL.3 intron showed medium enhancement levels of GUS expression. No expression was detected with BPSI.4 intron. 30 Table 8. Transient GUS expression testing for intron-mediated enhancement Intron candidates GUS expression' Zm.ublqultln promoter alone (negative control) GU 50e Zm.ublquitln promoter + Zm.ubiqultin intron1(positve control) +++ 100% Zm.ubiquitin promoter+ BPSI.1 (pBPSMM291) +++ 100% Zm.ubiquitin promoter + BPSI.2 (pBPSMM293) ++++ 100% Zm.ubiquitin promoter + BPSI.3 (pBPSMM294) +++ 80% Zm.ublquitin promoter+ BPSI4 (pBPSMM295) - 0% *GUS histochemical assays: a range of GUS activities (- no expression to ++++ high expres sion), **Relative GUS expression compared to the expression controlled by maize ubiquitin promoter fused with Zm.ubiquitin intron. 74 1.6.2 Analysisi of IME-intron candidates in stably transformed maize The binary vectors pBPSMM350, pBPSMM353, pBPSMM312, and pBPSMM310 (Ta ble 7), were transformed into maize using Agrobacterium-mediated transflormation (Ex ample 4.3). The levels and patterns of GUS expression controlled by BPSL1, BPSL,2, 5 BPSL3, or BPSIA intron were compared with those controlled by Zm.ubiquitin intron. BPSI.1, BPSI.2 and BPSI.3 introns enhanced expression in roots, leaves, and kernels throughout thevarious development stages at a similar level to that observed in tran sient assays (Table 9). Expression of Zm.ubiquitin promoter without intron was unde tectable in roots and leaves and was limited in kemels to the endosperm. Expression of 10 Zm.ubiqutin promoter with BPSL4 intron exhibited the same expression patterns as those controlled by Zm.ubiquitin promoter without intron. This result indicates that a transient assay can be used as a model system and is therefore one of the important screening systems to identify introns that function in intron-mediated enhancement (IME) in stable transformed plants. However, the results obtained with the transient 15 assays should be validated by the production of stable transformed transgenic plants. Table 9. GUS expression in transgenic maize plants Developmental Organs Zmubiquitin Zmubiquitin Zmubiquitin stage promoter::Zmu promoter:: promoter:: biquitin intron no intron BPSI.1 (pPSMM350) Five leaf Roots +++ - ++++ Leaves ++++ -+ Flowering Leaves ++ - +++ Late reproductive Kernels ++++ ++** +++ Developmental Organs Zmubiquitin Zmubiquitin Zmubiquitin stage promoter:: promoter: promoter: BPSI.2 BPSI.3 BPSl.4 (pBPSMM353) (pBPSMM312 (pBPSMM310) Five leaf Roots +++ ++ Leaves +++ ++ Flowering Leaves +++ +++ Late reproductive Kemels +++ ++++* *GUS histochemical assays: a range of GUS activities (- no expression to +.+ high expres 20 sion), ** only in endosperm, ND: not determined EXAMPLE 2. IME-introns located in the annotated DNA sequences 2.1 In silico screening system The in silicon intron-screening system for identifying introns that have the functional IME 25 comprises three major components: (1) Generate intron sequence database and screen for intron candidates using the functional IME criteria (indicated in Example 1.3); (2) Define the expression profiles of these candidate genes from which introns were selected; (3) Further examine the selected gene structures by conducting a map ping of EST sequences onto the genomic region where the candidate genes resided. 30 More than 30,000 annotated rice and maize genomic sequences were downloaded from NCBI. Intron, 5 - and 3 -UTR, promoter and terminator sequences were isolated (in silicon) from those annotated genes and their corresponding sequence databases 75 were generated (Table 10, 11). From the generated intron sequence database, more than 111,800 introns (i.e., 106049 rice introns, 4587 maize introns) were screened for potential Intron regulatory enhancement elements based on the functional IME criteria (see 1.3). A total of 108 potential intron candidates have been identified, and the pro 5 tein sequences of the intron candidate genes were retrieved from NCBl. The rice (we do not disclose maize sequences) homolog EST sequences weee identified from the cDNA libraries described In example I using the BLASTx algorithm (this program com pares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against protein sequences) at an E-value of I .0eh against those protein 10 sequences. Using the rice variant expression profiling data (see example 1), the in trons whose genes were homolog to the rice genes with desirable expression profiling, such as constitutive and tissue specific expression pattern, were selected as final in siico identified intron candidates for lab experimental test. 15 The rice UniGenes, which was derived from the EST sequence assembly, were up dated using the combined public rice EST data and the EST data obtained using the databases described in example 1, and the UniGene expression profiling data was generated using the rice variant expression profling data over the 23 different libraries described in example 1. The newly updated rice UniGene expression profiling data 20 were used to help select the final 108-intron candidates. Perl scripts have been written to isolate intron, 5 - and 3 -UTR, terminator, and promoter sequences from the entire NCBI rice and maize annotated gnomic DNA sequences for creating corresponding sequence databases, to screen for functional IME, and to compare the expression pro filing data (see example 5). The introns were retrieved from the CDS (coding se 25 quences) features of the annotated genes. A total of 106,049 rice introns and 4,587 maize introns have been retrieved (Table 10) from more that 30,000 annotated genes as the data summarized in Table II and 12. Table 10. Rice/maize sequence database summary Rice Maize Intron 106049 4587 6UTR 129 236 3'UTR 142 694 Terminator 7 5 Promoter 69 239 30 Table 11. Rice and maize gene summary* Average Rice Maize gene length 2471 3223 intron length 399 279 extron length 309 388 intron/gene 3.9 2.61 extron/gene 4 2.45 GC/intron 39% 40.8% GC/extron 54.8% 55.3% * Intron or extron without gene names were excluded from the calculation. 76 Table 12. Total number of genes in the database Species Gene Name Gene Identifier Rice 30059 30249 Maize 1281 3549 Furthermore, The full length coding sequences of all 108 candidate genes, in which introns were isolated, were downloaded from NCBI and blasted against the Hyseq rice 5 and maize UniGenes to identify Hyseq rice and maize homolog sequences, using BLASTN and 1.0e 20 cutoff E-value. Top hits of rice UniGenes were selected, and the gene expression profiling data was examined. The EST sequences, identified as ho molog to the coding sequences of selected intron candidate genes, were retrieved and mapped along with the intron candidate gene sequences to the rice genomic regions. 10 Based on the UniGene expression profiling data and the candidate gene structures, annotated and confirmed by the EST sequence alignments, nine introns were finally selected from a total of 108 intron candidates and are subject to the real time RT-PCR expression test. Among the nine introns, four showed a constitutive expression pattern, three preferably expressed in the early seed-developed stage, one preferably ex 15 pressed in root, and one was induced In the drought condition (Table 13). Table 13. Intron candidates selected based on the second in silico screening system Intron Rice GI Sequence homology number BPSI.5 (SEQ ID No. 5) 9624451 Sucrose transporter BPSI.6 (SEQ ID No. 6) 7523493 Similar to Arabidopsis thaliana chromosome 11 sequence from clones T22013, F12K2; putative lipase (AC006233) BPSL7 (SEQ ID No. 7) 20161203 putative cinnamyl-alcohol dehydrogenase BPSI8 (SEQ ID No. 8) 18921322 Putative ribonucleoprotein BPSI.9 (SEQ Ib No. 9) 12061241 putative mitochondrial carrier protein BPSI.10 (SEQ ID No. 10) 20160990 Putative protein Idnase BPS.11 (SEQ ID No. 11) 886404 5 LrTR intron (1) MADS3 box protein 2.2 Isolation of the intron candidates Genomic DNA from rice was extracted using the Qiagen DNAeasy Plant Mini Kit 20 (Qiagen). Genonic DNA regions containing introns of interest were isolated using con ventional PCR. Approximately 0.1 pg of digested genoimic DNA was used for the regu lar PCR reaction (see below). The primers were designed based on the rice genomic sequences. Five pL of the diluted digested genomic DNA was used as the DNA tem plate in the PCR reaction. PCR was performed using the TripleMaster PCR System 25 (Eppendorf, Hamburg, Germany) as described by the manufacturer. 77 Table 14. Primers used for amplification of widely expressed intron candidates Primers Sequence BPSI.5-5 (SEQ ID No. 61) 5 -oggggtecgagctctctggtggctgaggtaaIttctgttattacc BPSI.5-3 (SEQ ID No. 62) 5 -cggggatccggaagaaeacctgaaaacaggg BPSL6-5 (SEQ ID No. 63) 5 -cggggkccgagctcgacgattaggtaagtcattattgtctc BPSI.6-3 (SEQ ID No. 64) 5 -cggggatcocactgaaacctcaptgtagg BPSI.7-5 (SEQ ID No. 65) 5 -cggggtaccgagotetcctaaggtaagcactagtg BPSI.7-3 (SEQ ID No. 66) 5 -ggggatccgtaactcaacctgtttttttta BPSI.8-5 (SEQ ID No. 67) 5 -cggg agctccaatggoctaggtaagtatatgcttcc BPSI.8-3 (SEQ ID No. 68) 5 -cggggatcccccatcaagtacctgttttaag BPSI.9-5 (SEQ ID No. 69 ) 5 -cgggiaccgagotogaatacctaggtaagtocatctc BPSI.9-3 (SEQ ID No. 70) 5 -cggggatcccacacaagogacotggaaaaptaagc BPSI.10-5 (SEQ ID No.71) 5 -cgggtaccgagctccatctttttaggtaagWtttttgcg BPSI.10-3 (SEQ ID No. 72) 5 -cggggatccggtaaagasctgtttaatac BPSI.11-5 (SEQ ID No. 73) 5 -cggggkccgagctcgaacaggaaggtaagttctggctttcttgc BPSI.11-3 (SEQ ID No. 74) 5 -ggggatcctcagatcgacctggacacaaacgo Amplification was carried out in the PCR reaction (5 RL 1OX Advantage PCR Mix [Ep pendorf], 5 iL genomic DNA [corresponds to approximately 80 ng], 2.5 mM of each 5 dATP, dCTP, dGTP and dTTP [Invitrogen: dNTP mix], I ±L of 20 iM 5 -intron specific primer 20pM, 1 itL of 20 tM 3 intron specific primer, 1L TripleMaster DNA Poly merase mix [Eppendorf], in a final volume of 50 4L) under the optimized PCR program (1 cycle with 15 sec at 94'C and 1 min at 80"C 35cycles with 15 sec at 94*C, I min at 58*C and I min at 72*C) provided by Thermocycler (T3 Thermocyoler Biometra). 10 A QlAspin column was used to purify the PCR products as directed by the manufac turer (Qiagen, Valencia, CA), and the amplified introns were used directly for cloning into expression vectors, as described below. 2.3 Vector Construction 15 The base expression vector for these experiments was pBPSMM305, which comprises the maize lactate dehydrogenase (LDH) promoter without intron driving expression of the GUSint gene followed by the NOS terminator. The LDH promoter has been demon strated to direct undetectable levels of GUS expression by colorimetric staining in the absence of an intron capable of providing IME. 20 Intron PCR products were digested with Sacl & BamHl and cloned into pBPSMM305 linearized with Sacl & BamHl, generating the following LDH:intron:GUS expression vectors. 25 78 Table 15. GUS chimeric constructs containing introns in the 5 UTR pUC-based expression vector Composition of the expression cassette (promoter::intron: :reporter gene::terminator) pBPSJB041 (pBPSL017) ZmLDH promoter:BPSI.5::GUS::NOS3 pBPSJB042 (pBPSLI018) ZmLDH promoter::BPSL6::GUS::NOS3 pBPSJB043 (pBPSLI019) ZmLDH promoter.:BPSL7.::GUS::NOS3 pBPSJB044 (pBPSL020) ZmLDH promoter:BPSI.8::GUS::NOS3 pBPSJB045 (pBPSL021) ZmLDH promoter::BPSI.9::GUS::NOS3 pBPSJB046 (pBPSLI022) ZmLDH promoter::BPSIL10::GUS::NOS3 pBPSJB050 (pGPSLIO23) ZmLDH promoter::BPSL111::GUS::NOS3 Binary vector pBPSLI017 comprises the expression cassette containing the BPSI.5 5 intron and was generated by ligating in the Pmel-Pacl fragment from pBPSJB041 into pBPSLM139 linearized with Pmel and Pacl. Binary vector pBPSLI018 comprises the expression cassette containing the BPSI.6 intron and was generated by ligating in the Pmel-Pacl fragment from pBPSJB042 into 10 pBPSLM139 linearized with Pmel and Pacl. Binary vector pBPSLI019 comprises the expression cassette containing the BPSI.7 Intron and was generated by ligating in the Pmel-Pacl fragment from pBPSJB043 into pBPSLM139 linearized with Pmel and Pacl. 15 Binary vector pBPSL1020 comprises the expression cassette containing the BPSI.8 intron and was generated by ligating in the Pmel-Pacl fragment from pBPSJB044 into pBPSLM1 39 linearized with Pmel and Pacl. 20 Binary vector pBPSL021 comprises the expression cassette containing the BPSI.9 intron and was generated by ligating in the Pmel-Pacl fragment from pBPSJB045 into pBPSLM139 linearized with Pmel and Pacl. Binary vector pBPSLI022 comprises the expression cassette containing the BPSI.1O 25 intron and was generated by ligating in the Pmel-Pacl fragment from pBPSJB046 into pBPSLM 139 linearized with Pmel and Pacl. Binary vector pBPSLI023 comprises the expression cassette containing the BPSI 11 intron and was generated by ligating in the PmeI-Pacl fragment from pBPSJB050 into 30 pBPSLM139 linearized with Pmel and Pacl. 2.4 Transient assays for Identifying the Intron functioning IME These experiments were performed by bombardment of plant tissues or culture cells (Example 4.1), or by Agrobacterium-mediated transformation (Example 4.3), The target tissues for these experiments can be plant tissues (e.g. leaf or root), cultured cells (e.g. 35 maize BMS), or plant tissues (e.g. immature embryos) for Agrobacterium protocols. 79 Characterization of these introns for their ability to direct IME in conjunction with the LDH promoter was undertaken via transient expression by bombardment of expression vectors into maize leaf tissue and liquid-cultured BMS cells, respectively. 5 The maize lactate dehydrogenase promoter (ZmLDH) without any intronic sequence was used as basal expression (negative control). Introns of interest were cloned into the 5 UTR region of ZmLDH promoter. Maize ubiquitin intron was used as a positive control to measure the relative levels of expression enhanced by introns of interest based on GUS expression. 10 Due to the very low background (no detectable GUS expression) of the ZmLDH pro moter in the absence of Intron, the presence of any GUS staining! indicates that a par ticular intron is capable of providing IME. Of the introns tested, BPSI.10 and BPSI.11 introns consistently yielded the highest GUS expression, at a level comparable to the LDH::Zm.ubiquitin intron construct. In addition to these introns, BPSL5, BPSI.6, and 15 BPSI.7 introns consistently resulted in an intermediate level of GUS expression in be tween LDH alone and LDH::Zm.ubiquitin intron. Comparable results were obtained in maize leaves and BMS cells, indicating that the tested introns confer IME in green and non-green tissues (Table 16). 20 Table 16. Transient GUS expression testing for intron-mediated enhancement Intron candidates GUS expression* leaves BMS No intron (Zm.LDH promoter alone) - Zm.LDH + Zm.ubiquitin intron (positive control) ++++ ++ Zm.LDH promoter + BPS.5 ++ ++ Zm.LDH promoter + BPS.6 +++++ Zm.LDH promoter + BPSL7 1.1 +++ Zm.LDH promoter + BPSI.8 + Zm.LDH promoter + BPSI.9 - Zm.LDH promoter + BPSI.10 ++ ++ Zm.LDH promoter + B PSI.11 ++++ ND *GUS histochemical assays: a range of GUS activities (- no expression to ++++ high expression), ND: not determined. EXAMPLE 3. Identification of IME-introns located in the 5' untranslated region 25 3.1 In silicon screening system The in silicon intron screening system for identifying introns that have the functional IME located in the '5 UTR comprises three major components: (1) Genome mapping of the entire rice CDS, released from Institute of Genome Research on October 2, 2003 and the EST sequence collections; (2) identification and selection of the introns located in 30 the 5 UTR using both the functional IME criteria and the rice cDNA clone distribution profiles; (3) validation of the selected 5 UTR introns by examining the sequence align ments among the genomic DNA, CDS and ESTs, the gene model, sequence reading frame and intron splicing sites 80 A total of 56,056 annotated rice CDS were mapped onto the Japonica rice genome in which both rice CDS and genomic DNA sequences were obtained from The Institute of Genome Research. Additional 422,882 rice EST sequences of public and in-house sources were also mapped onto the rice genome. A splicing alignment software, 5 GeneSeqer (version September 2, 2003 from Iowa State University Research founda tion), was used to conduct the entire genome mapping. Since both EST and CDS were mapped onto their corresponding genomic regions, the sequence alignment coordina tors [coordinators are the start and/or end positions of the the genomic sequences where CDSIEST sequences aligned to] derived from the CDS mapping and the EST 10 mapping on the same genomic region provide opportunity to identify the alignment ex tension of the EST sequences along the genomic DNA beyond the start codon of the CDS. Such sequence alignment extension from the EST sequences beyond CDS indi cates the identification of the 5 UTRs, which have not been contained in the CDS, but in the EST sequences. The system selects these EST sequences, which extend the 15 sequence alignment beyong the CDS along the gnome for up to 5k base long for 5 URT intron screening. For any predicted exons, the last exon in the predicted 5 UTR region must aligned at the same position of the 1' exon of the CDS. The gnome map ping results have identified 461 genes that have their 5 UTR containing at least one intron. 20 Further stringent screen criteria that required at least 3 EST sequences confirming the same predicted 5 UTR introns were used to select the gene candidates, leading to identify 87 gene candidates. Those identified EST sequences, which were considered as the same transcript as the rice CDS, were used to retrieve the rice cDNA clone dis 25 tribution data or the microarray expression data in which either the clones of those identified EST sequences have been spotted on the rice microarray chip or homolog to those identified EST sequences were identified on the chip. For given the rice cDNA clone distribution profile, a gene, which has a cluster/variant size of more than 100 clones distributed over 23 cDNA libraries, was considered highly expressed. For given 30 the microarray expression, a gene, which has hybridization signal intensity exceeding the top 25% percentile within the same sample, was also considered highly ex pressed. In addition to the gene expression criteria used for gene candidate selection, the IME criteria (indicated in Example 1.3) were applied. 35 Furthermore, a validation of the selected candidate genes was conducted by examining the coincidence of the sequence alignments between EST, CDS sequences and ge nomic DNA sequence. Clearly the EST sequences needed to support the gene model predicted from the CDS. Any conflict of the sequence alignments between EST and 40 CDS would result in the deselecting the candidate genes. Using those criteria, a final list of 11 introns was selected (Table 17). 81 Table 17, Intron candidates selected based on the third in slico screening system Intron Rice GI number Sequence homology BPSI.12 (SEQ ID No. 12) 29620794 Putative adenosylmethionine decarboxy lase BPSI.13 (SEQ ID No. 13 ) 33666702 Aspartic proteinase BPSL14 (SEQ ID No. 14 ) 29678665 Lecl4b protein BPSI.15 (SEQ ID No. 15) 35009827 Putative mannose-binding rice lectin BPSI.16 (SEQ ID No. 16) 41883853 Putative reticulon BPSI.17 (SEQ ID No. 17) 2799981 Glycolate oxidase BPSI.18 (SEQ ID No. 18) 34763855 Similar to AT4g33690/T16L1_180 BPSI.19 (SEQ ID No. 19) 32533738 N/A BPS.20 (SEQ ID No. 20) 33657147 Hypothetical protein BPSI.21 (SEQ ID No. 21) 33800379 Putative membrane t ansporter BPSI.22 (SEQ I) No. 22) 2309889 Putative ACT domain repeat protein 3.2 Isolation of Introns Genomic DNA containing introns of interest is isolated using conventional PCR amplifi 5 cation with sequence specific primers (see 1.4) followed by cloning into a PCR cloning vector in the art. 3.3 Vector construction Introns are PCR amplified from rice genomic DNA using primers that engineer a Sacl 10 site on the 5 end of the intron and aBamHI site on the 3 end of the sequence. The PCR products are digested with Seac and BamHi and ligated into pBPSMM305 lin earized with Sacl and BamHl to generate pUC-based expression vectors comprising the Zm.LDH promoter:intron candidate:: GUSint: :NOS terminator. 15 Binary vectors for stable maize transformation are constructed by digesting the pUC expression vectors with Pmel and Pacl and ligating into pBPSLM139 digested with Pmel and Pacl. 3.4 Transient assays for identifying IME-Introns These experiments are performed by bombardment of plant tissues or culture cells 20 (Example 4.1), or by Agrobacterium-mediated transformation (Example 4.3). The target tissues for these experiments can be plant tissues (e.g. leaf or root), cultured cells (e.g. maize BMS), or plant tissues (e.g. immature embryos) for Agrobacterium protocols. EXAMPLE 4. Assays for identifying IME-Introns 25 These experiments are performed by bombardment of plant tissues or culture cells (Example 4.1), by PEG-mediated (or similar methodology) introduction of DNA to plant protoplasts (Example 4.2), or by Agrobacterium-mediated transformation (Example 4.3). The target tissue for these experiments can be plant tissues (e.g. leaf tissue), cul tured plant cells (e.g. maize Black Mexican Sweetcom (BMS), or plant embryos for 30 Agrobacterium protocols. 82 4.1 Transient assay using microprojectile bombardment The plasmid constructs are isolated using Qiagen plasmid kit (cat# 12143). DNA is precipitated onto 0.6 pM gold particles (Blo-Rad cat# 165-2262) according to the proto col described by Sanford et at (1993) and accelerated onto target tissues (e.g. two 5 week old maize leaves, BMS cultured cells, etc.) using a PDS-1000/He system device (Bio-Rad). All DNA precipitation and bombardment steps are performed under sterile conditions at room temperature. Black Mexican Sweet com (BMS) suspension cultured cells are propagated in BMS cell 10 culture liquid medium [Murashige and Skoog (MS) salts (4.3 g/L), 3% (w/v) sucrose, myo-inositol (100 mg/L), 3 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), casein hydro lysate (1 g/L), thiamine (1Omg/L) and L-proline (1.15 g/L), pH 5.8]. Every week 10 mL of a culture of stationary cells are transferred to 40 mL of fresh medium and cultured on a rotary shaker operated at 110 rpm at 27C in a 250 mL flask. 15 60 mg of gold particles in a siliconized Eppendorf tube are resuspended in 100% etha nol followed by centrifugation in a Mini centrifuge C1200 (National Labnet Co. Wood bridge, NJ) for 30 seconds. The pellet is rinsed once in 100% ethanol and twice in ster ile water with centrifugation after each wash. The pellet is finally resuspended in 1 mL 20 sterile 50% glycerol. The gold suspension is then divided Into 50 pL aliquots and stored at 4 0 C. The following reagents are added to one aliquot: 5 pL of 1 pg/pL total DNA, 50 pL 2.5M CaC 2 , 20pL 0.1M spermidine, free base. The DNA solution is vortexed for I minute and placed at -804C for 3 min followed by centrifugation for 10 seconds in a Mini centrifuge C1200. The supernatant is removed. The pellet is carefully resus 25 pended in I mL 100% ethanol by flicking the tube followed by centrifugation for 10 sec onds. The supernatant is removed and the pellet is carefully resuspended in 50 pL of 100% ethanol and placed at -80 0 C until used (30 min to 4 hr prior to bombardment). If gold aggregates are visible In the solution the tubes are sonicated for one second in a waterbath sonicator just prior to use. 30 For bombardment, two-week-old maize leaves are cut into pieces approximately 1 cm in length and Placed ad-axial side up on osmotic induction medium M-N6-702 [N6 salts (3.96 g/L), 3% (w/v) sucrose, 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (100 mg/L), and L-proline (2.9 gIL), MS vitamin stock solution (1 mL/L), 0.2 35 M mannitol, 0.2 M sorbitol, pH 5.8]. The pieces are incubated for 1-2 hours. In the case of BMS cultured cells, one-week-old suspension cells are pelleted at 1000 g in a Beckman/Coulter Avanti J25 centrifuge and the supernatant is discarded. Cells are placed onto round ash-free No 42 Whatman filters as a 1/16 inch thick layer using a 40 spatula. The filter papers holding the plant materials are placed on osmotic induction media at 2700 in darkness for 1-2 hours prior to bombardment. Just before bombard ment the filters are removed from the medium and placed onto on a stack of sterile filter paper to allow the call surface to partially dry. 45 Each plate is shot with 6 pL of gold-DNA solution twice, at 1,800 psi for the leaf materi als and at 1,100 psi for the BMS cultured cells. To keep the position of plant materials, a sterilized wire mesh screen is laid on top of the sample. Following bombardment, the filters holding the samples are transferred onto M-N6-702 medium lacking mannitol and 83 sorbitol and incubated for 2 days in darkness at 27 0 C prior to transient assays. Tran sient expression levels of the reporter genes are determined by GUS staining, quantifi cation of luminescence or RT-PCR using the protocols in the art. GUS staining is done by incubating the plant materials in GUS solution 1100 mM NaHPO4, 10 mM EDTA, 5 0.05% Triton XIOO, 0.025% X-Gluc solution (5-bromo-4-chloro-3-indolyl-beta-D glucuronic acid dissolved in DMSO), 10% methanol, pH 7.0] at 37 0 C for 16-24 hours. Plant tissues are vacuum-infiltrated 2 times for 15 minutes to aid even staining. Transient expression levels of the reporter genes are determined by staining, en 10 zyme assays or RT-PCR using the protocols in the art. 4.2 Transient assay using protoplasts Isolation of protoplasts is conducted by following the protocol developed by Sheen (1990). Maize seedlings are kept in the dark at 25 0 C for 10 days and illuminated for 20 hours before protoplast preparation. The middle part of the leaves are cut to 0.5 mm 15 strips (about 6 cm in length) and incubated in an enzyme solution containing 1% (w/v) cellulose RS, 0.1% (wlv) macerozyme RIO (both from Yakult Honsha, Nishinomiya, Japan), 0.6 M mannitol, 10 mM Mes (pH 5.7), 1 mM CaCI 2 , 1 MM MgCl 2 , 10 mM p mercaptoethanol, and 0.1% BSA (w/v) for 3 hr at 23"C followed by gentle shaking at 80 rpm for 10 min to release protoplasts. Protoplasts are collected by centrifugation at 100 20 x g for 2 min, washed once in cold 0.6 M mannitol solution, centrifuged, and resus pended in cold 0.6 M mannitol (2 x 10 6 mL). A total of 50 pg plasmid DNA in a total volume of 100 pL sterile water is added into 0.5 mL of a suspension of maize protoplasts (1 x 10 e cells/mL) and mix gently. 0.5 mL PEG solution (40 % PEG 4,000, 100 mM CaNO 3 , 0.5 mannitol) Is added and pre-warmed at 25 70c with gentle shaking followed by addition of 4.5 mL MM solution (0.6 M mannitol, 15 mM MgC 2 , and 0.1 % MES). This mixture is incubated for 15 minutes at room tem perature. The protoplasts are washed twice by pelleting at 600 rpm for 5 min and re suspending in 1.0 mL of MMB solution [0.6 M mannitol, 4 mM Mes (pH 5.7), and brome mosaic virus (BMV) salts (optional)] and incubated in the dark at 25"C for 48 hr. After 30 the final wash step, collect the protoplasts in 3 rnL MMB medium, and incubate in the dark at 25*C for 48 hr. Transient expression levels of the reporter gene are determined quantification of expression of reporter genes or RT-PCR using the protocols in the art in order to determine potentially intron candidates that function in intron-mediated en hancement. 35 4.3 Agrobacterium-mediated transformation in dicotyledonous and monocotyle donous plants 4.3.1 Transformation and regeneration of transgenic Arabidopsis thaliana (Co lumbia) plants To generate transgenic Arabidopsis plants, Agrobacterium tumefaciens (strain C58C1 40 pGV2260) is transformed with the various vector constructs described above. The Agrobacterial strains are subsequently used to generate transgenic plants. To this end, a single transformed Agrobacterium colony is incubated overnight at 28'C in a 4 mL culture (medium: YEB medium with 50 1 ig/mL kanamycin and 25 sg/mL rifampicin). This culture is subsequently used to inoculate a 400 mL culture in the same medium, 84 and this is incubated overnight (28 0 C, 220 rpm) and spun down (GSA rotor, 8,000 rpm, 20 min). The pellet is resuspended in infiltration medium (112 MS medium; 0.5 g/L MES, pH 5.8; 50 g/L sucrose). The suspension is introduced into a plant box (Duchefa), and 100 ml of SILWET L-77 (heptamethyltrisiloxan modified with polyal 5 kylene oxide; Osi Specialties Inc., Cat. P030196) is added to a final concentration of 0.02%. In a desiccator, the plant box with 8 to 12 plants is exposed to a vacuum for 10 to 15 minutes, followed by spontaneous aeration. This is repeated twice or 3 times. Thereupon, all plants are planted into flowerpots with moist soil and grown under long day conditions (daytime temperature 22 to 24 0 C, nighttime temperature 19"C; relative 10 atmospheric humidity 65%). The seeds are harvested after 6 weeks. As an alternative, transgenic Arabidopsis plants can be obtained by root transforma tion. White root shoots of plants with a maximum age of 8 weeks are used. To this end, plants that are kept under sterile conditions in I MS medium (1% sucrose; 100mg/L 15 inositol; 1.0 mg/L thiamine; 0.5 mg/L pyridoxine; 0.5 mg/L nicotinic acid; 0.5 g MES, pH 5.7; 0.8 % agar) are used. Roots are grown on callus-inducing medium for 3 days (1x Gamborg s B5 medium; 2% glucose; 0.5 g/L mercaptoethanol; 0.8% agar; 0.5 mg/L 2,4-D (2,4-dichiorophenoxyacetic acid); 0.05 mg/L kinetin). Root sections 0.5 cm in length are transferred into 10 to 20 mL of liquid callus-inducing medium (composition 20 as described above, but without agar supplementation), inoculated with 1 mL of the above-described overnight Agrobacterium culture (grown at 28*C, 200 rpm in LB) and shaken for 2 minutes. After excess medium has been allowed to run off, the root ex plants are transferred to callus-inducing medium with agar, subsequently to callus inducing liquid medium without agar (with 500 mg/L betabactyl, SmithKline Beecham 25 Pharma GmbH, Munich), incubated with shaking and finally transferred to shoot inducing medium (5 mg/L 2-isopentenyladenine phosphate; 0.15 mg/L indole-3-acetic acid; 50 mg/L kanamycin; 500 mg/L betabactyl). After 5 weeks, and after 1 or 2 me dium changes, the small green shoots are transferred to germination medium (1 MS medium; 1% sucrose; 100 mg/L inositol; 1.0 mg/L thiamine; 0.5 mg/L pyridoxine; 30 0.5 mg/L nicotinic acid; 0.5 g MES, pH 5.7; 0.8% agar) and regenerated into plants. 4.3.2 Transformation and regeneration of crop plants The Agrobacterlum-mediated plant transformation using standard transformation and regeneration techniques may also be carried out for the purposes of transforming crop 35 plants (Geivin& Schilperoort (1995) Plant Molecular Biology Manual, 2 "d Edition, Dordrecht: Kluwer Academic Publ. ISBN 0-7923-2731-4; Glick & Thompson (1993) Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, ISBN 0-8493-5164-2). For example, oilseed rape can be transformed by cotyledon or hypo cotyl transformation (Moloney (1989) Plant Cell Reports 8: 238-242). The use of antibi 40 otics for the selection of agrobacterla and plants depends on the binary vector and the Agrobacterium strain used for the transformation. The selection of oilseed rape is gen erally carried out using kanamycin as selectable plant marker. The Agrobacterium mediated gene transfer in linseed (Unum usitatissimum) can be carried out using for example a technique described by Mlynarova (1994) Plant Cell Report 13:282-285. 45 The transformation of soybean can be carried out using, for example, a technique de scribed in EP Al 0 424 047 or in EP Al 0 397 687, US 5,376,543, US 5,169,770. The transformation of maize or other monocotyledonous plants can be carried out using, for example, a technique described in US 5,591,616. The transformation of plants using 85 particle bombardment, polyethylene glycol-mediated DNA uptake or via the silicon car bonate fiber technique is described, for example, by Freeling &i Walbot (1993) 'The maize handbook' ISBN 3-540-97826-7, Springer Verlag New York). 5 EXAMPLE 5: Computer algorithm for retrieving sequence information from NCBI genebank file. The target feature keys are intron, terminator, promoter, UTR. The following script (writ ten in computer language Pearl) is giving an example for a computer algorithm of the 10 invention suitable to identify suitable intron sequences based of database information (see also Fig. 5a-f): #1/usr/local/bin/perl -w # intron.pl 15 open(IN,$ARGV[0) or die "can't find output"; while (defined(my $file=<IT> )) { 20 #start of a single annotation if ($file=~/LOCUS.*?\s+(\d+)\sbp(.*)/) { my $length=$1; my $mol=l; 25 $mol=O if $2 =~ /circular/; my @cdslist=(); my @start=(); my $order=0; # order=1: complementary coding. my @title=(); 30 my @titleO=(); my @intron=(); my $id="", my Terminatorr=(; my @promoter=(); 35 my @utr5=() my @utr3=(); my @origin=(); my $tab"" my $organism="; 40 while (defined(my $line=<IN> )) { $line=$tab.$line; if (Sline - /^VERSION.*?\s+(GI:\d+)/) { $id=$l; 45 }elsif ($line =- /^\s{2}ORGANISM\s+(.*)/){ if($1=-/Oryza sativa/i){ 86 $organism="rice"; )elsif($1=-/Zea mays/i) { $orqanism="maize"; }elsif($1=-/Glycine max/i){ 5 $organism-"soybean"; }else { $1=-/(\w+)/; $organism=$ 1; } 10 }elsif($line =~ /^\s{5}(CDS\s*)/){ #extract ods my Testt=$; my $gene="N/A"; my $start=1; my $product="N/A"; 15 my $gi-$id; my @cds=(); my @temp=(); if ($test =- /complement/) { $order=1 20 }else { $order = 0; } while ( my $in=<IN>) { if ($in =/\s\/(.*)/) { 25 $test-$test; if ($1=-/gene="(.*)"/) { $gene=$; $product=$1; 30 }else { last; } } else { $test=$test.$in; 35 } } #close while loop; $test =-s/\w+\d+\.\d;\d+\.\.\d+//g; $test =~ s/\D/ /g; 40 $test s/\s+/ /g; $test s/^\s+//; my @sort; if ($mol==0) { @sort=split(/ /,$test); 45 }else { @sort-sort {$a <=> $b} split(/ /,,$test); # tag complement cds 87 if ($order==1) { @cds = ("complement",@sort); } elsif ($order==O) { 5 @cds = @sort; I #close if loop; #retreave notation if intron exist; if (scalar(@cds) >= 4) { 10 while (my $in=<IN>) { $start=1; if ($in =- /codon_start=(\d+)/) { $start = $1; 15 }elsif ($in =- /\gene."(.*)"/){ $gene=$1; }elsif ($in =- /\/product=(.*)/){ $product=$1; $product=- tr/'"'//d; 20 }elsif ($in =- /db_xref="(GI:.*?)"/) { $gi =$1; last 25 } elsif ($in=- /\/(pseudo)/) { $product="pseudo"; last; } #close if loop } #close while loop; 30 push @start, $start; push @odslist, \@cds; # retreave 5'utr if start codon > 1; my @tem-(); for (my $i=l;$i<=($#cds-1)/2;$i++) { 35 my $title1=">$organismj$gilXntron_$i " my $title2=" $gene|$start|".($cds[2*$i 1+$order]+1)."..".(Scds[2*$i+$order]-1)."[Sproduct\n"; my @title=($title1,$title2); push @tem, \@title; 40 1 #close for loop push @title, \@tem; my $titleo=">$organism|$giF5UTR_0 Sgene|$start|".($cds{$order]-1)."..".($cds[$order]+$start 2). "j $product\n"; 45 push @titleO, titlee; } #close if @cds>4 loop 88 } elsif ($line =- /^\s{5}terminator/) { ($tab,my $note,my @term)=&getTerminator($line); 5 push terminator, $note; push @terminator, \@term; } elsif ($line =- /^\s{5}promoter/) { 10 ($tab,my $note,my @prom)-agetTerminator($line); push @promoter, $note; push @promoter, \@prom; 15 } elsif ($line -- /^\s15}5\DUTR/) { ($tab,my $note,my @temp)=&getTerminator($line) push @utr5,$note; 20 push eutr5,\@temp; } elsif ($line =- /^\s{5}3\DUTR/) { ($tabmy $note,my @temp)=&getTerminator( $line); 25 push @utr3,$note; push @utr3,\@tempy #get sequence @origin 30 } if ($line =~ /^(ORIGIN)/) { $line="' while (my $code-<IN>) { 35 if ($code /\/\//) { last; }else{ $1ine=$line.$code; } #close if loop 40 } #close while loop # $line -- s/\/\I/ /g; # print $line,"\n"; $line =- tr/0-9//d; $line tr/ //d; 45 $line tr/\n//d; @origin = split(//,$line); 89 for (my $i=o; $i<=$#cdslist;$i++) { if ($start[$i>2) { 5 my @first=(); my $first; if (${$cdslistt$i]}[OJ eq "complement") { my @utr=@origin[$cdslist[$i][1)-I ($cdslist[$i][1]+$start[$i]-2)]; 10 print @utr,"\n"; $first=scomplement ( @utr) ; } else { Ofirst=@origin[$cdslist[$i][Oj-l ($cdslistt$i][0]+$startt$il-2)]; 15 $first=join('"',first); I #close if loop for complement print $titleO{$i1,$first,"\n\n"; } #close if loop for $start>2; 20 if (${$cdslist[$i]}[0] eq "complement") { shift @{$cdslist[$i]}I for (my $j-l; $j<=($#{$cdslistt$i]}-l)/2;$j++) { my @int=6origin[$cdslist[$i][2*$j-1] 25 $cdslist[$i][2*$j]-2]; my $int1=&complement(@int); print $title[$i][$j-l][l,scalar(@int),$title[$i][$j 1]{1], $intl,"\n\n" if $#int<5000; } #close 2nd for loop for complement 30 } else { for (my $j=1; $j<=($#{$cdslist[$i]}-l)/2;$j++) { my @intm@origin[$cdslist[$iJ[2*$j-l) .. Scdslist[$i][2*$j] 2]; 35 if ($mol=O && $cdslist[$i][2*$j-1] > $cdslist[$i][2*$jI) { @int=(@origin[$cdslist[$i][2*$j-1] . $#origin], @origin[O .. $cdslist[$i][2*$jj-21); } 40 my $intljoin('',@int); print $title[$i][$j-l][o],scalar(@int),$title{$i][$j 1][1], $intl,"\n\n" if $#int < 5000; }#close 2nd for loop I #close else loop 45 } #close 1st for loop my $titlel=">$organism|$idlterminator" &getSequence(\@terminator,\@origin,$titlel); 90 $titlel=">$organismj$idpromoter" &getSequence(\@promoter,\@origin,$titlel); 5 $title1=">$organism|$id|5utr" &getSequence(\@utr5,\@origin,$titlel); Stitlel=">$organism|$idj3utr"; &getSequence(\@utr3,\@origin,Stitlel); 10 last; } else { $tab=""; } #close if $line loop 15 } #close while Sline loop next; } #close if $file loop 20 1 #close while $file loop close IN; #retreave complement sequnce sub complement{ 25 my @code=@ my @complemnt=(); for (my $i=O;$i<=$#code;$i++) { if ($code[$#code-$ij eq 1 t 1 ) { $complement[$i= "a"; 30 } elsif ($code[$#code-$i] eq "a") { $complement[$i]= "t"; } elsif ($code[$#code-$i) eq "c") { $complement[$i]= "g"; } elsif ($code[$#code-$i) eq "g") { 35 $complement[$i]= "c"; 3 else 4 Scomplement[$i]=$code[$#code-$iJ; }#close if loop } #close for loop 40 my $comp=join('',@complement); @complement=(); return Compp; } #close sub 45 #get sequence reference for feature keys sub getTerminator { my $line=$_[0]; my $order=0; 91 if ($line-/complement/) { $order=1; } else { } #close if loop 5 $line s/\d'UTR/; $line s/\D/ /g; $line s/\s+/ /g; $line s/^\s//; my @term=split(' ',$line); 10 §term=("c",@term) if $order=-1; my $in; read(IN,$in,6); my $note =" \n" 15 if ($ini-/\w/) { $note=<IN>; $note=-s/\s+\/// $note=-s/note=//; $note=- tr/""//d; 20 y #close if loop return ($in,$noter@term); } #close sub 25 #retreave sequence information for feature keys sub getsequence { my @array=@{$_[O]}; my @code=@{$_{1]); my $id=$_[2]; 30 for (my $i O; $i<($#array+1)/2;$i++) { my $note=$array[2*$i]; my @term=@{$array[2*$i+1} if ($term[OJ eq "c") { 35 shift @term; for (my $j=0; $j<=($#term-1)/2;$j++) { my @comp=@code[($term[2*$j]-1) .. ($term[2*$j+1]-1)]; my $int1=&complement(@comp); my $title=$id."_".($i+1)." ".scalar(@comp)." 40 $term[2*$j]..$term[2*$j+1]|$note"; print $title, $intl,"\n\n"; I #close 2nd for loop } else { for (my $j=0; $j<($#term+1)/2;$j++) { 45 my Oint=&code[(Sterm[2*$jJ-1) .. ($term[2*$j+1]-1)j; my $int1=join('',@int)y my $title=Sid."_".($i+1)." ".scalar(@int)." $term[2*$jj..Sterm[2*$j+1]|$note"; 92 print $title, $int1,"\n\n 1 '; } #close 2nd for loop } #close if loop I #close 1st for loop 5 } #close sub EXAMPLE 6 Expression of tissue-specific promoters in combination with IME introns 10 BPSI.1 and BPSI.5 have been fused with various monocot promoters and demon strated that most of these promoters without IME-intron did not show GUS expression, but IME-introns have enhanced expression. 6.1 Os.CP12 promoter::BPSI.1 intron::GUS::NOS terminator (pBPSMM355) 15 pBPSMM355 shows strong leaf-specific expression. This expression was detected in all tested developmental stages. No expression was detected in any other tissue tested. 6.2 Zm.HRGP promoter:: BPSI.1 intron::GUS: :NOS terminator (pBPSMM370) 20 pBPSMM370 is strongly expressed in roots. Significant expression was also detected in silk and in the outermost layers of the kernel that include the aleuron layer and seed coat. This expression was strongest around the base of the kernel. Staining in silk was strongest in the region dose to the attachment point with the kernel and was detected at very early developmental stages. 25 6.3 Os.CCoAMT1 promoter::BPSi.1 intron::GUS::NOS terminator (pBPSMM358) Os.Caffeoyl-CoA-O-methyltransferase (CCoAMT1) promoter in combination with BPSI.l (pBPSMM358) showed embryo-specific expression in T1 and T2 kernels. The expression level was low but very specific. No expression was detected in any other 30 tissue tested. 6.4 Zm.Globulin1 promoter:: BPSL1 intron::GUS::NOS terminator (EXS1025) EXS1025 is strongly expressed in the embryo. This expression starts between 5 days after pollination (DAP) and 10DAP. Expression is strongest in the scutellum and 35 weaker in the embryo axis (plumule with leaves and intemodes, primary root). Significant expression was also detected in the outermost layers of the kernel that in clude the aleuron layer. Expression is strongest at stages 15DAP to 25DAP and weaker at 30DAP. Weak expression was sometimes detected in the endosperm. No expression could be detected in any other organ including pollen. 40 6.5 Os.V-ATPase promoter::BPSI.1 Intron: :GUS::NOS terminator (pBPSMM369) pBPSMM369 is strongly expressed in roots. This expression was detected in all tested stages. Significant expression was also detected in all parts of the kernels and in pol len. Weak expression was detected in the leaves at early developmental stages and at 45 flowering. This expression was variable in strength and was in several plants at the detection limit. In general, expression was higher in homozygous T1 plants than in the heterozygous TO. 93 6.6 Zm.LDH promoter: :BPSI.1 intron::GUS::NOS terminator (pBPSMM357) pBPSMM357 shows weak activity in kernels. Expression in kernels was mainly located in and around the embryo. Very weak expression was also detected in roots. 5 6.7 Os.CB,7SI promoter::BPSI.1 intron:;GUS::NOS terminator (pBPSMM366) OsC-8,7-sterol-isomerase promoter containing BPSI.1 (pBPSMM366) shows weak activity in roots and good expression in kernels. 6.8 Os.Lea promoter::BPSI.1 intron::GUS::NOS terminator (pBPSMM371) 10 Os.Lea promoter in combination with BPSI.1 (pBPSMM371) showed strong embryo specific expression in kernels. Some expression could be detected in root tips but no expression was detected in any other tissue tested. 6.9 Zm.LDH promoter::BPSI.5 intron::GUS::NOS terminator (pBPSLM229) 15 pBPSLM229 shows weak expression in endosperm and aleuron layer, mainly at the top side of the kernel. No expression was detected in any other tissue tested. Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components 20 or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. 25 94

Claims (23)

1. A recombinant DNA expression construct comprising a) at least one promoter sequence functioning in plants or plant cells, and b) an intron consisting of the sequence described by SEQ ID NO: 2, or a functional equivalent thereof, and c) at least one nucleic acid sequence, wherein at least one of said promoter sequence and said intron sequences are functionally linked to at least one of said nucleic acid sequence and wherein said intron is heterologous to said nucleic acid sequence and/or to said promoter sequence.

2. The recombinant DNA expression construct of claim 1, wherein said functional equivalent comprises the functional elements of an intron and is characterized by a sequence 1. having at least 50 consecutive base pairs of an intron sequence described by SEQ ID NO: 2 or 2. having an identity of at least 80% over a sequence of at least 95 consecutive nucleic acid base pairs to a sequence described by SEQ ID NO: 2 or

3. hybridizing under high stringent conditions with a nucleic acid fragment of at least 50 consecutive base pairs of a nucleic acid molecule described by SEQ ID NO: 2. 3. The recombinant DNA expression construct of claim 1 or 2, further comprising one or more additional regulatory sequences functionally linked to said promoter.

4. The recombinant DNA expression construct of claim 3, wherein the regulatory sequence is selected from the group consisting of heat shock-, anaerobic responsive-, pathogen responsive-, drought responsive-, low temperature responsive-, ABA responsive-elements, 5'-untranslated gene region, 3'-untranslated gene region, transcription terminators, polyadenylation signals, and enhancers.

5. The recombinant DNA expression construct of any one of claims 1 to 4, wherein said nucleic acid encodes for i) a protein or ii) a sense, antisense, or double-stranded RNA sequence. 95

6. The recombinant DNA expression construct of any one of claims 1 to 5, wherein said nucleic acid sequence encodes a selectable marker protein, a screenable marker protein, an anabolic active protein, a catabolic active protein, a biotic or abiotic stress resistance protein, a male sterility protein, or a protein affecting plant agronomic characteristics.

7. The recombinant DNA expression construct of any one of claims 1 to 6, wherein said promoter sequence functioning in plants or plant cells is selected from the group consisting of a) the rice chloroplast protein 12 promoter as described by nucleotide 1 to 854 of SEQ ID NO: 113, or a sequence having at least 60% identity to said fragment, or a sequence hybridizing under stringent conditions to said fragment, or a sequence comprising at least 50 consecutive nucleotides of said fragment, and b) the maize hydroxyproline-rich glycoprotein promoter as described by nucleotide 1 to 1184 of SEQ ID NO: 114, or a sequence having at least 60% identity to said fragment, or a sequence hybridizing under stringent conditions to said fragment, or a sequence comprising at least 50 consecutive nucleotides of said fragment, and c) the p-caffeoyl-CoA 3-0-methyltransferase promoter as described by nucleotide 1 to 1034 of SEQ ID NO: 115, or a sequence having at least 60% identity to said fragment, or a sequence hybridizing under stringent conditions to said fragment, or a sequence comprising at least 50 consecutive nucleotides of said fragment, and d) the maize Globulin-1 [ZmGlbl] promoter (W64A) as described by nucleotide 1 to 1440 of SEQ ID NO: 116, or a sequence having at least 60% identity to said fragment, or a sequence hybridizing under stringent conditions to said fragment, or a sequence comprising at least 50 consecutive nucleotides of said fragment, and e) the putative Rice H+-transporting ATP synthase promoter as described by nucleotide 1 to 1589 of SEQ ID NO: 117, or a sequence having at least 60% identity to said fragment, or a sequence hybridizing under stringent conditions to said fragment, or a sequence comprising at least 50 consecutive nucleotides of said fragment, and f) the putative rice C-8,7 sterol isomerase promoter as described by nucleotide 1 to 796 of SEQ ID NO: 118, or a sequence having at least 60% identity to said fragment, or a sequence hybridizing under stringent conditions to said fragment, or a sequence comprising at least 50 consecutive nucleotides of said fragment, and g) the maize lactate dehydrogenase promoter as described by nucleotide 1 to 1062 of SEQ ID NO: 119, or a sequence having at least 60% identity to said fragment, 96 or a sequence hybridizing under stringent conditions to said fragment, or a sequence comprising at least 50 consecutive nucleotides of said fragment, and h) the rice Lea promoter as described by nucleotide 1 to 1386 of SEQ ID NO: 121, or a sequence having at least 60% identity to said fragment, or a sequence hybridizing under stringent conditions to said fragment, or a sequence comprising at least 50 consecutive nucleotides of said fragment.

8. An expression vector comprising a recombinant expression construct of any one of claims 1 to 7.

9. A transgenic cell or transgenic non-human organism comprising an expression vector as claimed in claim 8 or an expression construct of any one of claims 1 to 7.

10. The cell or non-human organism of claim 9, selected from the group consisting of bacteria, fungi, yeasts and plants.

11. The transgenic cell or non-human organism of claim 9 or 10, wherein said cell or organism is a monocotyledonous plant cell or organism selected from the group consisting of the genera Hordeum, Avena, Secale, Triticum, Sorghum, Zea, Saccharum and Oryza.

12. A cell culture, parts or propagation material derived from a transgenic cell or organism of any one of claims 9 to 11.

13. A method for providing an expression cassette for enhanced expression of a nucleic acid sequence in a plant or a plant cell, comprising the step of functionally linking at least one intron as described in claim 1 or 2 to said nucleic acid sequence.

14. A method for enhancing the expression of a nucleic acid sequence in a plant or a plant cell, comprising functionally linking at least one intron as described in claim 1 or 2 to said nucleic acid sequence.

15. The method as claimed in claim 13 or 14, wherein a promoter sequence functional in plants is linked to said nucleic acid sequence. 97

16. The method as claimed in claim 13 or 14, wherein the intron is linked to said nucleic acid sequence by insertion into the plant genomic DNA via homologous recombination.

17. The method of any one of claims 13 to 16, wherein said plant or plant cell is a monocotyledonous plant or plant cell.

18. The method of any one of claims 13 to 17, wherein said nucleic acid encodes for a protein as described in claim 6 or a sense, antisense, or double-stranded RNA.

19. The use of a cell or organism as claimed in any one of claims 9 to 11 or of cell cultures, parts of transgenic propagation material derived therefrom as claimed in claim 12 for the production of foodstuffs, animal feeds, seeds, pharmaceuticals or fine chemicals.

20. A recombinant DNA expression construct comprising: a) at least one promoter sequence functioning in plants or plant cells, and b) at least one intron, consisting of a sequence described by SEQ ID NO: 2, or a functional equivalent thereof, with expression enhancing properties in plants or plant cells characterized by at least the following features I) an intron length shorter than 1000 base pairs, and II) presence of a 5' splice site comprising the dinucleotide sequence 5'-GT-3' (SEQ ID NO: 78), and III) presence of a 3' splice site comprising the trinucleotide sequence 5'-CAG-3' (SEQ ID NO: 79), and IV) presence of a branch point resembling the consensus sequence 5'-CURAY-3' (SEQ ID NO: 75) upstream of the 3'splice site, and V) an adenine plus thymine content of at least 40% over 100 nucleotides downstream from the 5' splice site, and VI) an adenine plus thymine content of at least 50% over 100 nucleotides upstream from the 3' splice site, and VII) an adenine plus thymine content of at least 55%, and a thymine content of at least 30% over the entire intron, and c) at least one nucleic acid sequence, 98 wherein said promoter sequence and at least one of said intron sequences are functionally linked to said nucleic acid sequence and wherein said intron is heterologous to said nucleic acid sequence and/or to said promoter sequence.

21. A recombinant DNA expression construct according to claim 1, substantially as hereinbefore described with reference to the examples.

22. A method for providing an expression cassette according to claim 13, substantially as hereinbefore described with reference to the examples.

23. A recombinant DNA expression construct according to claim 20, substantially as hereinbefore described with reference to the examples. BASF PLANT SCIENCE GMBH WATERMARK PATENT AND TRADE MARK ATTORNEYS P29206AU01 99