WO2004113527A1

WO2004113527A1 - Gene involved in abscisic acid inactivation

Info

Publication number: WO2004113527A1
Application number: PCT/JP2004/008949
Authority: WO
Inventors: Eiji Nambara; Yuji Kamiya; Tetsuo Kushiro; Masanori Okamoto
Original assignee: Riken
Priority date: 2003-06-20
Filing date: 2004-06-18
Publication date: 2004-12-29

Abstract

Novel gene involved in ABA inactivation; and use thereof. In particular, a gene coding for: (a) protein consisting of an amino acid sequence of SEQ ID NO. 2, 4, 6 or 8, or (b) protein consisting of an amino acid sequence of SEQ ID NO. 2, 4, 6 or 8, having undergone deletion, substitution or addition of one or more amino acids, which has the activity of inactivating abscisic acid.

Description

Description Genes involved in abscisic acid inactivation

Technical field

The present invention relates to a gene involved in inactivating the plant hormone abscisic acid, a recombinant vector containing the gene, a transgenic plant, and a transformant containing the recombinant vector. For seeds collected from transgenic plants. BACKGROUND ART 'Apsidic acid (hereinafter abbreviated as ABA) is a plant hormone that is biosynthesized in plants and causes morphogenesis and physiological phenomena. It is thought that various morphogenesis and physiological phenomena are regulated by the concentration of ABA and the distribution of ABA in each tissue.

ABA is synthesized in vivo when plants are exposed to environmental stresses such as drying and low temperatures. As ABA is biosynthesized in plants, plants gain the ability to adapt to these environmental stresses. ABA is also biosynthesized during normal development. For example, ABA is synthesized in maturing seeds. The synthesized ABA induces dormancy in seeds and suppresses seed germination. As described above, various studies have been conducted on ABA, but sufficient studies have not been made on the reaction process of ABA degradation or on the enzymes' genes involved in ABA degradation. If research progresses on the reaction process of ABA degradation and on the enzymes and genes involved in ABA degradation, and if ABA degradation can be controlled in plants, it will be possible to elucidate the mechanism of adaptation to environmental stress and control germination.

Inactivation of ABA occurs through oxidative and branidation pathways (Cutler and rochko 1999, Zeevaart and Creelman 1988; both reviewed). Of these, the inactivation pathways related to the main physiology of ABA, such as plant drying and water absorption during seed germination, are oxidation pathways.In many plant species, seed germination and plant drying and re-absorption are important. The amount of phaseic acid (PA), an inactivation product of the oxidation pathway, and the amount of dihydrophaseic acid (DPA) generated by reduction of the ketone at the 4'-position of PA are observed. In contrast, metabolite analysis has rarely pointed out the importance of the physiological role of the conjugate pathway. There is only one report of a gene involved in ABA inactivation, and a glycosylase has been cloned from Azuki by Xu et al. (2002).

However, at present, there is almost no useful knowledge about the reaction process of ABA degradation or the enzymes and genes involved in ABA degradation.

Non-Patent Document 1 Cutler, A.J., and Krochko, J.E. (1999) .Formation and breakdown of ABA.Trens Plant Sci. 4, 472-478

Non-Patent Document 2 Zeevaart, J.A., and Creelman, R.A. (1988) .Metaboli sm and physiology of abscisic acid.Annu.Rev.Plant Physiol.Plant Mol.Biol. 39, 439-473.

Non-Patent Document 3 Xu, Z.-J., Nakajima, M., Suzuki, Y., and Yamaguchi, I. (2002) .Shir onirtg and characterization of the abscisic acid-specific glucosyl transferase gene from Adzuki bean seedlings. Plant Physiol. 129, 1285-1295.

Therefore, an object of the present invention is to provide a novel gene involved in ABA inactivation and its use. Disclosure of the invention

In view of the above-described circumstances, the present inventors have conducted intensive studies and as a result, have newly found a gene encoding a protein having ABA inactivating activity, and have completed the present invention. The present invention includes the following.

(1) A gene encoding the following protein (a) or (b):

(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8

(b) an amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8 in which one or several amino acids have been deleted, substituted or added, and have an activity of inactivating abscisic acid; Protein

(2) A gene containing the following DNA of (a) or (b). (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, or 7 (b) DNA consisting of the nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, or 7 DNA that codes for a protein that hybridizes under stringent conditions to

(3) The gene according to (1) or (2), which is derived from Arabidopsis, rice or tomato.

(4) A recombinant vector containing the gene according to (1) or (2).

(5) A transformant containing the recombinant vector according to (4).

(6) A transgenic plant containing the gene according to (1) or (2). (7) A seed collected from the transgenic plant according to (6).

(8) A plant in which the gene according to (1) or (2) is knocked out.

(9) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 4, or (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 A method of maintaining seed dormancy by inhibiting the inactivating activity of abscisic acid derived from a protein having the activity of inactivating abscisic acid.

(10) The method for maintaining dormancy of a seed according to (9), wherein the activity of inactivating abscisic acid derived from the protein is inhibited by Tetcyclacis (Tetcyclacis) and / or Uniconazole.

(1 1) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 4, or

(b) Absidine derived from a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having an activity of inactivating abscisic acid Seeds whose acid inactivating activity has been inhibited.

This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application Nos. 2003-176423 and 2003-367857, which are the priority documents of the present application. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows that the reaction solution obtained from the transformed yeast into which the CYP707A1 cDNA was introduced was subjected to HPLC. FIG. 9 is a characteristic diagram showing a result of the operation.

FIG. 2 is a characteristic diagram showing the results obtained when the reaction solution obtained from the transformed yeast into which the CYP707A3 cDNA was introduced was subjected to HPLC.

FIG. 3 shows the results when the reaction solution from the transformed yeast into which only empty pYeDP60 plasmid was introduced was subjected to HPLC.

FIG. 4 is a characteristic diagram showing the results when a reaction solution obtained from the transformed yeast into which the CYP707A2 cDNA was introduced was subjected to HPLC.

FIG. 5 is a characteristic diagram showing the results obtained when the reaction solution obtained from the transformed yeast into which the CYP707A4 cDNA was introduced was subjected to HPLC.

Fig. 6 is a characteristic diagram showing the inhibition of ABA 8'-hydroxylase (CYP707A1) activity by inhibitors (Tetcyclacis, Metyrapone and Uniconazole).

FIG. 7 is a diagram showing the expression of CYP707A1, CYP707A2, CYP707A3 and CYP707A4 in each organ by mRNA level.

FIG. 8 is a graph showing a change in the amount of endogenous ABA when absorbing dry seeds. :

FIG. 9 is a diagram showing the expression of CYP707A1, CYP707A2, CYP707A3, and CYP707A4 genes at the time of seed water absorption.

FIG. 10 is a graph showing a change in ABA amount at the time of drying and water absorption of a plant at the second week.

FIG. 11 is a graph showing changes in the amount of NCED3 mRNA during drying and water absorption of plants at 2 weeks. FIG. 12 is a graph showing changes in the amounts of CYP707A1, CYP707A2, CYP707A3, and CYP707A4raRNA at the time of drying and water absorption of plants at the second week.

FIG. 13 is a graph showing changes in CYP707A1, CYP707A2, CYP707A3 and CYP707A4 mRNA levels when plants were treated with ABA (30 M) and water on the second week.

1 4, Cyp707a2- 1 mutant gene, Cyp707a2_2 mutant gene is a schematic diagram showing the configuration of Cyp707a3- 1 mutant gene and cy _P 707a3- 2 mutant gene.

FIG. 15 is a characteristic diagram showing the relationship between the water absorption treatment and the germination rate in the CYP707A2 gene knockout gun and the CYP707A3 gene knockout line.

FIG. 16 is a characteristic diagram comparing the amounts of ABA, PA and DPA contained in dried seeds between the CYP707A2 gene knockout line and the wild type.

Figure 17 shows ABA in CYP707A2 gene knockout line and wild type seeds. It is a special use figure which shows a time-dependent change of quantity. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The gene according to the present invention encodes a protein having a function of inactivating ABA (hereinafter, ABA inactivating enzyme). Here, the function of inactivating ABA means a function of converting ABA to phaseinate (hereinafter abbreviated as PA). There are two known pathways for ABA inactivation. One is the glycosylation pathway in which glucose is added to the carboxyl group at the 1-position or the hydroxyl group at the 1'-position in ABA, and the other is the cyclic methyl group such as the 8'- or 7'-position of ABA that hydroxylates. Undergo oxidation pathway. Of these, the oxidative pathway is the irreversible pathway and is the essential inactivation pathway of ABA. Hydroxylation of the cyclic methyl group at position 8 'in ABA results in 8 hydroxy ABA. This 8, -hydroxy ABA is isolated as an equilibrium mixture with the more thermodynamically stable PA. Therefore, the function of converting ABA to PA more specifically means an ABA 8'-hydroxylase activity that hydroxylates the cyclic methyl group at the 8'-position of ABA.

1. Cloning of ABA inactivating enzyme gene

(1) Preparation of plant genome

Sources of genomic DNA include part of a plant or whole plant such as plant leaves, stems and roots. The target plant is not particularly limited, and includes Arabidopsis, rice, tomato, and soybean. Plants can be grown in the field by sowing seeds in soil, or can be grown under aseptic conditions by sowing in solid media such as GM and MS media. If necessary, a systemic acquired resistance (SAR) inducer such as PBZ (probenazole) can be added.

Preparation of genomic DNA from grown plants can be performed according to a conventional method. For example, first, a plant frozen with liquid nitrogen is ground in a mortar or the like, and the ground material is suspended in a buffer containing a surfactant such as TritonX-100 and filtered with gauze or the like. Subsequently, the cell nucleus is precipitated by centrifuging the filtrate, and a sodium lauroylpsychosinate solution or the like is added to the precipitate to digest the cell nucleus. Digestion fluid After the sample is subjected to, for example, centrifugation using a shim-brominated medium, the DNA layer is recovered, and the obtained DNA solution is dialyzed against a TE buffer or the like. Finally, the obtained DNA solution is precipitated by adding ethanol, and then dissolved in an appropriate amount of TE buffer or the like, whereby genomic DNA can be obtained.

(2) Cloning of ABA inactivating enzyme gene

The ABA inactivating enzyme gene can be cloned from genomic DNA prepared according to “(1) Preparation of plant genome”. For example, a genome can be prepared from plants such as Arabidopsis, rice, tomato, and soybean, and an ABA-inactivating enzyme gene derived from Arabidopsis, rice, tomato, and soybean can be cloned.

Arabidopsis thaliana ABA inactivating enzyme genes have four types of homologues, which are called CYP707A1, CYP707A2, CYP707A3 and CYP707A4, respectively. The nucleotide sequences of these CYP707A1, CYP707A2, CYP707A3, and CYP707A4 are shown in SEQ ID NOs: 1, 3, 5, and 7, respectively. The amino acid sequences of the proteins (ABA inactivating enzymes) encoded by these SEQ ID NOs: 1, 3, 5 and 7 are shown in SEQ ID NOs: 2, 4, 6 and 8, respectively.

The amino acid sequence of Arabidopsis ABA-inactivating enzyme (CYP707A1, CYP707A2, CYP707A3 and CYP707A4) is limited to the amino acid sequences shown in SEQ ID NOs: 2, 4, 6 and 8 as long as they have the function of inactivating ABA. However, it may be an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8. Here, several amino acids mean, for example, 2 to 50 amino acids, preferably 2 to 30 amino acids, and more preferably 2 to 10 amino acids.

In addition, based on the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, and 7, and the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, and 8, a database containing the genomic nucleotide sequences of various plants was searched. By doing so, ABA inactivating enzyme genes in various plants can be identified. For example, by searching a database (http: 〃 drnelson. Utmem. Edmem / edu / rice, html) that stores the nucleotide sequence of the rice (0. sativa (japonica)) genome, accession numbers AP004129.1 and AP004162 are obtained. ABA inactivating enzyme gene in rice identified in 1 can be identified. Also, Tomato Geno Database containing the base sequence of the system

By searching for (http: // drnelson. utmem. edu / toraato. html), the ABA inactivating enzyme gene in tomato identified by the accession numbers EST; AI489171 and EST247510 cLED17I4 can be identified. Furthermore, by searching the database (http: //drnelson.utmem.edu/soybean.html) containing the base sequence of the soybean genome, the soybean identified by accession numbers AI431116, AI735873, and AI966688 ABA inactivating enzyme genes can be identified. A database containing these rice, tomato, and soybean genome sequences can be accessed on Dr. David Nelson's Cytochrome P450 Homepage at the University of Tennessee.

Specifically, examples of the rice-derived ABA inactivating enzyme exhibiting high homology to Arabidopsis-derived ABA inactivating enzyme include those having the amino acid sequence shown in SEQ ID NOS: 24 and 25. it can. Examples of the tomato-derived ABA-inactivating enzyme having high homology to Arabidopsis-derived ABA-inactivating enzyme (CYP707A3) include those having the amino acid sequence shown in SEQ ID NO: 26. The amino acid sequences of ABA inactivating enzymes from Arabidopsis thaliana (CYP707A1, CYP707A2, CYP707A3 and CYP707A4), the amino acid sequences of ABA inactivating enzymes from rice (SEQ ID NOS: 24 and 25), and ABA inactivating from tomato Table 1 shows the results of comparing the homology with the amino acid sequence of the activating enzyme (SEQ ID NO: 26). The unit of the numerical values in Table 1 is%.

GYP707A1 CYP707A2 CYP707A3 CYP707A4 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26

CYP707A1 56.9 86.1 57.5 68.3 52.4 75.

CYP707A2 57.6 53.9 55.6 49.8 56.8

CYP707A3 57.4 66.9 52.4 74.4

GYP707A4 56.5 57.5 57.4 SEQ ID NO: 24 52.9 67.5 SEQ ID NO: 25 49.9

When the nucleotide sequence of the ABA inactivating enzyme gene in various plants is thus identified, the ABA inactivating enzyme gene can be isolated according to a conventional method. In other words, “(1) ABA inactivating enzyme gene can be isolated by PCR using a pair of appropriately designed primers with the genome prepared according to “Preparation of product genome” as type III. In addition, a cDNA library is prepared using total mRNA extracted from plant cells, and a cDNA containing the ABA inactivating enzyme gene is isolated from the cDNA library using a DNA probe designed based on the nucleotide sequence. can do.

On the other hand, the ABA inactivating enzyme gene is not limited to the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, and 7, but may be a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, or 7. On the other hand, DNAs that hybridize under stringent conditions and that encode a protein having a function of inactivating ABA are also included. Also, the stringent conditions refer to, for example, conditions at a sodium concentration of 800 to 1000 lηm, preferably 850 to 950 mM, and a temperature of 60 to 70 ° (: preferably 65 to 68 ° C).

The introduction of the mutation into the ABA inactivating enzyme gene can be carried out according to a standard method, using a known method such as the Kunkel method or the Gapped duplex method or a method similar thereto, for example, using a site-directed mutagenesis method. Mutation-introduced kits (such as Mutant-K (TAKARA) and Mutant-G (TAKARA)) or using the TAKARA LA PCR in vitro Mutagenesis series kit . 2. Production of recombinant vectors and transformants

(1) Preparation of recombinant vector

The recombinant vector of the present invention can be obtained by ligating (inserting) the gene of the present invention into an appropriate vector. The vector for introducing the gene of the present invention is not particularly limited as long as it can be replicated in a host, and examples include plasmid DNA and phage DNA.

Plasmid DNA includes plasmids for Escherichia coli host such as pBR322, pBR325, pUC118 and pUC119, plasmids for Bacillus subtilis such as pUB110 and pTP5, plasmids for yeast host such as YEpl3, YEp24 and YCp50, and plants such as pBI221 and pBI121. And phage DNA; and I phage. Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as vaccinia virus can also be used.

In order to introduce the gene of the present invention into a vector, first, purified DNA is appropriately A method of cutting with a restriction enzyme, inserting into an appropriate restriction enzyme site of vector DNA or a multicloning site, and ligating to a vector is employed.

The gene of the present invention needs to be incorporated into a vector so that the function of the gene is exhibited. Thus, the vector of the present invention includes a vector containing a promoter, a gene of the present invention, and, if desired, a cis element such as an enhancer, a splicing signal, a polyA addition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like. Can be linked. In addition, examples of the selection marker include an ampicillin resistance gene, a neomycin resistance gene, a dihydrofolate reductase gene, and the like.

(2) Preparation of transformant

The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express the gene of the present invention. For example, the genus Escherichia such as Escherichia coli, the genus Bachinoles such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida, and the lysozyme meliloti (Rhizobiu eli loti) and other yeasts belonging to the genus Rhizobium, such as Saccharomyces cerevis iae, Schizosaccharomyces pombe, and yeasts such as Pichia pastoris. Plant cells established from Arabidopsis, tobacco, corn, rice, carrot, etc .; protoplasts prepared from such plants; animal cells such as COS cells, CH0 cells; and insect cells such as Sf9, Sf21. Are mentioned.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the bacterium, and comprises a promoter, a ribosome binding sequence, a gene of the present invention, and a transcription termination sequence. Is preferred. Further, a gene that controls a promoter may be included.

Examples of Escherichia coli include Escherichia coli HMS174 (DE3), K12 and DH1, and examples of Bacillus subtilis include Bacillus subtilis Ml114 and 207-21. No.

Any promoter can be used as long as it can be expressed in a host such as E. coli. For example, those derived from Escherichia coli such as trp promoter, lac promoter, PL promoter and PR promoter and those derived from phage such as T7 promoter are used. Furthermore, artificially designed and modified promoters such as the tac promoter may be used.

The method for introducing the recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium ions [Cohen, SN, et al .: Proc. Natl. Acad. Sci., USA, 69: 2110-2114 (1972)], an electroporation method, and the like.

When yeast is used as a host, for example, Saccharomyces 'Celebiche, Schizosaccharomyces' bomb, Pichia's Pastris and the like are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast.Examples include gall promoter, gal10 promoter, heat shock protein promoter, MFal promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter. Promoter and A0X1 promoter.

The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, the electroporation method [Becker, DM, et al .: Methods. Enzymol., 194: 182- 187 (1990)], Spheroplast method [Hinnen, A. et al .: Proc. Natl. Acad. Sci., USA, 75: 1929-1933 (1978)], lithium acetate method [Itoh, H .: J Bacteriol., 153: 163-168 (1983)].

When a plant cell is used as a host, for example, a cell established from Arabidopsis thaliana, tobacco, sorghum, rice, carrot, etc., or protoplasts prepared from the plant are used. In this case, the promoter is not particularly limited as long as it can be expressed in plants, and examples thereof include a cauliflower mosaic virus 35S RNA promoter, an rd29A gene promoter, and rbcS proquita.

Methods for introducing a recombinant vector into a plant include a method using polyethylene glycol of Abel et al. [Abel, H., et al .: Plant J. 5: 421-427 (1994)] and an electroporation method. Is mentioned. When animal cells are used as the host, monkey cells COS-7, Vero, Chinese hamster ovary cells (CH0 cells), mouse L cells, rat GH3, and human FL cells are used. As a promoter, an SRa promoter, an SV40 promoter, an LTR promoter, a CMV promoter, or the like may be used, or an early gene promoter of a human cytomegalovirus may be used. Methods for introducing the recombinant vector into animal cells include, for example, the electoral poration method, the calcium phosphate method, and the lipofection method.

When insect cells are used as a host, Sf9 cells, Sf21 cells, and the like are used. As a method for introducing a recombinant vector into an insect cell, for example, a calcium phosphate method, a lipofection method, an electoral poration method and the like are used.

3. Production and functional analysis of ABA-inactivating enzyme

(1) Production of ABA-inactivating enzyme

The ABA inactivating enzyme of the present invention has an amino acid sequence encoded by the ABA inactivating enzyme gene of the present invention, or has an amino acid sequence in which the mutation is introduced into one or several amino acids in the amino acid sequence. And has an inactivating activity.

The ABA-inactivating enzyme can be obtained by culturing the transformant in a medium and collecting from the culture. The term “culture” means any of a culture supernatant, a cultured cell or a cultured bacterial cell, and a crushed cell or bacterial cell. When culturing the transformant in a medium, a usual method used for culturing a host can be applied. The medium for culturing the transformant obtained using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be used by the microorganism, to efficiently culture the transformant. Any of a natural medium and a synthetic medium may be used as long as the medium can be used. If plant cells are used as a host, the medium may be supplemented with vitamins such as thiamine and pyridoxine as necessary.If animal cells are used as the host, serum such as RPMI1640 may be used. Is added.

For example, carbon sources include carbohydrates such as glucose, fructose, sucrose and starch; organic acids such as acetic acid and propionic acid; ethanol and propanol. Such alcohols are used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium salts of inorganic or organic acids or other nitrogen-containing compounds, as well as peptone, meat extract, corn steep liquor, etc. Is used. As inorganic substances, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like are used. Furthermore, an antibiotic such as ampicillin or tetracycline may be added to the medium as needed.

The cultivation is usually carried out at 30 to 37 ° C for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and stirring culture. During the cultivation period, the pH is maintained at 7.0 to 7.5. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like.

When culturing a microorganism transformed with a vector containing an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using the Lac promoter, isopropyl- / 3-D-thiogalatatopyranoside (IPTG) is transformed with an expression vector using the trp promoter. When culturing the isolated microorganisms, indole ataryl acid (IAA) or the like may be added to the medium.

When the ABA-inactivating enzyme is produced in the cells or cells after the culture, the cells or cells are disrupted to extract the ABA-inactivating enzyme. When the ABA-inactivating enzyme is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Then, common biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., may be used alone or in appropriate combination. Thereby, the ABA-inactivating enzyme of the present invention can be isolated and purified from the culture.

(2) Functional analysis of ABA inactivating enzyme

A solution containing the ABA-inactivating enzyme and ABA obtained in “(1) Production of ABA-inactivating enzyme” is prepared and reacted for a predetermined time, and the amount of ABA and the ABA contained in the solution after the reaction are inactivated. Measure the amount of 8'-hydroxy ABA or PA obtained. As a result, ABA inactivating enzyme ABA inactivation function can be analyzed.

4. Production of plants in which the function of ABA inactivating enzyme is suppressed

ABA is a plant hormone biosynthesized in plants and is involved in plant morphogenesis and physiological phenomena. For example, it is known that dormancy is induced by biosynthesis of ABA in plant seeds, and germination is promoted by inactivation of ABA, that is, inactivation of ABA.

ABA-inactivating enzymes convert ABA to 8'-hydroxy ABA (or PA) and inactivate ABA. Therefore, morphogenesis and physiological phenomena caused by inactivation of ABA can be suppressed by functionally deficient of the ABA inactivating enzyme in the plant. For example, in a transgenic plant in which the ABA inactivating enzyme is functionally deficient, seed germination can be suppressed. In a transgenic plant modified so that the ABA inactivating enzyme can be expressed only under predetermined conditions, the timing of germination can be regulated.

In particular, among Arabidopsis thaliana-derived ABA inactivating enzymes, CYP707A2 (SEQ ID NO: 4) is relatively highly expressed in seeds and is greatly involved in seed germination. Therefore, in the seed in which the function of CYP707A2 (SEQ ID NO: 4) is inhibited, dormancy is maintained as compared with the wild-type seed. Here, maintaining dormancy means delaying the germination of the seed under conditions that induce germination such as the state of water absorption of the seed. Whether or not the dormancy of the seed has been maintained can be determined by measuring the time until germination after the seed is brought into a water absorbing state.

The function of the ABA-inactivating enzyme can be suppressed by, but not limited to, an antisense method or an RNA interference (RNAi) method. Alternatively, it can be carried out by knocking out an ABA inactivating enzyme by a homologous gene recombination method or the like. Furthermore, it can also be performed by introducing a mutant ABA inactivating enzyme gene into a plant.

In addition to artificially suppressing the function of ABA-inactivating enzyme, cereals with deep seed dormancy and strong resistance to stress are screened by screening plants with abnormal ABA-inactivating enzyme genes from crops. You can get a crop. For example, in rice, a transposon is randomly inserted into a gene, It is known that the function is lost, and a transposon is inserted into the ABA inactivating enzyme gene, so that a plant having a defective function can be screened.

In the antisense method, the target site is not particularly limited, and it is possible to target a protein coding region, a 5 ′ untranslated region, or the like. An antisense nucleotide that hybridizes to the base sequence of the gene encoding the ABA inactivating enzyme represented by SEQ ID NOS: 1, 3, 5, 7, or the like or any part of its complementary sequence may be used. . The antisense nucleotide is preferably an antisense nucleotide corresponding to at least 15 or more consecutive nucleotides in the nucleotide sequence of the gene encoding ABA inactivating enzyme. Antisense nucleotides include not only those in which all nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are complementary sequences, and those in which DNA or mRNA and nucleotides encode an ABA inactivating enzyme. As long as it can specifically hybridize to the nucleotide sequence, it includes those having a mismatch of one or more nucleotides. RNAi refers to a phenomenon in which, when double-stranded RNA (dsRNA) is introduced into a cell, the mRNA in the cell corresponding to the RNA sequence is specifically degraded, and the protein is not expressed. In the RNAi method, double-stranded RNA is usually used, but is not particularly limited. For example, double-stranded RNA formed in self-complementary single-stranded RNA can be used. The double-stranded region may be a double-stranded region in all regions, or even if some regions (for example, both ends or one end) have a single-stranded structure or the like. Good. The length of the oligo RNA used for RNAi is not limited, and is, for example, 25 bases or more (25 bp or more in the case of double strand).

Knockout of the gene encoding the ABA inactivating enzyme can be performed as follows. Knockouts are caused by foreign DNA and endogenous transposons. In the case of exogenous DNA, screening can be performed using a library of transgenic plants into which exogenous DNA has been randomly introduced, and in the case of endogenous transposons, can be screened using a plant library that has been regenerated through tissue culture that facilitates transposon flight. . Primers based on known sequences of foreign DNA and endogenous transposons

Perform PCR on genomic DNA prepared from a plant library using a combination of primers based on known sequences derived from the CYP707A gene (or its homolog). Thus, the introduced mutation can be confirmed by Southern hybridization using the DNA of the CYP707A gene or its homolog as a probe.

Transgenic plants in which the ABA inactivating enzyme is functionally deficient can be produced as follows. That is, first, a mutant ABA-inactivating enzyme gene that is deficient in ABA-inactivating enzyme activity is constructed. The mutant ABA inactivating enzyme gene can be prepared according to the mutation introduction method described in “1. (2) Cloning of ABA inactivating enzyme gene” above.

Next, the above-mentioned transgenic plant can be produced by introducing the mutant ABA inactivating enzyme gene into a plant host using genetic engineering techniques. Methods for introducing a mutant ABA-inactivating enzyme gene into a plant host include indirect methods such as the agrobacterium infection method and direct methods such as the particle gun method, polyethylene glycol method, ribosome method, and microinjection method. No.

When the Agrobacterium infection method is used, a transgenic plant into which a mutant ABA inactivating enzyme gene has been introduced can be prepared as follows.

(1) Construction of recombinant vector for plant introduction and transformation of Agrobacterium

The recombinant vector for plant introduction is prepared by cutting out the mutant ABA inactivating enzyme gene from the plasmid containing the mutant ABA inactivating enzyme gene using an appropriate restriction enzyme, and adding an appropriate linker to the obtained fragment as necessary. After ligation, it can be obtained by inserting into a cloning vector for a plant cell. As a cloning vector, a plasmid of a binary vector system such as pBI101, pBI121, pGA482, pGAH, or pBIG—an intermediate vector system plasmid such as pLGV23Neo, pNCAT, or pM0N200 can be used.

When a binary vector-based plasmid is used, the target gene is inserted between the boundary sequences (LB, RB) of the binary vector, and the recombinant vector is amplified in E. coli. Next, the amplified recombinant vector is introduced into Agrobacterium tumefaciens C58, LBA4404, EHA101, C58ClRifR, EHA105, etc. by a freeze-thaw method, an electoral poration method or the like, and the Agrobacterium teriformum is planted. Use for transduction of In addition to the above method, in the present invention, an agrobacterium for plant infection containing the gene of the present invention is prepared by a three-way conjugation method [Nucleic Acids Research, 12: 8711 (1984)]. Can be. That is, Escherichia coli having a plasmid containing a target gene, Escherichia coli having a helper plasmid (for example, pRK2013, etc.) and agrobacterium are mixed-cultured, and cultured on a medium containing rifampicillin and kanamycin. In order to express a foreign gene or the like in a plant, it is necessary to arrange a plant promoter or a terminator before and after the structural gene, respectively. Promoters that can be used in the present invention include, for example, 35S transcript derived from the force reflower mosaic virus (CaMV) [Jefferson, RA et al .: EMBO J 6: 3901-3907 (1987)], maize Ubiquitin [Christensen, AH et al .: Plant Mol. Biol. 18: 675-689 (1992)], nopaline synthase (N0S) gene, otatobin (OCT) synthase gene promoter, and the like. Examples of the terminator sequence include a terminator derived from cauliflower mosaic virus-derived ゃ nopaline synthase gene. However, the present invention is not limited to these promoters and terminators as long as they are known to function in plants.

If necessary, an intron sequence between the promoter sequence and the gene of the present invention, which has a function of enhancing gene expression, such as an intron of corn alcohol dehydrogenase (Adhl) [Genes & Development 1: 1183-1200. (1987)] can be introduced.

Furthermore, it is preferable to use an effective selectable marker gene in combination with the gene of the present invention in order to efficiently select a desired transformed cell. The selection markers used in this case include the kanamycin resistance gene (ΝΡΤΠ), the hygromycin phosphotransferase (htp) gene that confers resistance to the antibiotic hygromycin to plants, and bialaphos resistance. One or more genes selected from the phosphinothricin acetyltransferase (bar) gene and the like to be conferred can be used.

The mutant ABA inactivating enzyme gene and the selectable marker gene may be integrated together into a single vector, or two types of recombination, each incorporated into a separate vector. DNA may be used.

(2) Introduction of the gene of the present invention into a plant host

In the present invention, a plant host refers to a plant cultured cell, a whole plant of a cultivated plant, a plant organ (eg, leaf, petal, stem, root, rhizome, seed, etc.), or a plant tissue (eg, epidermis, phloem, soft tissue) , Xylem, vascular bundle, etc.). Hosts that can be used as plant hosts include rice, soybeans, tomatoes, tobacco, corn, Arabidopsis and the like.

When a plant culture cell, plant, plant organ or plant tissue is used as a host, the mutant ABA inactivating enzyme gene can be obtained by transfecting a vector into a collected plant section using an agrobacterium infection method, a particle gun method, or a polyethylene glycol method. Can be used to transform a plant host. Alternatively, transgenic plants can be prepared by introducing them into protoplasts by electroporation.

When a gene is introduced by the agrobacterium infection method, the step of infecting a plant with an agrobacterium containing a plasmid containing the target gene is essential. This is the vacuum infiltration method [CR Acad. Sci. Paris, Life Science, 316: 1194 (1993)]. That is, Arabidopsis thaliana grown in soil in which equal amounts of permikilite and perlite were combined in Arabidopsis thaliana was directly immersed in a culture solution of Agrobacterium containing a plasmid containing the gene of the present invention, and the Arabidopsis thawed. Place in a desiccator, suction with a vacuum pump until the pressure reaches 65 to 70 mmHg, and leave at room temperature for 5 to 10 minutes. Transfer the pot to a tray and cover with a wrap to keep humidity. Take the wrap the next day, let the plants grow and harvest the seeds.

Next, seeds are sown on an MS agar medium supplemented with an appropriate antibiotic in order to select an individual having the target gene. The seeds of the transgenic plant into which the gene of the present invention has been introduced can be obtained by transferring the plant grown in this medium into a pot and growing it.

Generally, a transgene is similarly introduced into the genome of a host plant, but a phenomenon called a position effect in which the expression of the transgene differs due to a different location of the transgene is observed. Northern method using DNA fragment of transgene as probe By performing the assay, a transformant in which the transgene is more strongly expressed can be selected.

To confirm that the gene of interest has been incorporated into the transgenic plant into which the gene of the present invention has been introduced and the next generation thereof, DNA can be extracted from these cells and tissues in accordance with a conventional method, using a known PCR method or The detection can be performed by detecting the introduced gene using Southern analysis.

The production of transgenic plants modified so that the ABA inactivating enzyme can be expressed only under predetermined conditions can be carried out according to the above-mentioned method.

The activity of the ABA-inactivating enzyme of the plant in which the function of the ABA-inactivating enzyme thus obtained is suppressed or the activity of the mutant ABA-inactivating enzyme is determined by the above-mentioned “3. Production and functional analysis of ABA-inactivating enzyme”. The analysis can be performed according to the method described above.

Seeds can be obtained by cultivating the plant produced as described above, in which the ABA inactivating enzyme gene is knocked out, or the transgenic plant, into which the mutant ABA inactivating enzyme gene is introduced, by an ordinary method. For example, in a seed collected from a transgenic plant into which a mutant ABA inactivating enzyme gene has been introduced, ABA inactivation is suppressed, so that a dormant state can be maintained for a long period of time. This makes the seed suitable for long-term storage and transportation.

In addition, ABA can be inactivated at a desired timing in a seed collected from a transgenic plant modified so that the ABA inactivating enzyme can be expressed only under predetermined conditions. This makes the seeds suitable for long-term storage and transportation, and also suitable for enterprise-type agriculture in which cereals and the like are cultivated systematically. Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples. '

(Example 1)

Arabidopsis cytochrome P450 CYP707A CYP707A2, CYP707A3 and

Functional analysis of CYP707A4 in yeast>

Arabidopsis-derived cytochrome provided by RIKEN Tsukuba Research Institute BioResource Center (3-1, Takanodai, Tsukuba, Ibaraki 305-0074, Japan) Using the full-length cDNAs (pda03938 and pda05432) of F450 CYP707A1 and CYP707A3, a cDNA fragment was prepared by PCR, in which Hpa I was introduced at the N-terminus and Eco RI restriction sites were introduced at the C-terminus for CYP707A1. For CYP707A3, a cDNA fragment was prepared in which Hpa I was introduced at the N-terminus and Kpnl restriction enzyme sites were introduced at the C-terminus. The nucleotide sequences of both the obtained cDNA fragments were confirmed by DNA sequencing, and were confirmed to be the same as the provided full-length cDNA and to be free of mutation.

Next, the resulting cDNA fragments are yeast expression vector pYeDP60 (Denis Pompon provide Mr. _{0 Pompon, D., Louerat, B.} , Bronine, A., and Urban, P. (1996). Yeast expression of Animal and plant P450s in optimized redox environment. Mothod. Enzymol. 272, 51-64). For the cDNA fragment of CYP707A1, the Bam HI site of pYeDP60 was blunt-ended, and ligation was performed using Eco RI-treated Bam HI site. For the cDNA fragment of CYP707A3, the BamHI site of pYeDP60 was blunt-ended and ligated using Kpnl-treated one.

Each of the obtained plasmids was used as a WAT11 strain of yeast (Saccharomyces cerevisiae) (Fe from Denis Pompon ft, Pompon, D., Louerat, B., Bronine, A., and Urban, P. (1996). Expression of animal and plant P450s in an optimized redox environment. Mothod. Enzymol. 272, 51-64) according to a standard method. The transformed yeast into which each plasmid was introduced was cultured in an SGI medium containing glucose, and then transferred to an SLI medium containing galactose. After culturing for 12 hours, the cells were collected. Then, the transformed yeast was suspended in a phosphate buffer (pH 7.6), and the cells were disrupted by a French press.

Next, this was ultracentrifuged to obtain a microsome fraction, and the obtained microsome fraction was suspended in a phosphate buffer (PH7.6). To the obtained microsomal fraction solution, 500 µM of NADPH and 38 µΜ of ABA (ethanol solution) were added, and reacted at 30 ° C for 1 晚.

After completion of the reaction, the reaction solution was extracted with ethyl acetate, concentrated to dryness, and the product was analyzed by liquid chromatography (HPLC). 0DS for HPLC (Senshu Pak

A PEGASIL-0DS) column (4.6 x 250 bars) was used. 10% methanol and 0.1% acetic acid Solution A and solution B consisting of 60% methanol and 0.1% acetic acid are prepared.Elute 0 to 3 minutes with 50% solution B, then elute with 50% B for 3 to 33 minutes. Elution was carried out with a gradient of ~ 100% B solution. The elution time is 16 minutes for the product PA and 30 minutes for the starting material ABA.

In the reaction solution obtained from the transformed yeast into which the CYP707A1 cDNA was introduced and from the transformed yeast into which the CYP707A3 cDNA was introduced, a peak was observed at 16 minutes, respectively. On the other hand, this peak was not detected in the reaction solution from the transformed yeast into which only empty pYeDP60 plasmid was introduced. The results are shown in FIGS. FIG. 1 shows the results obtained when the reaction solution obtained from the transformed yeast into which the CYP707A1 cDNA was introduced was subjected to HPLC. FIG. 2 shows the results obtained when the reaction solution obtained from the transformed yeast into which the CYP707A3 cDNA was introduced was subjected to HPLC. FIG. 3 shows the results when the reaction solution from the transformed yeast into which only empty pYeDP60 plasmid was introduced was subjected to HPLC. In FIGS. 1 to 3, the PA peak is shown as “PA” and the ABA peak is shown as “ABAJ”.

Next, in order to identify the obtained peak, analysis was performed using gas chromatography ^ -mass spectrometry (GC-MS). The column used was DB-1 (250 ra x 30 m). Analytical conditions: Initial temperature 80 ° C for 1 minute, stepl; 20 ° C / min to 220 ° C, step2; 5 ° C / min to 240 ° C, step3; 40 ° C / min to 300 ° C Up to step4; 5 minutes at 300 ° C. In the reaction solution obtained from the transformed yeast into which the CYP707A1 cDNA was introduced and from the transformed yeast into which the CYP707A3 cDNA was introduced, the peak was obtained at the same retention time (11 minutes) as that of PA, and the pattern of the mass spectrum was known. It completely matched the spectrum pattern of PA.

From the above results, it was clarified that the transformed yeast into which the CYP707A1 cDNA was introduced and the transformed yeast into which the CYP707A3 cDNA had been introduced had the activity of converting ABA to PA. That is, the cDNA of CYP707A1 and the cDNA of CYP707A3 could be identified as genes encoding ABA 8'-hydroxylase.

Similarly, in order to analyze the functions of CYP707A2 and CYP707A4, CYP707A2 cDNA and CYP707A4 cDNA were obtained from dried seeds and immature seeds according to a standard method. Using the cDNA of CYP707A2 and the cDNA of CYP707A4, the ability of the proteins encoded by CYP707A2 and CYP707A4 to inactivate ABA was examined according to the method described above. The results are shown in FIGS.

FIG. 4 shows the results obtained when the reaction solution obtained from the transformed yeast into which the CYP707A2 cDNA was introduced was subjected to HPLC. FIG. 4 shows the results obtained when the reaction solution obtained from the transformed yeast into which the CYP707A4 cDNA was introduced was subjected to HPLC. As can be seen from FIGS. 4 and 5, it was revealed that the transformed yeast into which the CYP707A2 cDNA was introduced and the transformed yeast into which the CYP707A4 cDNA was introduced also had an activity of converting ABA to PA. Was. That is, the cDNA of CYP707A2 and the cDNA of CYP707A4 could be identified as genes encoding ABA 8'-hydroxylase.

Biochemical analysis of CYP707A1 from Arabidopsis thaliana>

Next, the effects of ABA 8'-hydroxylase inhibitors were studied for the biochemical analysis of Arabidopsis ABA 8'-hydroxylase (here, CYP707A1). As the inhibitor, Tetcyclacis, Metyrapone and Uniconazole known as P450 inhibitors were used.

Specifically, に μΜ or 10μΜ of Tetcyclacis or 1 μM or! /, Is: 10 μM Metyrapone or ΙμΜ or 10 μΙ Uniconazole was added, and the amount of PA contained in the reaction solution was measured in the same manner. Figure 6 shows the results. As shown in Figure 6, when 10 Tetcyclacis and 10 μM Uniconazole were used as inhibitors, the amount of PA produced was significantly inhibited, and ABA 8'-hydroxylase activity could be inhibited. Was. On the other hand, when Metyrapone was used as an inhibitor, the amount of PA was hardly changed, and ABA 8'-hydroxylase activity could not be inhibited.

(Example 2

Expression analysis in Arabidopsis>

Next, the amount of accumulated CYP707A1, A2, A3 and A4 mRNA in Arabidopsis thaliana was analyzed by quantitative PCR. Seed Arabidopsis wild-type (Columbia strain) seeds on a 0.8% agar medium containing 1/2 Murashige & Skoog after surface sterilization. Grow for 2 weeks. Rosette leaves and roots of the grown plants were used for RNA extraction. Stems and inflorescences were sampled from soil-planted plants in which Bamiyuki Light (manufactured by Ryoku Sangyo) and Difmix (made by Sakata Seed) were mixed in equal amounts. The pod was used 10 days after flowering. Dried seeds were used about four weeks after harvest, two weeks after harvest. RNAqueous ™ (Ambion) was used to extract total RNA from pods and seeds. For extraction of total RNA from other organs, TRIZOL Reagent (Invitrogen) was used. The extraction was performed according to the manual attached to the extraction kit, and the total RNA was subjected to LiCl precipitation before the reverse transcription reaction. Reverse transcription was performed using Superscript ™ First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) according to the attached manual.

For quantitative PCR, ABI PRISM 7000 (Applied Biosystems) was used to quantify the reverse transcript by the Taqman probe method. The conditions of the thermal cycler were such that the enzyme was activated by treatment at 50 ° C for 2 minutes, followed by treatment at 95 ° C for 15 minutes. Subsequently, the denaturation reaction was performed at 95 ° C for 15 seconds, and the anneal / extension reaction was performed at 60 ° C for 1 minute, and the denaturation reaction and the anneal / extension reaction were repeated 45 times.

After completion of the reaction, prepared by diluting amplified cDNA into ^{10- 4 ~10- ¾ ο1 / ^ 1} , to prepare a calibration curve to quantify the mRNA of the target gene. For preparing a calibration curve for 18S RNA, a solution prepared by diluting 'salmon sperm DNA to lOng / 1 to Lpg / μ1 was used. Finally, each gene expression was shown as a relative value normalized with the value of 18S RNA. The reaction composition and primers used for Taqman PCR are as shown below.

<18S RNA measurement>

Reverse transcript 5 1

Pre-Developed TaqMan RNA Assay Reagents 18s ribosomal RNA (Applied Biosystems) 1.25 1

Quant iTect ™ Probe PCR Kit (QIAGEN) 12.5 μ 1

Water 6. 25 1

707A1-A4 mRNA measurement>

Reverse transcript 5 μ 1

Forward primer (ΙΟΟ μ Μ) 0.2 1

Repurse primer (100 / ζ Μ) 0.2 μ 1 TaqMan probe (50 M) 0.1 1

Quant iTect ™ Probe PCR Kit (QIAGEN) 12.5 / ^ 1

Water 7 μ 1

The forward primer, reverse primer and TaqMan probe used for quantification of CYP707A1 are as follows.

Forward primer; 5'-CTCACTCTCTTCGCCGGAAG-3 '(SEQ ID NO: 9)

Reverse primer; 5'-TTCCAAACTCCCACTCCCTCC-3 '(SEQ ID NO: 10)

TaqMan probe 5 'FAM- TGTCTAATCTCTCAGCGCCGCTTTGG- TAMRA3' (SEQ ID NO: 11)

The forward primer, reverse primer and TaqMan probe used for quantification of CYP707A2 are as follows.

Forward primer; 5'-CGTCTCTCACATCGAGCTCCTT-3 '(SEQ ID NO: 12) Reverse primer; 5, -CCAAAAGTCCATCAACACCCTC-3' (SEQ ID NO: 13)

TaqMan probe; 5 'FAM- TCCTCCAAACCCTTTCCTCTTGGACG- TAMRA3' (SEQ ID NO: 14)

The forward primer, reverse primer and TaqMan probe used for the quantification of CYP707A3 are as follows.

Forward primer; 5'-CTCTGTTTCTCTGTTTACTCCGATTTA-3 '(SEQ ID NO: 15) Reverse primer; 5, -TGCAGCAAAACAGAGAAGATACG-3' (SEQ ID NO: 16)

TaqMan probe; 5, FAM-CCGCCGTAGCTCCTCCACGAAAC-TA thigh A3 '(SEQ ID NO: 17)

The forward primer, reverse primer and TaqMan probe used for quantification of CYP707A4 are as follows.

Forward primer; 5'-CCTGAAACCATCCGTAAACTCAT-3 '(SEQ ID NO: 18) Reparse primer; 5'-TTCCTTACAATCTTGGGCCAA-3' (SEQ ID NO: 19)

TaqMan probe; 5 'FAM- CTGATATCGAGCACATTGCCCTT- TAMRA3' (SEQ ID NO: 20)

The forward primer, reverse primer and TaqMan probe used for quantification of AtNCED3 are as follows.

Forward primer; 5'-GCTGCGGTTTCTGGGAGAT-3 '(SEQ ID NO: 21)

Reparse primer; 5'-ATCGTCTTCTCAAAGCTCCGAC-3 '(SEQ ID NO: 22)

TaqMan probe; 5 'FAM-CTTGGTGGCAATCATACTCAGCCGC-TAMRA3' (SEQ ID NO: 23) As a result, the following was found. Arabidopsis CYP707A mRNA was detected in all organs examined, including rosette leaves, roots, stems, inflorescences, immature fruits, and dried seeds. In particular, immature fruits had higher CYP707A1 force S and dried seeds had higher CYP707A2 mRNA levels (Fig. 7).

Dried seeds accumulate a high concentration of ABA, and the amount of ABA decreases with water absorption, and the amount of PA and DPA increases accordingly (Fig. 8). This rapid decrease in ABA is thought to be an important step in the process of dormant seeds toward germination. Total RNA prepared from water-absorbed seeds that had been treated twice in a light place for 6, 12, 24 hours after absorption of dried seeds, potatoes, and CYP707A2 mRNA The amount of mRNA rapidly increased 6 hours after water absorption, and then decreased. On the other hand, CYP707A1 and CYP707A3 did not accumulate much mRNA in dried seeds, but tended to increase after 12 hours after water absorption. As for CYP707A4, mRNA could not be detected within 24 hours of water absorption (Fig. 9). In the studies showing the results in FIGS. 8 and 9, seeds 4 weeks after harvest were used.

On the other hand, when the plant is dried, the amount of ABA increases sharply, and when the dried plant is re-absorbed, the amount of ABA decreases rapidly (Fig. 10). The CYP707A gene mRNA level increased slowly during drying. Furthermore, when the plants dried for 6 hours were allowed to absorb water, the expression of all four CYP707A genes was activated (Fig. 12). The activation of CYP707A gene expression during re-absorption of dried plants is opposite to the expression of the NCED3 gene, which encodes a key enzyme of ABA synthesis during drying, and the NCED3 gene, which is strongly induced by drying, is re-absorbed. Therefore, its expression decreases sharply (Fig. 11). In addition, induction by 30 μΜ ABA was also observed in all CYP707A gene expression, and the induction of CYP707A1 by ABA was most prominent.

(Figure 13). In Figs. 10, 11, and 12, drying (indicated by white circles) is the result of drying the plant on a paper towel for the second week, and water absorption (indicated by black circles) is the same drying treatment for 6 hours. After the application, the paper towel was moistened to absorb water again. In the studies showing the results in FIGS. 10 to 12, seeds 2 weeks after harvest were used.

(Example 3)

In this example, knockout lines of the CYP707A2 gene and the CYP707A3 gene were created, and the phenotype related to the dormancy of the seed was analyzed. Arabidopsis Stock Center (ABRC) Using CYP707A2 gene and CYP707A3 gene obtained by obtaining T-DNA into the CYP707A3 gene obtained from (Alonso et al., Science, 301, 653-657 (2003)), homozygous strains were respectively produced.

Specifically, as shown in Figure 14, the T-DNA was inserted between the fifth exon and the fifth intron in the CYP707A2 gene, the cyp707a2-1 mutant gene, and the seventh intron in the CYP707A2 gene -Cyp707a2-2 mutant gene with inserted DNA, c-707a3-l mutant gene with T-DNA inserted into the first exon of CYP707A3 gene, and T- DNA inserted into the second etason of CYP707A3 gene The cyp707a3-2 mutant gene was obtained.

Four homozygous lines were created using these four mutant genes, and seeds were collected from the plant. Germination efficiency was examined using the collected seeds. Seeds were placed on a filter paper that had absorbed water, and the appearance of radicles was observed. The relationship between the number of days until germination and the germination rate is shown in Fig. 15 using the appearance of the radicle as the germination standard. As can be seen from Fig. 15, the germination rate of seeds in which the CYP707A3 gene was knocked out was slightly reduced as compared with the wild type. On the other hand, the germination rate of the seeds in which the CYP707A2 gene was knocked out was significantly reduced as compared with the wild type and the line in which the CYP707A3 gene was knocked out.

Fig. 16 shows the results of comparing the amounts of ABA, PA and DPA contained in the dried seeds in the wild type and in the line in which the CYP707A2 gene was knocked out. Furthermore, FIG. 17 shows the results of comparing the change in the amount of ABA contained in the seeds after the start of the water absorption treatment in the wild type and the line in which the CYP707A2 gene was knocked out. As shown in Figs. 16 and 17, in the line in which the CYP707A2 gene was knocked out, a larger amount of ABA was accumulated than in the wild type, and even after 24 hours from the start of water absorption, a larger amount of ABA was accumulated. Had been accumulated.

From these results shown in FIGS. 15 to 17, the CYP707A2 gene is a major ABA 8′-hydroxylase that determines the amount of ABA contained in seeds, and in Arabidopsis, ABA 8 ′ derived from the CYP707A2 gene -Inhibition of hydroxylase activity has been shown to maintain seed dormancy.

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial potential

According to the present invention, a novel gene having a function of inactivating ABA can be provided. Further, according to the present invention, an expression vector transformant having the novel gene, a transgenic plant and a seed can be provided.

Claims

The scope of the claims

1. A gene encoding the following protein (a) or (b):

(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8; (b) one or several amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, A protein comprising a substituted or added amino acid sequence and having abscisic acid inactivating activity

2. A gene containing the DNA of (a) or (b) below.

(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, or 7

(b) hybridizes under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, or 7, and inactivates abscisic acid Encoding a protein having activating activity

3. The gene according to claim 1, which is derived from Arabidopsis, rice or tomato.

4. A recombinant vector containing the gene according to claim 1 or 2.

5. A transformant containing the recombinant vector according to claim 4.

6. A transgenic plant comprising the gene according to claim 1 or 2.

7. A seed collected from the transgenic plant according to claim 6.

8. A plant in which the gene according to claim 1 or 2 is quartated.

9. (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or (b) an amino acid sequence wherein one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 A method for maintaining seed dormancy by inhibiting abscisic acid inactivating activity derived from a protein having abscisic acid inactivating activity.

10. The method for maintaining seed dormancy according to claim 9, wherein the activity of inactivating abscisic acid derived from the protein is inhibited by Tetcyclacis and / or Uniconazole.

1 1. (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 4, or (b) one or several amino acids are deleted in the amino acid sequence represented by SEQ ID NO: 4, Species comprising a substituted or added amino acid sequence, wherein abscisic acid-inactivating activity derived from a protein having abscisic acid-inactivating activity is inhibited.

ΎΔΟ A3