AU776291B2 - Novel gene regulating the synthesis of abscisic acid - Google Patents

Description

1 AR015

DESCRIPTION

NOVEL GENE CONTROLLING ABSCISIC ACID SYNTHESIS TECHNICAL FIELD The present invention relates to a novel gene. More particularly, the present invention relates to a novel gene encoding a protein having a function of controlling abscisic acid synthesis in a plant.

BACKGROUND ART Transposons are mutagenic genes which are ubiquitous in the genomes of animals, yeast, bacteria and plants. Transposons are classified into two categories according to their transposition mechanism. Transposons of class II undergo transposition in the form of DNA without replication. Examples of known class TI transposons include Ac/Ds, Spm/dSpm and Mu elements of maize (Zea mays) (Fedoroff, 1989, Cell 56, 181-191; Fedoroff et al., 1983, Cell 35, 235-242; Schiefelbein et al., 1985, Proc. Natl.

Acad. Sci. USA 82, 4783-4787), and Tam element of Antirrhinum (Antirrhinum majus) (Bonas et al., 1984, EMBO J, 3, 1015-1019). Class II transposons are widely used in gene isolation by means of transposon tagging. Such a technique utilizes a property of transposons. That is, a transposon transposes within a genome and enters a certain gene and, as a result, such a gene is functionally modified, whereby the phenotype controlled by the gene is changed. The affected gene may be isolated by detecting such a phenotype change (Bancroft et al., 1993, The Plant Cell, 5, 631-638; Colasantiet al., 1998, Cell, 93, 593-603; Grayet al., 1997, 2 AR015 Cell, 89, 25-31; Keddie et al., 1998, The Plant Cell, 877-887; Whitham et al., 1994, Cell, 78, 1101-1115).

Transposons of class I are also called retrotransposons. Retrotransposons undergo replicative transposition through RNA as an intermediate. A class I transposon was originally identified and characterized in Drosophila and yeast. A recent study has revealed that retrotransposons are ubiquitous and dominant in plant genomes (Bennetzen, 1996, Trends Microbiolo., 4, 347-353; Voytas, 1996, Science, 274, 737-738). It appears that most retrotransposons are an integratable but non-transposable unit. Recently, it has been reported that some retrotransposons of such a type are activated under stress conditions, such as injury, pathogen attack, and cell culture (Grandbastien, 1998, Trends in Plant Science, 3, 181-187; Wessler, 1996, Curr. Biol. 6, 959-961; Wessler et al., 1995, Curr. Opin. Genet. Devel. 5, 814-821). For example, such activation under stress conditions was found 2u in retrotransposons of tobacco, TntlA and Ttol (Pouteau et al., 1994, Plant 5, 535-542; Takeda et al., 1988, Plant Mol. Biol., 36, 365-376), and a retrotransposon of rice, Tosl7 (Hirochika et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 7783-7788).

The rice retrotransposon Tosl7 is a class I element in a plant which has been extensively studied. Tosl7 was cloned by RT-PCR using degenerate primers which had been prepared based on a conserved amino acid sequence of a reverse transcriptase domains of Tyl-copia group retroelements (Hirochika et al., 1992, Mol. Gen. Genet., 233, 209-216). Tosl7has alengthof 4.3 kbandhas two identical LTRs (long terminal repeats) of 138 bp and a PBS (primer 3 AR015 binding site) which is complementary to the 3' end of the initiator methionine tRNA (Hirochika et al., 1996, supra).

Transcription of Tosl7 is strongly activated by tissue culture, and the copy number of Tosl7 increases with time in culture. This initial copy number in Nipponbare, which is a japonica variety used as a genome research model, is two. In plants regenerated from tissue culture, the copy number is increased to 5 to 30 (Hirochika et al., 1996, supra).

Unlike class II transposons found in yeast and Drosophila, Tosl7 undergoes random transposition in a chromosome and induces mutation in a stable manner. Therefore, Tosl7 provides a useful tool for isolating genes in rice (Hirochika et al., 1997, Plant Mol. Biol. 35, 231-240; 1999, Molecular Biology of Rice, K. Shimamoto Ed., Springer-Verlag, 43- 48).

Abscisic acid is an important plant hormone. It is known that the hormone has various physiological actions in plants, such as acceleration of abscission, induction zu or aormancy, acceleration of aging, inhibition of growth, and stomatal closing (McCarty, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 71-93 (1995), Giraudat, J.

Curr. Opin. Cell Biol. 7: 232-240 (1995); Addicott, F.T.

"Abscisic Acid", Praeger Scientific, New York (1983)). It is also inferred that a reduction in the synthesis of abscisic acid leads to precocious germination and wilting of plants maize) (McCarty, Annu.

Rev. Plant Physiol. Plant Mol. Biol. 46, 71-93 (1995): Addicott, supra). Therefore, it is predicted that if abscisic acid synthesis can be controlled, it will become possible to regulate or confer plant expression, such as inhibition of precocious germination and drought resistance.

4 AR015 Abscisic acid is a type of apocartenoide.

Apocartenoides are compounds produced by oxidative cleavage of carotenoids, which exist widely in nature. Abscisic acid is produced by oxidative cleavage of an epoxycarotenoid.

Oxidative cleavage is the first reaction involved in abscisic acid biosynthesis, and has been proposed to be a rate-determining step in the reaction.

To date, two genes involving in abscisic acid biosynthesis have been isolated from plants. One of the two genes is VP14 isolated from a tobacco (Marvin, E. et al., EMBO 15: 2331-2342, 1996), and the other is VP14 isolated from maize (Tan, B-C et al., Proc. Natl. Acad. Sci.

USA, 94, 12235-12240, 1997; Scheartz, S.H. et al., Science 276, 1872-1874), both of which were isolated by utilizing transposons. It is believed that there are other various genes which are involved in abscisic acid biosynthesis.

There is a demand for further study of enzymes for abscisic acid biosynthesis.

The VP14 gene is a gene encoding abscisic acid synthetase. The product of the gene, which is an 9-cisepoxycarotenoid dioxygenase, cleaves 9-cis-epoxycarotenoids 9'-cis-violaxanthin and 9'-cisneoxanthin) to produce C 2 apoaldehyde and xanthoxin.

Xanthoxin is a precursor of abscisic acid in higher plants.

Expression of abscisic acid is induced by drying (Zeevaart, J.A.D. and Creelman, Annu. Rev. Plant Mol. Biol. 39, 439 (1988)). Further, mutations of the VP14 gene result in a decrease in abscisic acid synthesis. Therefore, it has been proposed that the VP14 gene is a rate-determining enzyme for abscisic acid synthesis (Scheartz, et al., Science 5 AR015 276, 1872-1874, 1997). To date, VP14 has been identified only in maize.

DISCLOSURE OF THE INVENTION The present invention provides a novel plant gene provided using Tosl7.

The inventors have diligently studied and systematically analyzed the phenotypes of plants having a newly transposed Tosl7 copy and a sequence flanking a Tosl7 target site. As a result, the inventors obtained a rice mutant having a precocious germination mutation due to Tosl7 insertion and examined the Tosl7 target site to find a novel gene controlling abscisic acid synthesis, thereby completing the present invention.

The present invention relates to an oligonucleotide encoding a plant gene capable of controllinq abscisic acid synthesis, comprising an oligonucleotide encoding an amino acid sequence from position 1 to 164 of SEQ ID NO. 2 in SEQUENCE LISTING, or an oligonucleotide encoding a second amino acid sequence having one or several amino acid deletions, substitutions, or additions in the amino acid sequence.

Preferably, the oligonucleotide is derived from rice.

Preferably, the oligonucleotide is represented by SEQ ID NO. 1 in SEQUENCE LISTING.

Preferably, the oligonucleotide encodes a gene 19/07 '04 MON 16:03 FAX 61 3 9288 1567 FREEHILLS CARTER SMITH B 004513879 6 capable of regulating expression of a VP14-like gene.

According to one aspect, the present invention relates to a vector comprising the above-described oligonucleotide, in which the oligonucleotide is operatively linked to a control sequence.

Preferably, the present invention relates to the PBI101-Hm-VS1 vector.

According to one aspect, the present invention relates to a plant transformed using the above-described vector.

Further, the present invention relates to a method for controlling abscisic acid synthesis in a plant, comprising the step of introducing into a plant the abovedescribed oligonucleotide.

It will be understood that the term "comprises" or its grammatical variants as used herein is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

BRIEF DESCRIPTION OF THE DRAWINGS 15 Figure 1 shows photographs indicating phenotypes of precocious germination mutants. The left phenotype In the photograph shows a precocious germination mutant (hetero) line and the right phenotype in the photograph shows a normal line.

Figure 2 is a diagram showing linkage analysis between precocious 20 germination mutation and Tosl7. In the figure, mutation indicates samples derived from precociously germinated individuals and WT indicates samples derived from normal plants. Lanes 1 to 10 indicate the precocious germination mutants and lanes 11 to 19 0006 COMS ID No: SBMI-00833390 Received by IP Australia: Time 16:10 Date 2004-07-19 7 AR015 indicate the normal lines. An arrow indicates homo bands of Tosl7. In the precocious germination mutants, the Tosl7 bands are homozygous.

Figure 3 shows the sequence of a TAIL-PCR product of a sequence in the vicinity of a Tosl7 insertion site (SEQ ID NO. 3).

Figure 4 shows an about 6.0 kb sequence in the vicinity of the Tosl7 insertion site. The sequence was obtained by screening in Example 3 (SEQ ID NO. 4).

Figure 5 is a diagram showing the nucleotide sequence (SEQ ID NO. 1) of an oligonucleotide according to the present invention and the corresponding amino acid sequence. The base sequence and amino acid sequence of cDNA, which was obtained using RACE and a primer extension method based on the sequence in the vicinity of the Tosl7 insertion site obtained using TAIL-PCR, was determined. In the figure, zu a sequence of nucleotides from position 383 to 877 encodes an ORF consisting of 164 amino acids (SEQ ID NO. In the figure, bold characters indicate the nuclear localization signal (bold character), and an underline indicates a glycine-rich amino acid sequence. A downward arrow indicates a Tosl7 insertion site in the mutant line A0150 (position 274).

Figure 6 is a schematic diagram showing the T-DNA plasmid used in a complementation test. A 2390 bp fragment obtained by BglII digestion, which includes an ORF into which Tosl7 is inserted, was inserted into a BamHI site of pBI101-Hm.

8 AR015 Figure 7 shows a portion of the sequence of a rice VP14-like gene (SEQ ID NO. This sequence was used as a probe to analyze expression of the VP14 gene.

Figure 8 is a diagram showing expression of a VP14-like gene in a vsl mutant strain. Expression of the VP14-like rice gene was analyzed by northern blotting. WT indicates a sample from a normal line and vsl indicates a sample from a precocious germination mutant. In "low humidity" indicated in the figure, indicates growth at normal humidity and indicates growth at low humidity. An arrow indicates the band position of the RNA which disappeared in the precocious germination mutant vsl.

Figure 9 is a diagram showing expression analysis of a VS1 gene using a primer extension method. RNA prepared from rices grown at high humidity (control) or low humidity (low humidity) was used to perform primer extension.

Induction due to drying was not observed.

2U BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a novel plant gene provided using Tosl7, a vector including the novel gene, a plant transformed using the novel gene, and a method for improving a plant comprising the step of transforming a plant using the novel gene.

The present invention provides an oligonucleotide encoding a plant gene capable of controlling abscisic acid synthesis. The term "capable of controlling abscisic acid synthesis" as used herein refers to prevention or enhancement of a gene involved in abscisic acid biosynthesis 19/07 '04 MON 16:03 FAX 61 3 9288 1567 FREEHILLS CARTER SMITH B 004513879 9 in a plant. The term "plant" comprises both monocotyledons and dicotyledons.

Representative examples of an oligonucleotide according to the present invention, which encodes a plant gene capable of controlling absclsic acid synthesis, include an oligonucleotide encoding an amino acid sequence from position 1 to 164 of SEQ ID NO. 2 in SEQUENCE LISTING, and an oligonucleotide encoding an amino acid sequence having one or several amino acid deletions, substitutions, or additions in that amino acid sequence.

The oligonucleotide of the present invention, which encodes a plant gene capable of abscisic acid synthesis comprises an oligonucleotide having at least an 80% sequence identity with a sequence encoding an amino acid sequence from position 1 to 164 of SEQ ID NO. 2 in SEQUENCE LISTING, preferably at least an 85% sequence identity, more preferably at least a 90% sequence identity, even more preferably at least a 95% sequence identity, and most preferably at least a 99% sequence identity, as long as the oligonucleotide is capable of controlling abscisic acid synthesis in a plant. The term "sequence identity" refers to that two oligonucleotides of interest have the same sequence. The level of sequence identity between two oligonucleotide sequences are optimally aligned; sequence positions having the same nucleotide base (eg. A, T, C, G, U, or I) between the sequences are counted and the total number of matching positions is 20 called the matching position number; and the matching position number is divided by the total number of bases of the two oligonucleotides and the result is multiplied by 100. The sequence identity may be, for *o

OD

1007 COMS ID No: SBMI-00833390 Received by IP Australia: Time 16:10 Date 2004-07-19 10 AR015 example, calculated using the following sequence analyzing tools: Unix-based GCG Wisconsin Package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive Madison, Wisconsin, USA53711; Rice, (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 1RQ, England); the ExPASy World Wide Web Server for Molecular Biology (Geneva University Hospital and University of Geneva, Geneva, Switzerland); and MacVector 6.0 (Teijin System Technology).

Herein, peptide variation includes amino acid addition, deletion, substitution,.or modification. Amino acid addition refers to addition of one or more amino acids to an original peptide chain. Amino acid deletion refers to deletion of one or more amino acids from an original peptide. Amino acid substitution refers to substitution of one or more amino acids in an original peptide. Amino acid or peptide modification includes, but is not limited to, zu single or multiple, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydration, acylation with an acetyl group or aliphatic group), and the like. An amino acid to substitute or be added may be a naturally-occurring amino acid or alternatively a non-naturally-occurring amino acid. A naturally-occurring amino acid is preferable. In another embodiment of the present invention, the peptide and its variant of the present invention may be in the form of a salt of a peptide, such as ammonium salt (including alkylor aryl-ammonium salt), sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate, bicarbonate, benzonate, sulfonate, thiosulphonate, mesylate (methylsulfonate), 11 AR015 ethylsulfonate, and benzenesulfonate.

(Variant having an equivalent biological function) Hereinafter, biological-functionally equivalent substitution in a peptide will be briefly described. When biological-functionally equivalent substitution is performed in the peptide of the present invention or the DNA segment encoding the peptide, a protein or a functional molecule encoding a peptide, which still has a desired property, is obtained. Hereinafter, a change in amino acid of protein for producing an equivalent or an improved second generated molecule will be discussed. Amino acid substitution may be conducted by chemical synthesis or by using a genetic engineering technique in which a codon encoding an amino acid in a DNA sequence is changed. The present invention is not limited to these.

A certain amino acid may be substituted with another amino acid without explicitly reducing or eliminating zu interactive bonding ability, for example, in a protein structure having a cationic region or a binding site of a substrate molecule. Biological functional activity of a certain protein defines its interactive ability. Therefore, even when substitution of a specific amino acid sequence is performed in a protein sequence and DNA coding a sequence therefor, a protein still having an original property can be obtained. Therefore, it is intended by the inventors that various modifications may be performed in the disclosed peptide sequence or a DNA sequence encoding the peptide without explicitly reducing or eliminating the biological utility or activity thereof.

When such variation is produced, the hydrophobic 12 AR015 index of an amino acid may be taken into consideration. The importance of the hydrophobic amino acid index of a protein is taken into consideration in the art when an attempt is made to confer an interactive biological function to the protein (Kyte, J and Doolittle, J. Mol. Biol. 157(1): 105-132, 1982). It is recognized that the hydrophobic properties of its amino acid contributes to the secondary structure of a produced protein. The secondary structure defines interaction between the protein and other molecules a plasma membrane molecule, an enzyme, a substrate, a receptor, DNA, an antibody, and an antigen). A hydrophobic index is assigned to each amino acid based on the hydrophobicity and electric charge of the amino acid: isoleucine valine leucine phenylalanine cysteine/cystine methionine alanine glycine threonine serine tryptophan tyrosine proline histidine glutamic acid glutamine aspartic acid asparagine lysine and arginine (Kyte, J and Doolittle, R.F., 1982).

It is well known in the art that even when a certain amino acid is substituted with another amino acid having a similar hydrophobic index or value, a protein can still have a similar biological activity a protein having an equivalent biological function). In such variation, amino acid substitution within ±2 of hydrophobic index is preferable, amino acid substitution within ±1 of hydrophobic index is more preferable, and amino acid substitution within of hydrophobic index is even more preferable. It is understood in the art that amino acid substitution is effectively performed based on hydrophilicity. US Patent 13 AR015 No. 4,554,101 describes that the greatest local average hydrophilicity of a protein dominated by the hydrophilicity of a flanking amino acid relates to the biological properties of the protein. As described in US Patent No. 4,554,101, the following hydrophilic indices are assigned to amino acid residues: arginine lysine aspartic acid glutamic acid serine asparagine glutamine glycine(0); threonine proline alanine histidine cysteine methionine valine leucine isoleucine tyrosine phenylalanine tryptophan It is to be understood that an amino acid may be substituted with another amino acid having a similar hydrophilic index so that a protein remains a biological equivalent (particularly, an immunologically equivalent) protein. In such variation, amino acid substitution within ±2 of hydrophilic index is preferable, amino acid substitution within ±1 of hydrophilic index is more preferable, and amino acid substitution within zu or nyarophilic index is even more preferable.

Herein, "conservative substitution" refers to amino acid substitution in which an original amino acid has a hydrophilic index or/and a hydrophobic index similar to those of a substituting amino acid. A preferable conservative substitution is amino acid substitution within ±2 of hydrophilic index or/and hydrophobic index, more preferably ±1 of hydrophilic index or/and hydrophobic index, and even more preferably ±0.5 of hydrophilic index or/and hydrophobic index.

As briefly described above, amino acid substitution is performed based on the above-described indices.

14 AR015 Examples of illustrative substitution based on the consideration of the above-described various properties, include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine, which are well known to those skilled in the art. The present invention is not limited to these.

A sequence of nucleotides may be changed by utilizing a degenerate sequence of a genetic code. Such a degenerate sequence is well known to those skilled in the art. It is apparent to those skilled in the art that degenerate sequences encode identical amino acids.

The term "control sequence" as used herein refers to a DNA sequence, such as a functional promoter and any other relevant transcriptional element an enhancer, a CCAAT box, a TATA box, and SPI site).

zu The term "operatively linked" as used herein refers to that an oligonucleotide is linked to a regulatory element which regulates gene expression, such as a promoter and an enhancer, in such a manner that a gene encoded by the oligonucleotide can be expressed in a host cell.

It is well known to those skilled in the art that the types and kinds of a control sequence vary depending on the host cell. Examples of control sequences well known to those skilled in the art include the CaMV35S promoter and the nopaline synthase promoter. A gene may be introduced into a plant body using a known method. Examples of such known methods include a method mediated by Agrobacterium or a method of directly introducing a gene 15 AR015 into a cell. An example of a method mediated by Agrobacterium is Nagel et al.'s method (Micribiol. Lett.,67,325(1990)). In this method, for example, an expression vector is first introduced into Agrobacterium using electroporation, and the transformed Agrobacterium is then introduced into a plant cell in accordance with a method described in Plant Molecular Biology Manual Gelvin et al., Academic Press Publishers). Examples of known methods for directly introducing a gene into a cell include the electroporation method and the gene gun method.

A gene-introduced cell is selected with reference to drug resistance, such as hygromycin resistance, and thereafter, is regenerated into a plant body using a commonly used method.

Generally, names and laboratory protocols as used herein are well known in the art. Recombinant techniques, polynucleotide synthesis, and microorganism culture and transformation electroporation) are used within standard technologies. These techniques and protocols are described in various general publications in the art and in this specification (generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, These publications are herein incorporated by reference.

The oligonucleotide of the present invention is representatively obtained by a method described herein.

Alternatively, the oligonucleotide of the present invention may be obtained by chemical synthesis based on the sequence disclosed herein. For example, the oligonucleotide of the 16 AR015 present invention may be synthesized using an oligonucleotide synthesizer (manufactured by Applied Bio Systems) in accordance with the specification provided by the manufacturer.

PCR amplification methods are well known in the art (PCR Technology: Principles and Applications for DNA Amplification, edited by HA Erlich, Freeman Press, New York, NY (1992); PCR Protocols: A Guide to Methods and Applications, edited by Innis, Gelfland, Snisky, and White, Academic Press, San Diego, CA (1990); Mattila et al. (1991) Nucleic Acids Res. 19: 4967; Eckert, K.A. and Kunkel, T.A. (1991) PCR Methods and Applications 1: 17; PCR, McPherson, Quirkes, and Taylor, IRL Press, Oxford). These publications are herein incorporated by reference.

EXAMPLES

Hereinafter, the present invention will be zu described by way of examples. The present invention is illustrated by the examples described below and is not limited to the examples.

Chemicals and kits used in the following Examples are available from Iwai Chemicals Company unless otherwise specified.

(Example 1) Activation of Tosl7 by culture and characterization of a resultant mutant A fully mature seed of Nipponbare, which is a japonica variety, was used as a starting material to conduct callus initiation culture and cell suspension culture as described previously (Hirochika et al., 1996, supra).

17 AR015 Tosl7 was activated in accordance with Otsuki's method (1990) (Rice-protoplast culture, Agriculture, Forestry and Fisheries Technical Information Society). Briefly, a fully mature rice seed was cultured in MS medium containing 2,4-dichlorophenoxyacetic acid (Otsuki (1990), supra) (25 0 C, one month) to induce callus. The resultant callus was cultured in N6 liquid medium containing 2,4-D (Otsuki (1990), supra) for 5 months and was then transferred to regeneration medium (Otsuki (1990), supra), thereby obtaining regenerated rice (a first generation (RI) plant).

A second generation of the resultant regenerated rice 500 line was disseminated in a field. The R2 group was germinated and, one week after the germination, seeds were produced. After maturation, each line was examined in detail and precocious germination was found in one line.

This strain is designated as VS1. To confirm the precocious germination property, a panicle whose precocious germination had been confirmed was incubated at a humidity of 100% and at a temperature of 25 0 C. As shown in Figure 1, iL was found that in tne vsi strain, about one quarter of the seeds on a panicle were germinated. In a normal line, no germination was observed under the same conditions. Such a result indicates that the VS1 strain has a precocious germination mutation. It was observed that on one panicle, the ratio of the number of seeds having precocious germination to the number of seeds having no precocious germination was 1:3. This indicates that the mutation in precocious germination is recessive mutation.

(Example 2) Analysis of a precocious germination mutant As described above, the gene analysis revealed that the mutation in precocious germination was a recessive 18 AR015 mutation. An individual plant which had had precocious germination was further grown, and wilt as observed for a plant lacking in moisture, was observed. The phenomena of the precocious germination mutation and the wilt suggested a reduction in an abscisic acid content (McCarty, Annu.

Rev. Plant Physiol. Plant Mol. Biol. 46, 71-93 (1995)).

Thereafter, the abscisic acid content of a normal line and a mutant line were measured. The measurement was conducted under a high humidity condition(i.e., in a sealed test tube) and then under a low humidity condition six hours after the cap of a test tube was removed). The measurement of an abscisic acid content was conducted using an abscisic acid immunoassay detection kit (Sigma). The result is shown in Table 1. In this measurement, the abscisic acid content of a young seedling was measured for each line. Whereas the abscisic acid content of a control line was increased due to drying, the abscisic acid content of the precocious germination mutant was not increased under thei sani uiidliunis. As a result, it was concluded that the isolated precocious germination mutant of this example had an abnormality in abscisic acid synthesis.

Table 1: Abscisic acid content (nmol/g fresh weight) Growth Normal Mutant Ratio of abscisic acid condition content (Normal/mutant) High humidity 77.5 18.3 4.2 (sealed test tube) Low humidity 5025 28.5 176 (six hours after the cap of the test tube was removed) 19 AR015 (Example 3) Isolation and structural analysis of a causative gene for the precocious germination mutant To identify and isolate a causative gene for the precocious germination mutant, linkage analysis with the Tosl7 gene was conducted using a population in which precocious germination mutants segregate. The linkage analysis was conducted in accordance with a typical method well known to in the art. Specifically, precocious germination mutants and normals were distinguished from one another by the naked eye, and DNA was extracted from each group using a CTAB method (Murray, G.C. and Thompson, W.F., Nucl. Acids Res. 8: 4321-4325 (1980)). The resultant DNA was digested using a restriction enzyme, followed by 0.7% agarose gel electrophoresis. Thereafter, the DNA was adsorbed to a nylon membrane (Schleicher Schnell). Tosl7 was digested using restriction enzymes, XbaI and BamHI, and the resultant DNA fragments were labeled with 32 P-dCTP, followed by Southern blotting. The result is shown in Figure 2. Lanes 1 to 10 indicate DNA obtained from the precocious germination mutants, and lanes 11 to 19 indicate DNA obtained from the normals. Bands of Tosl7 indicated by an arrow were observed for all of the precocious germination mutants. Although the Tosl7 bands were observed for a part of the normals, the darkness of the bands were weak (the amounts of DNA loaded on the lanes are not uniform. To avoid this problem, the darkness of a band was compared with that of a band immediately below the arrow). As a result, the Tosl7 band indicated by the arrow was homozygous in the precocious germination mutants, and heterozygous in the normals. This indicates that the Tosl7 bands indicated by the arrow and the precocious germination mutation have complete linkage. Similar analysis was applied to a segregated population consisting of 60 individuals to 20 AR015 confirm the above-described results. These results suggest that Tosl7 indicated by the arrow in Figure 2 induced a precocious germination mutation.

After the agarose electrophoresis of the mutant DNA treated with the above-described method, a portion of the gel corresponding to a region around a size indicated by the arrow in Figure 2 was dissected, followed by DNA extraction. The DNA extraction was conducted using QIAquick Gel Extraction Kit (Qiagen). Using this DNA as a template, TAIL-PCR (Liu, and Whiffier, Genomics 674-681 (1995)) was conducted to clone a sequence flanking Tosl7, a portion of a causative gene for the precocious germination mutation. TAIL-PCR was conducted in accordance with a method described in Miyao et al., Plant Biotechnology 15: 253-256 (1998). The resultant sequence is shown in Figure 3 (SEQ ID NO. This sequence was used as a probe to conduct screening of a rice genomic library (obtained from Makoto Takano of the National Institute of 2G AyLub'uluyicdal Resources). Tne screening was conducted in accordance with Sambrook et al. (supra) By this screening, an about 6 kbp sequence in the vicinity of a Tosl7 insertion portion of the resultant clone was determined. The sequence is shown in Figure 4 (SEQ ID NO. RACE primer extension (Frohman, M.A. et al., Proc. Natl. Acad. Sci. USA 8998-9002 (1988)) was conducted based on the base sequence of the gene to determine the base sequence of cDNA encoded in the Tosl7 insertion site (Figure 5, SEQ ID NO. This cDNA has a full length of 894 bp (Figure 5, SEQ ID NO. 1) and encodes an ORF consisting of 164 amino acids (Figure nucleotides from position 383 to 877; SEQ ID NO. This ORF includes a glycine-rich motif (underlined in Figure and a nuclear localization signal (bold characters in 21 AR015 Figure The Tosl7 insertion site is indicated by an arrow (Figure (Example 4) Complementation test by gene introduction 1. Construction of a complementary vector and transformation of a precocious germination mutant using Agrobacterium tumefaciens In Example 3, a 2390 bp BglII fragment including a full-length open reading frame of a gene was integrated into the pBI101-Hm vector (obtained from Kenzo Nakamura of the School of Agricultural Sciences of Nagoya University) (Figure The resultant vector was designated as pBI101-Hm-VS1. Using this recombinant vector, an Agrobacterium tumefaciens EHA strain was transformed by electroporation in a selective medium containing 50 mg/l of kanamycin and hygromycin (obtained from Kenzo Nakamura of the School of Agricultural Sciences of Nagoya University).

2G The L tsultant Agrobacrerlum strain was cryopreserved until future use.

The seed of the precocious germination mutant was sterilized with 1% sodium hypochlorite, and washed with sterilized distilled water five times. The seed was placed on N6 medium containing 3% sucrose, 0.3 g/l casein hydrolysate, 2.8 g/l proline, and2.0 mg/1 2,4-D, which was solidified with 5.0 g/l gel gum (Chu et al., 1975, Sci.

Sinica, 18, 659-668). ThepHof the medium hadbeen adjusted to pH 5.8 before being autoclaved. The seed was grown in the dark at 28 0 C for three weeks, thereby obtaining a small callus having a size of about 2 mm. The small callus was transferred to a callus inducing medium and cultured in the 22 AR015 light for four days. The resultant callus was subjected to Agrobacterium infection.

The above-described cryopreserved Agrobacterium was cultured in the dark at 22 0 C for three days in AB medium containing 50 mg/l kanamycin and 50 mg/l hygromycin, which was adjusted to pH 7.2 and solidified with 15 g/l agar (Chilton et al., 1974, Proc. Natl. Acad. Sci. USA, 71, 3672-3676). Agrobacterium bacteria were collected and suspended in liquid AAM medium (Hiei et al., 1994, Plant 6, 271-282) containing 20 xg of acetosyringone (Hiei, 1994, Plants, 6, 271-282). The resultant suspension was co-incubated with the above-described induced callus in the dark at 22 0 C for seven days so that the callus was infected with Agrobacterium. The resultant callus was washed five times with a liquid callus inducing medium containing 500 mg/l of carbenicillin, followed by drying on a sterilized Whatman No. 1 filter paper. The callus was cultured for three weeks on a callus inducing medium containing 50 mg/l hygromycin to select a hygromycinresistance callus, which was then transferred to a regeneration medium containing MS basal medium (pH 5.8) (Murashige and Skoog, 1962, Physiol. Plant., 15, 473-497) containing 30 g/l sucrose, 30 g/l sorbitol, 2 g/l casein hydrolysate, 2g/l kinetin, 500 mg/l carbenicillin, 50 mg/l hygromycin and 5 /1 gel gum.

The transformant was easily regenerated in a regeneration medium containing hygromycin without auxin or NAA, and transferred to soil. The transformant grew normally without exhibiting a wilt phenomenon which is observed in the precocious germination mutant. This result leads to the conclusion that the mutation of the gene 23 AR015 isolated in Example 3 is responsible for the precocious germination mutation. This gene was designated as the VS1 gene.

(Example 5) Isolation of a VP14-like gene from rice As described above, the VP14 gene is believed to control the rate-determining step in abscisic acid synthesis. To date, this gene has been isolated only from maize. Therefore, in order to isolate the VP14 gene from rice, the VP14 gene isolated from maize and the VP14-like gene isolated from Arabidopsis were compared with each other with respect to a conserved sequence using the genetic analysis software MacVector 6.0 (Teijin system technology) to identify the conserved sequence. A 5'-end primer and a 3'-end primer corresponding to two portions of the conserved sequence were prepared. Using these two primers, the rice VP14 gene was amplified. The template used in this amplification was prepared as follows: a rice leaf was subjected to drying (a leaf was dissected and left at room temperature for three hours); RNA was extracted from the leaf; the RNA was treated with reverse transcriptase (TaKaRa) to obtain a cDNA sample. By this PCR, an about kbp product was obtained. The sequence of this product was partially determined to reveal a high identity between the rice VP14 and the maize VP14 The partial sequence is shown in Figure 7 (SEQ ID NO. Thereafter, this product was used as a probe to conduct expression analysis of the rice VP14-like gene. DNA extracted from normal rice was used as a sample to conduct Southern blotting analysis.

Five bands were obtained, suggesting that at least five VP14-like genes are present in rice.

24 AR015 (Example 6) Functional analysis of the VS1 gene Thereafter, whether or not the VS1 gene can regulate expression of the VP14-like gene was studied. In Example 6, initially, the rice VP14-like gene was isolated and was used as a probe to conduct northern blotting analysis. mRNA was isolated from normal line rice and VS1-mutant rice, which had been grown at high and low humidities, using ISOGEN (Nippon gene). This mRNA was isolated using agarose gel and was absorbed on a nylon membrane (Schleicher Schnell). Thereafter, the mRNA was subjected to northern blotting using a 32 P-dCTP labeled VP14 probe (Sambrook et al., supra). X-ray film was obtained from Kodak. The result is shown in Figure 5. As is seen from Figure 5, in the case of high humidity (indicated by in Figure 8), expression of VP14 was not found in both the normal line (WT) and the mutant (vsl). In the case of low humidity two bands having different sizes (2.0 kb and 2.5 kb) were detected in the normal line while a band disappeared in the vsl mutant (vsl). Therefore, VS1 is considered to be a gene causing down regulation of the VP14 or VP14-like gene.

Thereafter, expression of the VS1 gene was analyzed using a primer extension method. The primer used in the primer extension method was TTCGAGTTTTTGTGTTAGGG (SEQ ID NO. 6) corresponding to the VS1 gene, whose 5' terminus was labeled with "P-y-ATP using polynucleotidekinase (TOYOBO).

RNA was prepared from a leaf subjected to dry induction treatment or an untreated leaf using ISOGEN (Nippon gene), This RNA which was used as a template was placed on a sequencing gel (Sambrook et al., supra), followed by electrophoresis and exposure of X-ray film (Kodak). The details of the experiment were in accordance with Hirochika 25 AR015 (EMBO 12, 2521-2528, 1993). The result is shown in Figure 9. As is seen from Figure 9, it is apparent that VS1 is expressed similarly at low and high humidities.

The above-described Examples illustrate and describe various aspects of the present invention, and how the specific oligonucleotide of the present invention is prepared and utilized, but are not intended to limit the scope of the present invention.

INDUSTRIAL APPLICABILITY A novel oligonucleotide capable of controlling abscisic acid synthesis, which is applicable to plant breeding, is provided. When the oligonucleotide is introduced into a plant to control abscisic acid synthesis, a plant having useful traits, such as drought resistance and suppressed precocious germination, is provided.

EDITORIAL NOTE APPLICATION NUMBER 37355/99 The following sequence listing pages 1 17 are part of the description. The claims pages follow on pages 26 28 WO 00171727 WO 0071727PCT/JP99/02734 (010 National Institute of Agrobiological Resources, Ministry of Agricu Iture, Foretry. and Fisheries, and Bio-oriented Technology Research Adva ncement Institution (120> A novel gene controlling abscidic acid synthesis (130 F5-99PCT289/AROL5PCT (140> (141> (160> 6 (170 Patentln Ver. (21 0> (211> (21 2> (213> 1 894

DNA

Oryza sativa (220> (221> (222>

CDS

(383) (877) (220> 1/17 WO 00/71727 PCT/JP99/02734 <221> CDS <222> (66) <220> <221> CDS <222> (134) <220> <221> miscfeature <222> (274) <223> Tosl7 insertion site <220> 929.11 micer foaturo <222> (698) <223> glycine rich <220> <221> misc_feature <222> (624)..(662) <223> nucleus localization signal <220> <221> CDS <222> (242) 2/17 WO 00/71727 WO 0071727PCT/JP99/02734 (220> <221> CDS (222> (196) 220> (221> CDS (222> (255). (275) (220 (221> CDS (222> (353) (400> 1 aagattcttg atacagggaa gaaatctgta ccttagagma taan~a ggprttcrtut RA ttttgagctt aaccatgacc aacatgagaa citcactctg gacccaaagt gcatcagact 120 caaaactggt ttgaactata acaagcttgt tttcgggggt gttgagaata ctcccactgg 180 tctatggcct gtlagaaaag catttgacaa cttctggagg catgctggtg gtgaaaacat 240 aaatatatca gaagatgttg caatatttgc Igtgattata tctgaggcag caagattacg 300 cccggtgcat gactacatca aagattcttt cctttctgag aatccgggtt taagcaaatt 360 ggggaaacat ccctccaacc cc atg tat gtg aag aag tac aag aaa att tct* 412 3/17 WO OV71727 WO 0071727PCTIJP99/02734 Met Tyr Val Lys Lys Tyr Lys Lys Ilie Ser gct cac ata atg Ala His Ilie Met ga a Glu aat gtt gac ctc Asn Val Asp Leu atg aaa cag ggc cia Met Lys Gin Gly Leu 20 gag cca Glu Pro tcg gct Ser Ala 460 508 aaa cca tic Lys Pro Phe cat gag aaa cit His Glu Lys Leu ala gat tct atg Ilie Asp Ser Met ctt agt Leu Ser gic agg atc tta Val Arg Ilie Leu agg gat gca tl Arg Asp Ala Phe aac As n tca gga tta 556 Ser Gly Leu lit gag Phe Giu cat gag aaa cci.

His Clu Lys Pro ccc Pro 65 aag ccc aic tt Lys Pro Ilie Phe ga a Glu aac aca ttg gat Asn Thr Leu Asp 604 cgt Arg cag Gin gag gil aag Ciu Val Lys cac His 80 aaa aga aag cag Lys Arg Lys Gin aag cgg aaa aag Lys Arg Lys Lys aaa 652 Lys tcg 700 Se r gga aag aag gga gga Gly Lys Lys Gly Gly ggg gga tca gga ggg Gly Gly'Ser Gly Gly 100 4/17 gga ggt Gly Gly gga ggl gga Gly Gly Giy 105 WO 00171727 WO 0071727PCr/JP99/02734 att ggg gaa.

Ilie Gly Gin gaa gta cca. aag gga Gin Val Pro Lys Gly 110 gga gga gil cga gaa Gly Gly Val Arg Giu 115 aga ctg aag Arg Leu Lys 120 gga gca Gly Ala tac aag cat git Tyr Lys His Val cit tta gtc c Lea Leu Val Lea atg Met 135 ggc tca. ltg 796 Gly Ser Lea cat alt tta. tgi tit acc His Ilie Lea Cys Phe Thr 140 Lea His Phe Asp Val Leu 155 160 tca.

Ser 145 gaa ala, tic tta Gi Ilie Phe Leu gig acg tgc ala Val Thr Cys Ilie 844 Lea Ilie Tyr Ilie t ct er ft- t r f f f rT j f QaA (210> 2 (211> 164 (212> PRT (213> Oryza saliva (400> 2 Met Tyr Val Lys Lys Tyr Lys Lys Ilie 1 5 Ser Ala His Ilie Met Gin Asn 10 1 5/17 wo oon1727 WO 0071727PCT/JP99/02734 Val Asp Leu Met Lys Gin Gly Leu Lys Leu Val Ilie Asp Ser Met Clu 40 Glu Pro Lys Pro Phe Lys His Giu Val Arg Ilie Ser Ala Leu Ser Ilie Leu Phe Arg Asp Ala Phe Asn Pro Lys Pro Ilie Phe Glu Asn 70 Lys Arg Lys Gin Asp Lys Arg Ser Gly Leu Phe His Glu Lys Pro Thr Leu Asp Lys Lys Lys Arg 75 Gin Giu Vai Lys His Giy Lys Lys Gly Gly Giy

I

Gly Ser Gly Lys Gly Gly 115 Gly Gly 100 Gly Gly Giy Gly Ser 105 Ilie Gly Glu Clu Val Pro 110 Tyr Lys His Gly Vai Arg Glu Arg Leu Lys Gly Ala 120 Val Ser Leu Leu Val Leu 130 Met i135 Gly Ser Leu His Ilie 140 Leu Cys Phe Thr Ser 145 GuI lie Phe Leu Leu 150 Val Thr Cys Ilie Leu 155 His Phe Asp Val Lea 160 6/17 WO 00n 1727 WO 0071727PCT/JP99/02734 Leu Ilie Tyr Ilie (210 3 330 (212> DNA (213> Oryza saliva (400> 3 at tatatctg aggcagcaag attacgcccg gtgcatgaci acatcaaaga ttctttcctt rInr 2a Iz r raaattf I2n fa 2nt taacra 2r ~dt pr j ra P -prP, ft a f$jtTI,T~ I )(I C O- ooaagtacaaga aaatttctgc tcacataatg gaaaatgttg acctcatgaa acagggccta 180 gagccaaaac catlcaagca tgagaaactt gtcatagatt ctatggaatc ggctcttagt 240 attgtcagga tcttattcag ggatgcattt aactcaggat tat ttgagca tgagaaacct 300 cccaagccca tctttgaaaa cacattggat 330 (210> 4 (211> 6032 <212> DNA 7/17 WO 00171727 WO 0071727PCTIJP99/02734 (213> Oryza sat iva (400> 4 gtggtttttn aacctcgcag cgagctcccc ggtatgctca tcgaactttt ctcacaccgc cccaacagca agacgctcgt cgaggtcgtc ccggtcatgg tggaggagct ctacatccga 120 aaggtagacg acgaggcgct ccgctacaag gccalggccg ccgccgtccc cgccaacgtc 180 gtcgagtcta gcattgatcc ggataccacc gtggtgaaca aagaaggcca cgtcctcacg 240 tccaagtaat ccatctacga cttcatccac atgctcaagt gcttcattcc tcacaagctc 300 aacctcagca tcatccgtgt cctgtcgcgc tcaccec~ma cgran Rte rtrwrrrCe7 RG tggtctcggt cgtcagggag aaccaccagg gccactccat cgtggagctc ccggacggaa 420 gcgcttgatg tcgccgcgac gaggctgctc gtcacctgtc aatgcacatg gggcacacca 480 tcgccgagag gctctgcaag acgcagagag ccgccggcgc cggcgaaaga ggcagacgag 540 gggcggaggg ccgcgctgct gcaggcggtt tgcggtcgtg ggcggtggca tgtcggcggc 600 gtcgggctcc gcgtgcgggt gtttgctccg ggaccatagt gcgtcgggcg gcgtcgtcgt 660 2 ctcgccgcgg actggtgatg agcagatgca acagctggag gtcggccggc gtagagccgc 720 8/17 WO 00/71727 WO 0071727PCT/JP99/02734 cgccgccggt ggcagctcct ccctcgccgg ccgctgcccg cgcccgcaac gcccatctgt 780 tctctgcagc cgccgtggag aggggaggag agaggaagag agatggtgag gggaggggaa 840 gaagtggctg acaigtggga cccacgtggg tcccacgctg agtcatctgc catalcagac 900 aaaaccggag tcaaaaccgt Icagggatac tagittatic tggttttata agttgaggga 960 cgcattgtat ctggtttigt ggttcaagga cgattcaaca agttgaggga cctccggtgt 1020 act ttttccc taaagttatt atgtcacatt tgagtacgga cggcccacta atctcgcgat 1080 gCCLLgaaaaL. gagC~g gaC5aLL..I CCCa*C. aag.L.bIL.IL UU6L.UIUL 1 cgcagcggca ttcttccgag ccccctcttc gccatttcgc CtgCtccgtg atcgccgtcg 1200 cgcaccattt cgccgccctg ccccgcccca gcaacgattg cgtgcgggat ctcaaaccct 1260 agggttccct cggcgctgac gggagcaacc tctcgccgcc gacgccgcta tcgcccggca 1320 gattcgttga cgatggggcg ctcacggccc tcgcctccgc ctcctcacgc gagcgaggta 1380 atcgtctccc cctttaaccg ttcgttgttg tttgtgttca gccgtggagc ggagalgacg 1440 cggtatcgaa taccaattgg cttagatgcg attttggaaa cagaacttac gtcattccgc 1500 9/17 WO OOf7l727 WO 0071727PCT/JP99/02734 ggcagaattt tactttatcc tttactttat ccacagcgga ttcatgatac agagttagcg 1560 ataatatgtt gattglgcat lattagagat ttttgtttcc gattatgtg cagcgctagt 1620 aagattccct gagcatgtgc aacttatgat ccacctgccg aactgatttt gtcttgctaa 1680 Itagtgcata aatttcctag gtaggacaac ctattgaatt cgtltcagtc gttcctatta 1740 attgtattat ggtgattgtt ttttittttt gaaacctaga tcttgctggt tttatattaa 1800 gagagggaaa tgccatacaa aattatggtg attggtttac ttggatatga tttccaataa 1860 actattgaca gagtattaga ctgtatattg gatccaaaaa tgtgagaaca gaatatggtt 1980 cagcagtcca tagttitgct gctgttgctt alaatcttgc aaggttatca gccagattgt 2040 tacagttaac ttgcaaggct atatatcagc cacattgtta ctctgaactt ggaagtttaa 2100 tatgcctgga atctttcaca ggattgggag Ittggatata ccctaatgaa ctacaactga 2160 ccattaagca gcactgcctg ttgaaaagtt gctagtttac tcattlaact tccatatatg 2220 aatgtacaca cctgtaattt tgtttagcag atcaggaata gggggctcac ctcaaagcat 2280 10/17 WO 00n 1727 WO 0071727PCT/JP99/02734 gtgaccacaa tttgccttga tggaactgac aaggaagcgt tagcaagcta tcttagtggc 2340 attgaagagg taattaattg g(catatgaa gatggaatta aggiggatat ctcagatatt 2400 tgtggacagt gccatgaggt aticactaca agagctggag gctcaatgga agtggaattc 2460 gtgtacatgg gtcggtctat aagaltcltg atacagggaa gaaatcigta ccttagagga 2520 lggaaagcaa gggattgtgt ttttgagctt aaccatgacc aacatgagaa cttcactctg 2580 gacccaaagt gcatcagact caaaactggt ttgaactata acaagcttgt tttcgggggt 2640 gt toauaat~ a trreqr I g lr t nt crrr! aft uammna pn f f rn n^ f^ "-n catgctggtg gtgaaaacat aaatatatca gaagatgttg caatatttgc tgtgattata 2760 tctgaggcag caagattacg cccggtgcal gactacatca aagattcrtt ccl ttcigag 2820 aatccgggtt taagcaaatt ggggaaacat ccctccaacc ccatgtatgt gaagaagtac 2880 aagaaaattt ctgctcacat aatggaaaat gttgacctca tgaaacaggg cctagagcca 2940 aaaccattca agcatgagaa act tgtcata gattctatgg aatcggctct tagtattgtc 3000 2 aggatct tat tcagggatgc atttaactca ggattatttg agcatgagaa acctcccaag 3060 11/17 WO OOM727 WO 0071727PCr/JP99/02734 cccatctttg aaaacacatt ggatcgtcag gaggttaagc acaaaagaaa gcaggacaag 3120 cggaaaaaga aaggaaagaa gggaggaggg ggatcaggag ggggaggtgg aggtggatcg 3180 attggggaag aagtaccaaa gggaggagga gttcgagaaa gactgaaggg agcaagttac 3240 aagcatgtta gtcttttagt cctcatgggc tcattgcata ttttatgttt tacctcagaa 3300 atattcttac tggtgacgtg catattgcat tttgatgtat tattaatata tatatgatgt 3360 gtctgtttga tcctagtgca cctgaagttg acaataactt gacgctgatc agcttttcat 3420 0. C,~00- cctttgttca acaaaatgat tttgcataat ttcctgattc tcagtgtgac atctatatat 3540 ttgcagcatg tgacgatgtg aaaciagtca ccggtcaata Itatgagttg ttagatacat 3600 aaatgaaact ttatcctgct gaattattta gcgatcatcc tctgatttga gatgtgacat 3660 ttgattcacc cattgtttca tcgaaactgt tcatagtgtt atgctgttgc gtgctccatt 3720 gttttccttg tttgcaactc aacatctcgt ttacagaatt ccatttatct ttcatatact 3780 taccgtaata gcttttgaaa ctagtataca aaagatacgt gttggaataa cacccactct 3840 1 2/17 WO OOM727 WO 0071727PCT/JP99/02734 tttgtttggt aatcctcaca ggccagagac attgacagca agagtattgg ccccagcagt 3900 aatggctctg atgatgagaa agtgaactgg aaggctctaa aagatgtcat atacaaaggt 3960 aatcaaccct acttgaggct ttcattgaag aitccgttgt gctcatcgca tgataatgga 4020 tcaggcgaag tggaiggitg gaccittatt actgattttl aagcattgtt attcctaaag 4080 tatctcagct ttgcatagaa gatgctcaaa agcaagtgga tcttttttct ttgctctgtt 4140 gcagagtgtt ttattcattc tggtttgaag atcttatcca ttctctctga tgtccgtctg 4200 tggtgacatg tctcaaggtt tatcggttca gtaacggtta atgaactgat gatactattt 4320 cactgaacag atttgtcaga aggtaggatc gagggctgca attctgcaaa aicttgtcaa 4380 ccccattgag gtgttcgtag atgatgaatg tgctgggtat actactgtgc ctttaagtta 4440 acaattttga tiagttcttt tcaaagaata actatacaaa catataattt ctatatgcct 4500 tcctctgatt attttaggaa accagctccc caagtgccaa agagttcaaa accatctatt 4560 2 atgaagctta ggcaagcaac aagcagaaac ctcaagcggt aagaatagtt tagcttatac 4620 13/17 WO 00171727 WO 0071727PCTIJP99/02734 tattatgttt ccacaggitc tgttcctttc tcagtttttt ttatgcacac ttgctttttt 4680 tgaacaggga aattgaactg cttatggaga acccacagcg caacttccct ctaaccaact 4740 tctgtcttga cccctcaaag gtatgcttgt tttttacaat atgtaattat aaactcaata 4800 cttttttttt tgaaaccgaa gctaggctaa gctggagtta tattaaggag aatagaaaag 4860 tatttacaat atcaccaaag aaggtgaagg agaaagaaac aagggaaact agggaagaaa 4920 acataaaaga aacacacact aaatgaaacc taagcggata ttctcgcgat gttttttgct 4980 Ccacacaitgc tccatatlt galigic igidiiiiga igagiagiig cgacaiggtt MU ttctcttgat tttggaacac cctcctgttt cgttcacacc atatttccta tgccaccaac 5100 aagtggattg acttgagtgc tttagccttg tttttccctc cttgaaatgt actcaatact 5160 tgctaacatg tatattgtta atgaactaat gataatgttt tggaccagta aggagatgaa 5220 gcttagcttt cgttgtatat ttacttgtitc ttaggttgat tttgcttaag atctctgtct 5280 cttgtgctat atgggtttgt gagagatgtc agcgagttgc atgatcaaat agaattagtg 5340 talgacacag tgcgaacagt tccatttggt ctactgattl tgcttgagaa aagtgcttgt 5400 14/17 WO 00/71727 PTJ9123 PCT/JP99/02734 gaaaacagca atttcttgac tgtccctgtc tgtaaacaac atcaatccat tgaaaaggaa 5460 aagcttttaa acctcacagc tattgttgac tttgctcctt ttttatgcat gcagttgatt 5520 aaatctagca ctatggtttc ccctgtcalt tatgcgcaac gtcgcctgtc atatctgatg 5580 aaiggcatgg gaagacaaca agctctcggt gatgcaatgc aagcttggag acggccaacc 5640 acttttaatt ttctggatgc tgctctgcct tctttaggca tggaaatiga atctgaagaa 5700 gcaatcgagg gtgcatcaaa ttttcagctt gagctaaaga gaagggggat gttctccagc 5760 tcacttatgc accaagcaag cgtacgcaac aaaggctgtt tgttttcagg cagcgctctt 5880 caaaagaatt tccatttatg gattgcacga ggtctcaggi tgctacgctg atctgactgc 5940 gcgataactt tcatatgccg ggaattcatg cttaaatagc caattaagcc tgatccgaat 6000 tcgttccctt tagtgagggt taattccgcg gc 6032 (210> (211) 492 (212) DNA 15/17 WO OOM727 WO 0071727PCT/JF'99/02734 (213> Oryza sat iva (400> aagaactcag taattcggaa gtcgtggatc atggtgggct ggtcgagcgg gatctcgacg tcggccgact tggtgccgtc gggcgcgaag tagaagtact tgaggtacgg cttcttgatc 120 acgtcgtagc tgagcgcgtg gagctctccg gtggccgggt cgagct~ggg gtgcgcgatc 180 atggcgcagc cgagctgccc gtcgaagtcg tagcggccga cggtctcgag gtcgccgtcg 240 gcggtgacgc gcacctggta ggggaggtcg tcctccgaca tggcgaggag cctgccgttg 300 O-tf 6& 6%5%6Lg gigccgigtg acgggtcgag gaggccgagg 360 Cggcgcgcgc gtagaacaga gcaaggcgcg cgatgcccgg agtggccatg gagctcacct 420 atcgcctfag gaaaactgga agggcgaatt czgagatatc catncactgg cggccgtcaa 480 catnatctaa ag 492 (210> 6 (211> 2 5 <212>

DNA

(213> Artificial SeQuence 16/17 WO 00/7727 PTJ9123 PCT/JP99/02734 (220>- (223> Description of artificial sequence: Primer for primer extension an alysis 400> 6 ttcgagtttt tgtgttaggg 1 7/17

Claims (17)

1. An oligonucleotide encoding a plant gene capable of controlling abscisic acid synthesis, comprising an oligonudeotide encoding an amino acid sequence from position 1 to 164 of SEQ ID NO. 2 in SEQUENCE LISTING, or a functional homologue of SEQ ID NO. 2.

2. An oligonucleotide according to claim 1, wherein the oligonucleotide is derived from rice.

3. An oligonucleotide according to claim 1, wherein the oligonucleotide is represented by SEQ ID NO. 1 in SEQUENCE LISTING.

4. An oligonucleotide encoding a plant gene capable of controlling ethylene synthesis comprising: an oligonucleotide which is capable of hybridising with an oligonucleotide according to claim 1; or an oliaonucleotide which is a aenetic variant of an oliaonucleotide 15 according to claim 1, further characterised in that it encodes an amino acid sequence having S. substantially the same function as the sequence of SEQ ID NO. 2.

5. An oligonucleotide according to any one of claims 1 to 4 capable of regulating expression of a VP14-like gene. 20 6. A vector comprising an oligonucleotide according to any one of claims 1 to 5, wherein the oligonucleotide is operatively linked to a control sequence.

7. A vector according to claim 5, wherein the vector is pBI101-Hm-VS1.

8. A plant transformed using a vector according to claim 5 or 6. COMS ID No: SBMI-00838565 Received by IP Australia: Time 14:02 Date 2004-07-22 22/07 '04 THU 14:00 FAX 61 3 9288 1567 004516684v2 FREEHILLS CARTER SMITH B o1008 27

9. A method for controlling abscisic acid synthesis in a plant, comprising the step of introducing into a plant an oligonucleotide according to any one of claims 1 to An oligonucleotide encoding a plant gene capable of controlling ethylene synthesis substantially as described herein with reference to the examples.

11. A plant transformed with a vector comprising an oligonucleotide according to claim 9 operatively linked to a control sequence.

12. A method for controlling ethylene synthesis substantially as described herein with reference to the examples.

13. An oligonucleotide encoding a plant gene capable of controlling abscisic acid synthesis, comprising an oligonucleotide having at least an 80% sequence identity with a sequence encoding an amino acid sequence from position 1 to 164 of SEQ ID NO. 2. a a a. 44 a a 4 a S 4 a.

14. An oligonucleotide according to claim 1 S at t FFio/, An oligonucleotide according to claim at least

16. An oligonucleotide according to claim at least 20 17. An oligonucleotide according to claim at least 99%. 13, wherein the sequence identity is 13, wherein the sequence identity is 13, wherein the sequence identity is 13, wherein the sequence identity is

18. An oligonucleotide according to any one of claims 1 to 4 capable of controlling abscisic acid synthesis, the gene having one or more conservative substitutions wherein a substitute amino acid has a hydrophilic Index and hydrophobic index similar to that of the original amino acid- COMS ID No: SBMI-00838565 Received by IP Australia: Time 14:02 Date 2004-07-22 22/07 '04 THU 14:00 FAX 61 3 9288 1567 FREEHILLS CARTER SMITH B ~009 004516684v2 28

19. An oligonucleotide according to any one of claims 1 to 4 capable of controlling absdsic acid synthesis, the gene having one or more conservative substitutions (as herein defined), each substitution being within 2 of the hydrophilic index and hydrophobic index.

20. An oligonucleotide according to any one of claims 1 to 4 capable of controlling abscisic acid synthesis, the gene having one or more conservative substitutions (as herein defined), each substitution being within 1 of the hydrophilic index and hydrophobic index.

21. An oligonucleotide according to any one of claims 1 to 4 capable of controlling abscisic acid synthesis, the gene having one or more conservative substitutions (as herein defined), each substitution being within 0.5 of the hydrophilic index and hydrophobic index. National Institute of Agrobiological Sciences Bio-Oriented Technology Research Advancement Institution By their Registered Patent Attorneys Freehills Carter Smith Beadle 22 July 2004 «c a *A e• 9- COMS ID No: SBMI-00838565 Received by IP Australia: Time 14:02 Date 2004-07-22