AU2007221926B2 - Seed-specific gene promoters and uses thereof - Google Patents

Description

AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant(s): NATIONAL INSTITUTE OF AGROBIOLOGICAL SCIENCES Invention Title: SEED-SPECIFIC GENE PROMOTERS AND USES THEREOF The following statement is a full description of this invention, including the best method for performing it known to me/us: - 2 SEED-SPECIFIC GENE PROMOTERS AND USES THEREOF FIELD OF THE INVENTION This invention relates to seed-specific gene 5 promoters and uses thereof. BACKGROUND OF THE INVENTION Recombinant DNA technology is being implemented as a way of improving plant breeds. Using this technology, 10 plants with additional functions such as herbicide resistance, pest insect resistance, and the like have been created, and progress is being made in their practical application. The use of recombinant technologies to improve plant breeds not only aims to add new functions to 15 plants: there has been much research and development into the expression of useful proteins in plants, by introducing these plants with a foreign gene. Such research uses plants as factories to produce useful proteins. Production of recombinant proteins in plants has many 20 advantages, the most evident of which are the reduced cost compared to systems that utilize transgenic animals; the ease with which scale of production can be adjusted to suit market size; and the absence of any risk of contamination by animal-borne pathogens such as viruses and prions 25 (Daniell et al., Trends Plant Sci., 6, 219-226 (2001); Fischer and Emans, Transgenic Research, 9, 279-299 (2000); Giddings et al., Nature Biotech., 18, 1151-1156 (2000)). Recently, systems using seeds for production of recombinant protein in plants have been shown to be more 30 advantageous than those using leaves or roots (Delaney, 2002, Plants as Factories for Protein Production (Hood, E.E. and Howard, J.A) pp. 139-158 (2002). Netherlands: Kluwer Academic; Howard and Hood, Plants as Factories for Protein Production (Hood, E.E. and Howard, J.A) pp. vii-x 35 (2002). Netherlands: Kluwer Academic). Seeds are storage organs, in which a special organelle called a protein body H.\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU_des.doc 28/10/04 - 3 stably stores a large amount of a small number of storage proteins. This feature has been employed by using seeds as ideal bioreactors for producing recombinant protein. Recombinant proteins accumulated in seeds are very stable, 5 and can be administered orally without any need for further processing or purification. Antibodies or vaccines expressed in seeds are reported to be highly stable, and can be stored for years, even at room temperature, without decomposition. Moreover, vaccines administered via seeds 10 are thought to trigger antibody production by the mucosal immune system, without processing or purification (Walmsley and Arntzen, Curr. Opin. Biotech., 11, 126-129 (2000)). When producing proteins using recombinant technology, the yield of a protein of interest is affected by many 15 factors, including transcription factors. The most important and easily controlled of these factors is the choice of promoter. In order to use rice seeds as a platform for recombinant protein production, it is important to use a promoter suited to the needs of 20 individual proteins and their use in biotechnology. This is because the promoter controls the timing, location and level of expression. However, analyses of the cis-regulatory factors involved in endosperm-specific expression are limited to 25 those of a small number of glutelin genes, using different species (transgenic tobacco) and the same species (transgenic rice). (Croissant-Sych and Okita, Plant Sci., 116, 27-35 (1996); Takaiwa et al., Plant Mol. Biol., 16, 49-58 (1991a); Takaiwa et al., Plant Mol. Biol., 30, 1207 30 1221 (1996); Wu et al., Plant J., 14, 673-983 (1998a); Wu et al., Plant J., 23, 415-421 (2000); Yoshihara et al., FEBS Lett., 383, 213-218 (1996); Zhao et al., Plant Mol. Biol., 25, 429-436 (1994); Zheng et al., Plant J., 4, 357 366 (1993)). Studies of a few other rice storage protein 35 promoters were no more than observations of their spatial expression patterns (Wu et al., Plant Cell Physiol., 39, H,\soniam\keep\SPECIFICATIONS\PS4849 MOA-A0307-AUdes.doc 28/10/04 -4 885-889 (1998b)). SUMMARY OF THE INVENTION The present invention has been made considering the 5 above circumstances. Therefore the present invention provides promoters with seed-specific promoter activity, and methods of expressing foreign proteins in seeds. The present invention also provides promoters with specific promoter activity in a particular site, such as the seed 10 endosperm, embryo, and aleurone layer. The present inventors have isolated a number of promoters of rice genes expressed in seeds, and constructed binary vectors in which each promoter was inserted upstream of GUS reporter gene. The present 15 inventors then transformed rice using the Agrobacterium method. Then, for each promoter, the inventors used GUS expression as an index to examine the site of expression, the expression pattern during seed maturation, and the level of expression in seeds. They thus discovered 20 promoters with an activity of expression specific to a particular site in seeds, and with higher activity than constitutive promoters and known seed-specific promoters. As described above, it is useful for a seed expressing a foreign gene product to be taken as food. However, for 25 this to be possible, the foreign gene must be expressed in an edible part of the seed. For example, in one of the main cereals, rice, the endosperm is normally eaten, and therefore the above goal would be achieved by using an endosperm-specific promoter in rice. Furthermore, 30 promoters specific for a particular site in a seed will enable expression of a foreign gene at a desired place in a seed. Therefore, these promoters can be valuable tools for metabolic engineering using seeds. For example, using a promoter that directs expression in the aleurone 35 layer or embryo may control fatty acid metabolism. Thus, the present invention relates to promoters 259630_1 (GHMalerm) P54849.AU.1 - 5 specific to a particular site in a seed, and to uses thereof. More specifically, it provides: (1) an isolated DNA having promoter activity in seeds, wherein the DNA consists of a nucleotide sequence 5 according to SEQ ID NO: 2; (2) a DNA comprising a gene functionally linked downstream of the DNA according to (1); (3) a vector comprising the DNA according to (1) or (2); (4) a transformed plant cell comprising the DNA according 10 to (2); (5) a transformed plant cell comprising the vector according to (3); (6) a transformed plant comprising the cell according to (4) or (5); 15 (7) a reproductive material of the plant according to (6); (8) the reproductive material according to (7), wherein the reproductive material is a seed; and (9) a method of expressing a gene in a seed generated 20 from a plant cell, comprising the steps of: (a) introducing the DNA according to (2) or the vector according to (3) into the plant cell; (b) regenerating a plant from the plant cell; and (c) growing the plant to produce seeds. 25 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a diagram of the construction of the chimeric gene used for rice transformation. The 5' flanking regions of various genes encoding rice seed 30 storage proteins and non-storage proteins were fused into a region between two restriction sites, selected from HindIII, SalI, and SmaI sites. The GUS reporter gene and 2596360_1 (GHMatter) P54849.AU.I - 6 the Nos terminator were then fused. The promoter was shown to promote the genes: 1.3 kb GluB-1, 2.3 kb GluB-1, GluB-2, GluB-4, 10 kDa prolamin, 13 kDa prolamin (PG5), 16 kDa prolamin, 26 kDa Glb-1, REG2, Ole18, soybean P-conglycinin, 5 AlaAT, GOGAT, PPDK, AGPase, and SBE1. Fig. 2 is a series of pictures depicting the results of histochemical analysis of GUS expression induced by the gene promoters of various seed storage proteins and non storage proteins. The GUS protein was detected by 10 vertically dissecting transgenic seeds by hand, and incubating the sections in a solution containing X-Gluc. a, 1.3 kb GluB-1 promoter; b, 2.3 kb GluB-1 promoter; c, GluB-2 promoter; d, GluB-4 promoter; e, 10 kDa prolamin promoter; f, 13 kDa prolamin (PG5a) promoter; g, 16 kDa 15 prolamin promoter; h, 26 kDa Glb-1 promoter; i, REG2 promoter; j, Ole18 promoter; k, P-conglycinin promoter; 1, AlaAT promoter; m, GOGAT promoter; n, AGPase promoter; o, PPDK promoter; and p, SBE1 promoter. Fig. 3 is a series of pictures showing the results of 20 histochemical analysis of GUS expression in vegetative tissues. lf, leaf; ls, leaf sheath; sk, stalk; rt, root; and ed, eudodersis. a, 10 kDa prolamin promoter; b, PPDK promoter; and c, AGPase promoter. Fig. 4 is a series of pictures showing the time 25 course of changes in GUS activity induced by seed promoters, over the maturation stages of seed development. It shows the results of histochemical staining, using X Gluc, of the vertical sections of transgenic rice seed at 7, 12, and 17 DAF. a, 1.3 kb GluB-1 promoter; b, 2.3 kb 30 GluB-1 promoter; c, GluB-2 promoter; d, GluB-4 promoter; e, 10 kDa prolamin promoter; f, 13 kDa prolamin (PG5a) promoter; g, 16 kDa prolamin promoter; h, 26 kDa Glb-1 promoter; i, REG2 promoter; and j, Ole18 promoter. Fig. 5 continues from Fig. 4 and is a series of 35 pictures. k, P-conglycinin promoter; 1, AlaAT promoter; m, GOGAT promoter; n, AGPase promoter; o, PPDK promoter; p, H:\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU_des.doc 28/10/04 - 7 SBE1 promoter; and q, ubiquitin promoter. Fig. 6 shows the results of measuring the GUS activity expressed by the various promoters in maturating seed at 17 DAF. GUS activity is expressed in pmol 4MU/min/ptg protein 5 units. DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel DNAs with promoter activity in seeds. This invention, as described above, is 10 based on the discovery by the present inventors of promoters with promoter activity specific to particular sites in seeds, and that exhibit greater activity than constitutive promoters and known seed-specific promoters. Specifically, the above DNAs of the present invention 15 include DNAs with promoter activity that comprise a sequence of SEQ ID NO: 2. The present inventors identified rice derived DNAs comprising promoter activity, and grouped them into the following three groups: (A) Promoter DNAs specific to the endosperm (the 20 nucleotide sequences of the respective DNAs are shown in SEQ ID NOs: 1 to 4). (B) Promoter DNAs specific to embryo or aleurone tissue (the nucleotide sequences of the respective DNAs are shown in SEQ ID NOs: 5 and 6). 25 (C) Promoter DNAs for expression in the entire seed (the nucleotide sequence of the DNA is shown in SEQ ID NO: 7). Group (A), the endosperm-specific promoter DNA group, comprises the rice glutelin GluB-1 gene promoter (SEQ ID NO: 30 1), rice glutelin GluB-4 gene promoter (SEQ ID NO: 2), 10 kDa prolamin promoter (SEQ ID NO: 3), and 16 kDa prolamin promoter (SEQ ID NO: 4). Expression of this group can be observed in aleurone and sub-aleurone tissues at seven days after flowering, and progressively spreads into the inner endosperm 35 region during maturation. This expression pattern does not change during the maturation process. 2431551.1 (GHMatters) - 8 Group (B), the embryo or aleurone tissue-specific promoter DNA group, comprises the rice embryo globulin gene promoter (SEQ ID NO: 5), and rice oleosin promoter (SEQ ID NO: 6). This group shows expression in the aleurone tissue in the 5 early stages of maturation (seven days after flowering), and the expression spreads into the embryo and aleurone tissue during maturation, but not to the endosperm. Group (C), promoter DNAs for expression in the entire seed, comprises the rice ADP-glucose pyrophosphorylase gene 10 promoter (SEQ ID NO: 7). This promoter first shows expression in the embryo in the early stage of maturation, and then in the entire seed during maturation (expression in the embryo is also extremely high in the late stage of maturation). One skilled in the art can use conventional methods to 15 prepare DNAs comprising the seed-specific promoters of groups (A) to (C) (hereinafter abbreviated as "the DNAs of this invention"). For example, the DNAs can be prepared by designing an appropriate pair of primers based on a nucleotide sequence of any of SEQ ID NOs: 1 to 7 (for example, SEQ ID 20 NOs: 9 to 22), and performing PCR using a rice genomic DNA as the template, and screening a genomic library with the resulting amplified DNA fragment as a probe. Moreover, a commercially available DNA synthesizer may be used to synthesize a desired DNA. 25 The DNAs of this invention may be used to obtain (isolate) DNAs comprising promoter activity. In the first step of isolating a DNA, a DNA of this invention or its part may be used as a probe, or an oligonucleotide that specifically hybridizes with a DNA of the invention may be 30 used as a primer to isolate a DNA comprising high homology with the above DNA from a desired organism. The DNAs of the invention also comprise DNAs that hybridize with DNAs comprising a nucleotide sequence of SEQ ID NO: 2, which can be isolated using standard hybridization techniques 35 (Southern E.M., J. Mol. Biol., 98, 503 (1975)) or PCR methods (Saiki R.K. et al., Science, 230, 1350 (1985); Saiki R.K. et 2431551_1 (GHMatters) - 9 al., Science, 239, 487 (1988)). Thus, it is feasible for one skilled in the art to isolate from a desired organism a DNA high homologous to a DNA comprising a nucleotide sequence of SEQ ID NO: 2, using a DNA comprising a nucleotide sequence of 5 SEQ ID NO: 2 or its part as a probe, or an oligonucleotide that specifically hybridizes with a DNA comprising a nucleotide sequence of SEQ ID NO: 2 as a primer. In order to isolate such DNAs, hybridization is preferably performed under stringent conditions. Hybridization may be performed with 10 buffers that permit the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. At high stringency, hybridization complexes will remain stable only where the nucleic acid molecules are almost completely complementary. Many factors determine the stringency of 15 hybridization, including G+C content of the cDNA, salt concentration, and temperature. For example, stringency may be increased by reducing the concentration of salt or by raising the hybridization temperature. Temperature conditions for hybridization and washing greatly influence stringency and 20 can be adjusted using melting temperature (Tm). Tm varies with the ratio of constitutive nucleotides in the hybridizing base pairs, and with the composition of the hybridization solution (concentrations of salts, formamide and sodium dodecyl sulfate). In solutions used for some membrane based 25 hybridizations, addition of an organic solvent, such as formamide, allows the reaction to occur at a lower temperature. Accordingly, on considering the relevant parameters, one skilled in the art can select appropriate 2431551_1 (GHMaters) - 10 conditions to achieve a suitable stringency based experience or experimentation. Herein, stringent hybridization conditions mean conditions using 6 M urea, 0.4% SDS, and 0.5x SSC, or those using 0.1% SDS (60 0 C, 0.3 M 5 NaCl, 0.03 M sodium citrate), or conditions providing an equivalent stringency. Under more stringent conditions, for example, performing hybridization in 6 M urea, 0.4% SDS, and 0.lx SSC, one can expect to isolate DNAs with higher homology. High homology means sequence identity 10 over the entire nucleotide sequence of preferably 50% or higher, more preferably, the isolated DNA is at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical. To determine the percent identity of two DNAs, the is sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. The percent identity between two sequences can be determined using conventional techniques such as to those 20 described herein, with or without allowing gaps. In calculating percent identity, typically exact matches are counted. For example, when an isolated DNA of the present invention is longer than or equivalent in length to a prior art sequence, the comparison is made with the full length 25 of the inventive sequence. Alternatively, when an isolated DNA of the present invention is shorter than the prior art sequence, the comparison is made to a segment of the prior art sequence of the same length as that of the inventive sequence (excluding any loop required by the homology 30 calculation). Identity between nucleotide sequences can be determined by using the BLAST algorithm developed by Karlin and Altschul (Proc. Natl. Acad. Sci. U.S.A., 87, 2264-2268 (1990); Karlin S. and Altschul S.F., Proc. Natl. Acad. Sci. 35 U.S.A., 90, 5873). A program called BLASTN has been developed based on the BLAST algorithm (Altschul S.F. et H:\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU dea.doc 28/10/04 - 11 al., J. Mol. Biol., 215, 403 (1990)). When analyzing nucleotide sequence using BLASTN, parameters may be set as score = 100 and wordlength = 12, for example. When using the BLAST and Gapped BLAST programs, the default parameters for 5 each program may be chosen. Specific procedures for these analyses are publicly known (http://www.ncbi.nlm.nih.gov/). Another example of a mathematical algorithm that may be utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. 10 The DNAs of this invention are normally derived from plants, preferably from monocotyledons, and more preferably from Poaceae, but are not limited to any particular origin, as long as the DNA has seed-specific promoter activity. In addition, this invention provides DNAs that are 15 structurally similar to the above DNAs, and comprise promoter activity. Such DNAs include DNAs with seed specific promoter activity that comprise a nucleotide sequence wherein one or more nucleotides are substituted, deleted, added, and/or inserted into a nucleotide sequence of SEQ ID NO: 2, wherein 20 the DNA is homologous to the nucleotide sequence of SEQ ID NO: 2 with a sequence identity of 70% or more. Such DNAs can also be used to isolate a DNA of this invention that comprises promoter activity. Methods for preparing such DNAs are well known to those skilled in the art, and include the 25 hybridization techniques and polymerase chain reaction (PCR), as described above. Furthermore, the above DNAs may be prepared by introducing mutations into DNAs comprising a nucleotide sequence of SEQ ID NO: 2, for example, by using site-directed mutagenesis method (Kramer W. and Fritz H.J., 30 Methods Enzymol., 154, 350 (1987)). One skilled in the art can determine whether or not the DNAs prepared as above comprise promoter activity by using methods such as known reporter assays using reporter genes. Reporter genes are not limited to any particular gene, as long 35 as their expression is detectable. For example, reporter genes may be those routinely used by 2431551_1 (GHMatters) - 12 those skilled in the art, such as CAT gene, lacZ gene, luciferase gene, P-glucuronidase (GUS) gene, and GFP gene. The reporter gene expression level can be measured by the methods commonly known to those skilled in the art, 5 depending on the type of reporter gene. For example, when CAT gene is used as the reporter gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When lacZ gene is used as the reporter, expression level can be 10 measured by detecting the color of a dye compound produced by the catalytic function of the gene product. In the case of luciferase gene, expression level can be measured by detecting fluorescence from a fluorescent compound produced by the catalytic function of the gene product; the GUS 15 reporter gene expression can be measured by detecting luminescence of Glucuron (ICN), or the color of 5-bromo-4 chloro-3-indolyl-p-glucuronide (X-Gluc) as the result of catalytic function of the gene product. Furthermore, GFP expression can be measured by detecting the fluorescence of 20 GFP protein. In addition, if a gene other than those described above is used as a reporter, the expression level of the gene can be measured by methods known to those skilled in the art. For example, mRNAs may be extracted by standard 25 methods, and then used as templates to perform Northern hybridization or RT-PCR to measure the transcription level of the gene. Furthermore, DNA array technology may be used to measure gene transcription levels. In addition, fractions containing proteins encoded by the genes may be 30 recovered by standard methods, and expression of the protein of the present invention may be detected by electrophoresis, such as SDS-PAGE, to measure the translation level of the gene. Furthermore, the expression of the protein encoded by a gene may be detected by Western 35 blotting, using an antibody against the protein to measure its translation level. The antibody used to detect the H:\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AUdes.doc 28/10/04 - 13 protein encoded by the gene can be any antibody, and is not particularly limited as long as it is detectable. For example, both monoclonal antibodies or polyclonal antibodies may be used. The antibody can be prepared by 5 methods known to those skilled in the art. Furthermore, the present invention provides DNAs in which an arbitrary gene is functionally linked downstream of an above promoter DNA. The DNAs of the invention enable specific expression of a desired protein or peptide, 10 encoded by an arbitrary gene, in seeds, by activating a promoter DNA. Herein, "functionally linked" means that a DNA of the invention and a gene are linked to each other such that expression of the downstream gene is triggered by binding 15 of a transcription factor to a DNA with promoter activity of the present invention. Thus, even if the gene is linked with another gene, and forms a fusion protein with the product of the other gene, it is collectively considered to be "functionally linked" as long as expression of the 20 fusion protein is induced when a transcription factor binds to the DNA of this invention. The present invention also provides vectors comprising a DNA (referred to below as "the above DNA"), wherein an arbitrary gene is functionally linked to an 25 above promoter DNA or downstream of it. The vectors of this invention are useful for maintaining the above DNA in host cells, or for expressing a protein of interest and such by transforming plants. The vectors used for insertion of the above DNA are 30 not limited to any particular vector as long as they enable expression of the inserted gene in plant cells. For example, vectors comprising a promoter for constitutive gene expression in plant cells (for example, cauliflower mosaic virus 35S promoter), or vectors comprising a 35 promoter that can be activated by an external stimuli in an inducible manner, can be used. Vectors comprising the H:\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AUdeo.doc 28/10/04 - 16 as is. As used herein, an "isolated promoter" is a promoter removed from its original environment (e.g., the natural environment if naturally occurring) and thus, altered by the 5 "hand of man" from its natural state. Seeds are essentially storage organs, and contain a large space for accumulating foreign gene products. Enzymes, antibodies, and the like that are accumulated in seeds are stable for more than one year, even when stored at room 10 temperature. In addition, there is no need to purify such proteins if they are taken as food. Thus, useful products can be produced for extremely low costs. Furthermore, no special production facilities are required, other than agricultural fields, and they are safe from the risk of contamination by is animal viruses and such. The promoters of the present invention are especially valuable as promoters for the production of useful products using seeds. For example, these promoters enable the large scale production of medicinal products (e.g., vaccines, 20 antibodies, blood products, and interferons) or industrial enzymes in seeds. In addition, they can be used to express allergen epitopes in plants, creating plant crops for the treatment of allergies such that eating the seed of such a plant can treat pollinosis, house dust allergies, and the 25 like. These promoters also enable the expression in a seed of a foreign gene whose product is highly nutritious, thus improving the nutritional value of the seed. Furthermore, it is possible to use the above promoters to create functional seeds by expressing functional peptides or functional proteins 30 that comprise the effect of reducing high blood pressure, serum cholesterol, blood sugar, or such in seeds. In addition, the set of promoters of this invention, which enable the expression of a gene in a desired region of a seed and a desired stage of seed development, can be used as 35 important tools for metabolic engineering utilizing 2431551_1 (GHMatter) - 15 Kamm et al. (Plant Cell, 2, 603 (1990)), and potato may be regenerated by the method of Visser et al. (Theor. Appl. Genet., 78, 594 (1989)). Arabidopsis may be regenerated by the method of Akama et al. (Plant Cell Reports, 12, 7-11 5 (1992)), and eucalyptus may be regenerated by the method of Doi et al. (JP-A Hei 8-89113). Furthermore, the present invention provides not only plants carrying cells into which the above DNA has been introduced, but also the reproductive materials of those 10 plants. After obtaining transformed plants that have the above DNA or vector introduced in their genome, reproductive materials (for example, seeds, fruits, cuttings, tubers, tuberous roots, shoots, calluses, and protoplasts) can be obtained, and the plants can be mass 15 produced from these sources. In particular, in addition to being a reproductive material, seeds are places where the above introduced promoter causes accumulation of foreign gene product. Furthermore, the present invention provides methods 20 of expressing an arbitrary gene in plant seed cells. The methods of the invention comprise the steps of introducing plant cells with a DNA in which an arbitrary gene is functionally linked downstream of the above promoter DNA of the invention, or the above vector of this invention, and 25 regenerating a plant from the plant cells. The steps of introduction into plant cells and regeneration of a plant can be performed by the above methods. The methods of the invention may be used to obtain a desired gene product from a plant, or to obtain seeds that accumulate the desired 30 gene product. Plants regenerated using a desired gene by a method of this invention bear seeds that accumulate the product of the desired gene. Thus, the gene product can be obtained by purification from these seeds, or such. In addition, if the desired gene product is a medicinal 35 compound or the like, seeds can be used as a final form, omitting purification steps, because seeds can be ingested Hs\8oniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU_des.doc 28/10/04 - 16 as is. As used herein, an "isolated promoter" is a promoter removed from its original environment (e.g., the natural environment if naturally occurring) and thus, altered by 5 the "hand of man" from its natural state. Of the promoters isolated by the present inventors, 10 kDa rice prolamin promoter resulted in an interesting observation. In testing seed specific promoter activity, it was found that when the Nos terminator was linked 10 downstream of 10 kDa prolamin promoter, gene expression was induced not only in seeds but also in the phloem of roots, stalks, and the like. However, when the original 3' untranslated region (0.3 kb; SEQ ID NO: 8) was linked, gene expression other than in seeds was clearly suppressed. 15 Thus, the inventors discovered that the 3'-untranslated region of the rice 10 kDa prolamin promoter can suppress gene expression in tissues other than seed endosperm, and is therefore required for endosperm-specific gene expression. The inventors had previously found that a 20 foreign gene product could be expressed in seeds at a high level by inserting the 5'-untranslated region between a promoter ensuring expression in seeds and a foreign gene (JP-A 2002-58492). This seed storage protein gene is the first case for which both the 3'-flanking region and the 25 5'-flanking region have been identified as necessary for seed specific expression. Endosperm-specific gene expression, where gene expression in tissues other than endosperm is suppressed, is made possible by inserting the 3'-untranslated region 30 downstream of a promoter that comprises activity in tissues in addition to endosperm. Thus, the present invention further provides (1) DNAs comprising a 3'-untranslated region of SEQ ID NO: 8, (2) vectors comprising the 3'-untranslated region, (3) vectors 35 comprising a promoter and the 3'-untranslated region, (4) vectors comprising a promoter, a gene, and the 3'- - 17 THIS PAGE IS LEFT INTENTIONALLY BLANK 24315511 (GHMatter) - 18 seeds. For example, by using a promoter that directs expression to the aleurone layer or embryo, a metabolic process of interest in fatty acid metabolism can be controlled. 5 Any patents, patent applications, and publications cited herein are incorporated by reference. EXAMPLES This invention will be explained in detail below with 10 reference to Examples, but it is not to be construed as being limited thereto. [EXAMPLE 1] Construction of the promoter-GUS gene chimeric constructs is and isolation of transgenic plants The expression patterns and promoter activity of a number of genes expressed in seeds were characterized, instead of examining the genes presumed to be regulatory factors. Fifteen different promoters ranging in size from 20 0.8 to 2.4 kb were isolated by PCR using genomic DNA or genomic clones as a template. The genes and the size of their corresponding promoters are as follows: rice 10 kDa prolamin, 0.8 kb; rice 13 kDa prolamin (PG5a), 0.9 kb; rice 16 kDa prolamin, 25 0.9 kb; rice glutelin GluB-4, 1.4 kb; rice embryo globulin (REG2), 1.3 kb; rice 18 kDa oleosin (Ole18), 1.3 kb; rice glutamate synthase gene (GOGAT), 0.8 kb; rice pyruvate orthophosphate dikinase (PPDK), 0.8 kb; rice ADP-glucose pyrophosphorylase (AGPase), 2.0 kb; rice starch branching 30 enzyme (SBE1), 2.0 kb; and soybean P-conglycinin, 1.0 kb. Rice glutelin GluB-1, 1.3 kb, 2.3 kb; rice glutelin GluB-2, 2.4 kb; rice alanine aminotransferase (AlaAT), 1 kb; rice 26 kDa globulin (Glb-1), 1.0 kb; and maize ubiquitin promoter, 2 kb. 35 Of these, the promoter sequences of 2.3 kb GluB-1, GluB-4, 10 kDa prolamin, 16 kDa prolamin, rice embryo H:\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AUdes.doc 28/10/04 - 19 globulin, rice oleosin, and rice ADP-glucose pyrophosphorylase are shown by SEQ ID NOs: 1 to 7, and the sequences of the primer pairs used to isolate these promoters are shown by SEQ ID NOs: 9 to 22, respectively. 5 Fragments of various promoters were inserted into the modified binary vector pGPTV-35S-HPT, which comprises the hygromycin phosphotransferase (HPT) gene as a selection marker (Fig. 1). The modified vector was constructed from the pGPTV-HPT binary vector (Becker et al. (1992)) using 10 the Nos promoter as the HPT gene promoter instead of the 0.8 kb CaMV35S promoter. The seed gene promoters to be tested were introduced upstream of the UdiA gene encoding P-glucuronidase (GUS) in the modified binary vector. Transgenic rice plants (Oryza sativa cv Kitaake) were 15 created using Agrobacterium-mediated transformation. The plasmids constructed as above were introduced into EHA105 strain Agrobacterium tumefaciens by electroporation. Five week-old calluses derived from mature rice seeds were treated with the transformed A. tumefaciens for three days. 20 Each of the infected calluses was continuously cultured for four weeks in N6 selection media comprising hygromycin, and MS regeneration media. Regenerated young plants were transferred to an incubator (Goto et al., Nature Biotech., 17, 282-286 (1999)). 25 More than 20 different lines of independent transgenic plants were generated for each construct. The presence of the promoter fusions of interest was confirmed by PCR using genomic DNAs isolated from the leaves of independent transgenic rice lines, and the positive lines 30 were used to characterize the promoter. [EXAMPLE 2] Activity of the seed storage protein gene promoters in seeds 35 Transgenic rice seeds were examined by histochemical staining to identify the site of GUS reporter gene HM\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU_dea.doc 28/10/04 - 20 expression, which was induced by the seed storage protein promoters. For histochemical analysis, maturing seeds in a stage 17 days after flowering (DAF) were sectioned along their longitudinal axis with a razor blade, and the 5 sections were incubated in 50 mM sodium phosphate buffer (pH 7.0) containing 0.5 mM X-Gluc (5-bromo-4-chloro-3 indolyl-glucuronide) and 20% methanol at 37 0 C. The optimal incubation time for the staining reaction varied from 30 minutes to overnight, depending on the level of GUS 10 activity. Fig. 2 shows the detected expression patterns. Rice glutelin promoters (1.3 kb and 2.3 kb GluB-1, GluB-2, and GluB-4; Fig. 2 a to d) and prolamin promoters (10 kDa, 13 kDa, and 16 kDa; Fig. 2 e to g) induced GUS gene expression 15 in endosperm. GUS gene expression by the glutelin promoters and prolamin promoters was also detected in aleurone layer and subaleurone tissues, but not in embryos. Further detailed examination of the maturing seeds of transgenic rice carrying the glutelin promoters and 20 prolamin promoters revealed that the peripheral endosperm regions showed the highest GUS activity, while the inner regions showed weak activity. The GluB-1 promoters (both 1.3 kb and 2.3 kb) showed significantly higher activity in endosperm regions close to the embryo. GUS expression 25 induced by 13 kDa prolamin promoter (PG5a) was strictly restricted to the peripheral endosperm regions. The 26 kDa globulin Glb-1 promoter induced GUS expression in the inner starchy endosperm tissue (Fig. 2 h). GUS expression induced by the embryo storage protein promoters (REG2, 30 Ole18, and 0-conglycinin; Fig. 2 i to k) was restricted to the embryo and aleurone tissues, and was not observed in endosperm at all. The patterns of GUS gene expression induced by these embryo storage protein promoters were almost identical. Interestingly, despite a number of 35 reports on differential expression between monocotyledonous and dicotyledonous plants (Chowdhury et al., Plant Cell H:\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU dea.doc 28/10/04 - 21 Rep., 16, 277-281 (1997); Rathaous et al., Plant Mol. Biol., 23, 613-618 (1993)), the -conglycinin promoter from soybean, which is a dicotyledonous plant, maintained embryo-specific expression in rice, a monocotyledon. 5 Notably, GUS expression induced by the P-conglycinin promoter was extremely low in rice, in sharp contrast to the high expression by the same promoter in the embryos and cotyledons of the dicotyledonous plant, tobacco. Overall, GUS activity was not detected in any leaves, 10 leaf sheaths, stalks, or roots of transgenic rice comprising a fusion with a seed storage protein promoter (data not shown). The only exception was the 10 kDa prolamin promoter, which induced some expression in vegetative organs (Fig. 3). These results support the 15 conclusion that endosperm storage protein genes (except the 10 kDa prolamin) are expressed in an endosperm-specific manner, and the expression of embryo storage protein genes are restricted to the embryo and aleurone layer. Although the seed storage protein promoters resulted 20 in specific gene expression in the endosperm or embryo, the promoters of non-storage proteins exhibited different expression patterns (Fig. 2). The GUS gene controlled by the AlaAT promoter was expressed in the center of starchy endosperm, and its activity was higher in the endosperm 25 region close to the embryo (Fig. 2 1). The expression pattern of the PPDK-GUS transgene was similar to that of the endosperm storage proteins (Fig. 2 o). The GUS gene controlled by the AGPase promoter was expressed over the entire seed, including the pericarp, and was highly 30 expressed in the inner starchy endosperm and embryo in particular (Fig. 2 n). In contrast, the GOGAT and SBE promoters induced GUS gene expression mainly in the scutellum (the of embryo and endosperm boundary) (Fig. 2-m and 2-p). 35 [EXAMPLE 31 H:\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU des.doc 28/10/04 - 22 GUS expression pattern in the vegetative organs Most of the examined promoters showed either endosperm- or embryo-specific GUS gene expression. However, GUS activity was also detected in the vegetative 5 tissues of transgenic rice comprising the promoters of the 10 kDa prolamin, PPDK, and AGPase genes (Fig. 3), and those comprising the AlaAT promoter (Kikuchi et al., Plant Mel. Biol., 39, 149-159 (1999)). In these transgenic rice plants, GUS activity was detected in leaves, leaf sheaths, 10 and the phloem of vascular bundles in stalks, in addition to in the endosperm or over the entire seed (Fig. 3 a to c). GUS activity was also detected in the endodermis of the roots of the transgenic rice. However, the expression pattern obtained with the AGPase promoter was slightly 15 different from those obtained with the PPDK and 10 kDa prolamin promoters. In particular, the AGPase promoter induced high level GUS expression in the apical meristem, whereas the latter two induced ubiquitous staining in the root. Furthermore, the AGPase promoter showed distinct GUS 20 activity in the root, and it was stronger than PPDK and 10 kDa prolamin promoters. In its natural state, the 10 kDa prolamin gene is normally expressed in endosperm undergoing maturation, and not detectable in vegetative tissues. The ectopic 25 expression of the GUS fusion product observed herein was reversed to a normal endosperm-specific expression pattern by substituting the Nos terminator with the 0.3 kb region located downstream of the stop codon of the 10 kDa prolamin gene in its natural state (data not shown). Notably, this 30 substitution of the 3'-transcription termination region had almost no influence on the activity of the promoter. These results indicate that the endosperm specific expression of 10 kDa prolamin gene requires both 5'- and 3'-flanking regions. 35 SEQ ID NOs: 23 and 24 show the primer pair used to isolate the 3'-transcription termination region. H:\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU_des.doc 28/10/04 - 23 [EXAMPLE 4] Promoter activity during seed development The expression pattern of introduced genes in 5 developing seeds was examined by the histochemical staining of vertical sections of seeds collected at 7, 12, and 17 DAF. Specifically, seeds undergoing maturation in stages 7, 12, and 17 days after flowering (DAF) were sectioned along their longitudinal axis with a razor blade, and the 10 cut sections were incubated in 50 mM sodium phosphate buffer (pH 7.0) containing 0.5 mM X-Gluc (5-bromo-4-chloro 3-indolyl-glucuronide) and 20% methanol at 37 0 C. The optimal incubation time for the staining reaction varied from 30 minutes to overnight, depending on the level of GUS is activity. The expression pattern during seed maturation was examined for each transgenic line, and Figs. 4 and 5 show the results of each representative line for each seed promoter. Interestingly, the site where GUS expression was 20 first detected differed for each construct. The glutelin promoter and prolamin promoter first showed blue GUS staining in the peripheral endosperm regions, i.e., the aleurone and subaleurone tissues. In the glutelin promoter and 16 kDa prolamin promoter, staining then spread to the 25 inner starchy endosperm as the seed matured (17 DAF), while this was not observed for the 10 kDa and 13 kDa prolamin promoters (Fig. 4 a to g). This expression pattern was in marked contrast to the pattern with the 26 kDa Glb-1 promoter, where blue GUS staining was first detected in the 30 inner starchy endosperm cells close to the embryo, and did not change during the development process (Fig. 4 h). GUS gene expression induced by the REG2, Ole18, and P-conglycinin gene promoters was detected by seven days after flowering (DAF). Their activity tended to be 35 observed first in the aleurone layer, and later in the embryo. Expression by these promoters was restricted to H:\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU_des.doc 28/10/04 - 24 the aleurone tissue and embryo (Fig. 4 i, j, and Fig. 5 k). Figs. 5 1 to p show the temporal expression patterns of non-storage protein promoters during seed maturation in representative transgenic lines. GUS expression by the 5 AlaAT promoter was first observed in the inner starchy endosperm tissue, and eventually spread through the entire endosperm, although the embryo remained unstained (Fig. 5 1). GUS activity by the SBE1 promoter was also restricted to the inner starchy endosperm tissue, and in particular, 10 the tissue close to the embryo (Fig. 5 p). However, because of the extremely low level of GUS activity, the blue staining was not detectable until 12 DAF. In contrast, when the AGPase gene promoter fusion was introduced, GUS staining first appeared in the embryo, and 15 later spread into the center of the endosperm. Blue GUS staining was finally observed all throughout seeds undergoing maturation, with the most intense staining found in the embryo (Fig. 5 n). This expression profile during seed development was very similar to that observed for the 20 ubiquitin promoter (Fig. 5 q). In contrast, the expression pattern with the PPDK promoter was similar to those with the glutelin promoter and prolamin promoter (Fig. 5 o). GUS activity by the GOGAT promoter was restricted to the scutellum, and there was no particular change during seed 25 development, except that GUS activity was not detectable at 7 DAF (Fig. 5 m). [EXAMPLE 5] Quantitative analysis of the promoter activity 30 To evaluate the activity of various promoters, GUS fluorescence was assayed by the method of Jefferson (1987). Maturing seeds at 17 DAF were homogenized in GUS extraction buffer (50 mM NaPO 4 [pH 7.0], 10 mM 2-mercaptoethanol, 10 mM Na 2 -EDTA, 0.1% SDS, 0.1% Triton X-100). After 35 centrifugation, 10 l of the supernatant was mixed with 90 tl of assay buffer containing 1 mM 4-methylumbelliferyl-p H.\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU_des.doc 28/10/04 - 25 D-glucuronide (MUG). After incubating for one hour at 37*C, 900 ptl of 0.2 M Na 2

CO

3 was added to the mixture to terminate the reaction. Values obtained using a fluorometer were compared with those obtained from serial dilutions of 4 5 methylumbelliferone (4MU). The protein amount was determined using a Bio-Rad Protein Assay kit, with serum albumin as the standard. Three seeds were assayed for each transgenic plant. As shown in Fig. 6, significant differences were 10 found between the promoter activities. The tested seed promoters were classified into four groups based on their activity. The group showing high GUS activity comprises the following four promoters: GluB-4, 10 kDa prolamin, 16 kDa prolamin, and Glb-1 promoters. The average GUS 15 activities of these promoters were 44.8±16.5, 38.8±10.8, 27.1±12.7, and 28.6±11.8 pmol 4MU/min/gg protein, respectively. The group with moderate GUS activity includes the following 2.3 kb GluB-1 and AGPase gene promoters. Their GUS activity is lower than that observed 20 for the high activity group, but is much higher than for the other groups. The average GUS activities of 2.3 kb GluB-1 and AGPase gene promoters were 21.3±7.0 and 10±4.7 pmol 4MU/min/ptg protein, respectively. Seven promoters, i.e., 1.3 kb GluB-1, GluB-2, 13 kDa prolamin, REG-2, Ole18, 25 AlaAT, and PPDK promoters, were tentatively grouped into a group with relatively low GUS activity. The average GUS activities of these promoters were 2.1±1.2, 5.5±2.2, 7.4±5.5, 2.4±1.2, 2±4.6, 5.9±4.0, and 4.0±3.0 pmol 4MU/min/ptg protein, respectively. The remaining three 30 promoters, the GOGAT, SBE1, and P-conglycinin gene promoters, were grouped into the low GUS activity group. The GUS expression induced by these promoters was very faint, with activity below 1 pmol 4MU/min/tg protein. The GUS activity of the control ubiquitin promoter was an 35 average of 7.4±8.5 pmol 4MU/min/tg protein (in maturing seeds). Although the ubiquitin promoter has been used in H.\soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AU des.doc 28/10/04 - 26 many applications as a general promoter, its level was about the same as those obtained with the promoters of the group with relatively low GUS activity. For purposes of comparison, the activities of the 5 PPDK promoter and AGPase promoter in vegetative tissues were also examined. The average GUS activities for PPDK promoter in leaf, stalk, and leaf sheath were 8.7±6.8, 3.7±3.6, and 16.3±13.9 pmol 4MU/min/pig protein respectively, and 12.5±5.0, 40.2±28.5, and 23.2±16.6 pmol 10 4MU/min/pg protein for AGPase promoter, respectively. The level of these promoter activities was about the same or even higher than those obtained with maturing seeds. In contrast, while 10 kDa prolamin promoter showed expression in vegetative tissues, its GUS activity (3.1±1.1, 6.0±2.9, 15 and 2.3±1.0 pmol 4MU/min/pg protein in leaf, stalk, and leaf sheath, respectively) was significantly lower than that observed with maturing seeds. While the PPDK, AGPase, and 10 kDa prolamin genes were expressed constitutively, their expression levels in various tissues varied depending 20 on the gene. All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the 25 references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does 30 not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. In the claims which follow and in the preceding description of the invention, except where the context 35 requires otherwise due to express language or necessary implication, the word "comprise" or variations such as H \soniam\keep\SPECIFICATIONS\P54849 MOA-A0307-AUdes.doc 28/10/04 - 27 "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. The entire disclosure in the complete specification of parent Australian Patent Application No. 2004224913 is by this cross-reference incorporated into the present specification.

Claims (10)

1. An isolated DNA having promoter activity in seeds, 5 wherein the DNA consists of a nucleotide sequence according to SEQ ID NO: 2.

2. A DNA comprising a gene functionally linked downstream of the DNA according to claim 1. 10

3. A vector comprising the DNA according to claim 1 or claim 2.

4. A transformed plant cell comprising the DNA according to 15 claim 2.

5. A transformed plant cell comprising the vector according to claim 3. 20

6. A transformed plant comprising the cell according to claim 4 or claim 5.

7. A reproductive material of the plant according to claim 6. 25

8. The reproductive material according to claim 7, wherein the reproductive material is a seed.

9. A method of expressing a gene in a seed generated from a 30 plant cell, comprising the steps of: (a) introducing the DNA according to claim 2 or the vector according to claim 3 into the plant cell; (b) regenerating a plant from the plant cell; and (c) growing the plant to produce seeds. 35

10. A DNA according to claim 1 or claim 2, vector according to claim 3, transformed plant cell according to claim 4 or 25930_1 (GHMaters) PS4849 AU I - 29 claim 5, transformed plant according to claim 6, reproductive material according to claim 7, or method according to claim 9, substantially as herein described with reference to any one of the examples and/or drawings. 5 2598380_1 (GHMattes) P54849 AU 1