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WO2004113527A1 - Gene intervenant dans l'inactivation de l'acide abscisique - Google Patents

Gene intervenant dans l'inactivation de l'acide abscisique Download PDF

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WO2004113527A1
WO2004113527A1 PCT/JP2004/008949 JP2004008949W WO2004113527A1 WO 2004113527 A1 WO2004113527 A1 WO 2004113527A1 JP 2004008949 W JP2004008949 W JP 2004008949W WO 2004113527 A1 WO2004113527 A1 WO 2004113527A1
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aba
gene
seq
amino acid
plant
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Japanese (ja)
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Eiji Nambara
Yuji Kamiya
Tetsuo Kushiro
Masanori Okamoto
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RIKEN
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RIKEN
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8267—Seed dormancy, germination or sprouting
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • 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.
  • BACKGROUND ART 'Apsidic acid hereinafter abbreviated as ABA
  • ABA BACKGROUND ART 'Apsidic acid
  • 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).
  • 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.
  • 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).
  • 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.
  • an object of the present invention is to provide a novel gene involved in ABA inactivation and its use. Disclosure of the invention
  • 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.
  • 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.
  • 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.
  • 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.
  • the gene according to the present invention encodes a protein having a function of inactivating ABA (hereinafter, ABA inactivating enzyme).
  • the function of inactivating ABA means a function of converting ABA to phaseinate (hereinafter abbreviated as PA).
  • PA phaseinate
  • 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.
  • SAR systemic acquired resistance
  • PBZ probenazole
  • 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.
  • a surfactant such as TritonX-100 and filtered with gauze or the like.
  • the ABA inactivating enzyme gene can be cloned from genomic DNA prepared according to “(1) Preparation of plant genome”.
  • 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.
  • 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.
  • 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.
  • a database containing the genomic nucleotide sequences of various plants was searched.
  • 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.
  • accession numbers AP004129.1 and AP004162 are obtained.
  • ABA inactivating enzyme gene in rice identified in 1 can be identified.
  • Tomato Geno Database containing the base sequence of the system
  • the ABA inactivating enzyme gene in tomato identified by the accession numbers EST; AI489171 and EST247510 cLED17I4 can be identified.
  • 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.
  • 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 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%.
  • GYP707A4 56.5 57.5 57.4 SEQ ID NO: 24 52.9 67.5 SEQ ID NO: 25 49.9
  • the ABA inactivating enzyme gene can be isolated according to a conventional method.
  • “(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.
  • 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.
  • 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.
  • DNAs that hybridize under stringent conditions and that encode a protein having a function of inactivating ABA are also included.
  • the stringent conditions refer to, for example, conditions at a sodium concentration of 800 to 1000 l ⁇ m, 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)
  • TAKARA LA PCR in vitro Mutagenesis series kit .
  • 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.
  • 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 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.
  • a selection marker include an ampicillin resistance gene, a neomycin resistance gene, a dihydrofolate reductase gene, and the like.
  • 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.
  • the host is not particularly limited as long as it can express the gene of the present invention.
  • the genus Escherichia such as Escherichia coli
  • the genus Bachinoles such as Bacillus subtilis
  • the genus Pseudomonas such as Pseudomonas putida
  • 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.
  • Escherichia coli examples 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.
  • 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.
  • 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.
  • a method using calcium ions [Cohen, SN, et al .: Proc. Natl. Acad. Sci., USA, 69: 2110-2114 (1972)], an electroporation method, and the like.
  • yeast When yeast is used as a host, for example, Saccharomyces 'Celebiche, Schizosaccharomyces' bomb, Pichia's Pastris and the like are used.
  • 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.
  • 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)].
  • a plant cell for example, a cell established from Arabidopsis thaliana, tobacco, sorghum, rice, carrot, etc., or protoplasts prepared from the plant are used.
  • 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.
  • 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.
  • 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.
  • Sf9 cells When insect cells are used as a host, Sf9 cells, Sf21 cells, and the like are used.
  • 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.
  • 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.
  • culture means any of a culture supernatant, a cultured cell or a cultured bacterial cell, and a crushed cell or bacterial cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an inducer may be added to the medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • isopropyl- / 3-D-thiogalatatopyranoside (IPTG) is transformed with an expression vector using the trp promoter.
  • IPTG isopropyl- / 3-D-thiogalatatopyranoside
  • IAA indole ataryl acid
  • the ABA-inactivating enzyme 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNAi RNA interference
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • a transgenic plant into which a mutant ABA inactivating enzyme gene has been introduced can be prepared as follows.
  • 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.
  • 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.
  • the target gene is inserted between the boundary sequences (LB, RB) of the binary vector, and the recombinant vector is amplified in E. coli.
  • 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.
  • 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)].
  • a plant promoter or a terminator 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.
  • the terminator sequence include a terminator derived from cauliflower mosaic virus-derived ⁇ nopaline synthase gene.
  • the present invention is not limited to these promoters and terminators as long as they are known to function in plants.
  • 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.
  • Adhl corn alcohol dehydrogenase
  • 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 ( ⁇ ), the hygromycin phosphotransferase (htp) gene that confers resistance to the antibiotic hygromycin to plants, and bialaphos resistance.
  • htp hygromycin phosphotransferase
  • bar phosphinothricin acetyltransferase
  • 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.
  • 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.).
  • a plant organ eg, leaf, petal, stem, root, rhizome, seed, etc.
  • a plant tissue eg, epidermis, phloem, soft tissue
  • Xylem vascular bundle, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. '
  • 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).
  • the Bam HI site of pYeDP60 was blunt-ended, and ligation was performed using Eco RI-treated Bam HI site.
  • 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.
  • 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.
  • FIGS. 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.
  • FIGS. 1 to 3 shows the results when the reaction solution from the transformed yeast into which only empty pYeDP60 plasmid was introduced was subjected to HPLC.
  • the PA peak is shown as “PA”
  • the ABA peak is shown as “ABAJ”.
  • 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.
  • 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.
  • CYP707A2 cDNA and CYP707A4 cDNA were obtained from dried seeds and immature seeds according to a standard method.
  • 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.
  • 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.
  • the cDNA of CYP707A2 and the cDNA of CYP707A4 could be identified as genes encoding ABA 8'-hydroxylase.
  • ABA 8'-hydroxylase inhibitors were studied for the biochemical analysis of Arabidopsis ABA 8'-hydroxylase (here, CYP707A1).
  • CYP707A1 Arabidopsis ABA 8'-hydroxylase
  • Tetcyclacis, Metyrapone and Uniconazole known as P450 inhibitors were used as the inhibitor.
  • CYP707A1, A2, A3 and A4 mRNA in Arabidopsis thaliana was analyzed by quantitative PCR.
  • RNAqueous TM (Ambion) was used to extract total RNA from pods and seeds.
  • TRIZOL Reagent (Invitrogen) was used for extraction of total RNA from other organs. 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 TM First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) according to the attached manual.
  • 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.
  • the forward primer, reverse primer and TaqMan probe used for quantification of CYP707A1 are as follows.
  • the forward primer, reverse primer and TaqMan probe used for quantification of CYP707A2 are as follows.
  • the forward primer, reverse primer and TaqMan probe used for the quantification of CYP707A3 are as follows.
  • the forward primer, reverse primer and TaqMan probe used for quantification of CYP707A4 are as follows.
  • the forward primer, reverse primer and TaqMan probe used for quantification of AtNCED3 are as follows.
  • 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).
  • 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.
  • CYP707A1 and CYP707A3 did not accumulate much mRNA in dried seeds, but tended to increase after 12 hours after water absorption.
  • 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.
  • 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.
  • 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.
  • Fig. 15 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention porte sur un nouveau gène intervenant dans l'inactivation de l'acide abscisique (ABA) et ses utilisations, et en particulier sur un gène codant pour: (a) une protéine consistant en une séquence d'acide aminé de SEQ ID NO. 2, 4, 6, 8, ou (b) une protéine consistant en une séquence d'acide aminé de SEQ ID NO. 2, 4, 6, 8 ayant subi une suppression, substitution ou addition d'un ou plusieurs acides aminés, et qui a pour effet l'inactivation de l'acide abscisique.
PCT/JP2004/008949 2003-06-20 2004-06-18 Gene intervenant dans l'inactivation de l'acide abscisique Ceased WO2004113527A1 (fr)

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WO2007045040A1 (fr) 2005-10-20 2007-04-26 Commonwealth Scientific And Industrial Research Organisation Céréales à dormance modifiée
CN110577938A (zh) * 2019-11-11 2019-12-17 中国农业科学院生物技术研究所 ABA 8’-羟化酶基因OsABA8ox2在植物光形态建成和根发育中的应用
CN114540407A (zh) * 2022-01-13 2022-05-27 安庆市长三角未来产业研究院 SlCYP707A基因作为负调控因子在促进番茄亚低温抗性中的应用

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EP1033405A2 (fr) * 1999-02-25 2000-09-06 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007045040A1 (fr) 2005-10-20 2007-04-26 Commonwealth Scientific And Industrial Research Organisation Céréales à dormance modifiée
US8269082B2 (en) 2005-10-20 2012-09-18 Commonwealth Scientific And Industrial Research Organisation Cereals with altered dormancy
AU2006303820B2 (en) * 2005-10-20 2013-06-20 Commonwealth Scientific And Industrial Research Organisation Cereals with altered dormancy
CN110577938A (zh) * 2019-11-11 2019-12-17 中国农业科学院生物技术研究所 ABA 8’-羟化酶基因OsABA8ox2在植物光形态建成和根发育中的应用
CN110577938B (zh) * 2019-11-11 2020-03-10 中国农业科学院生物技术研究所 ABA 8’-羟化酶基因OsABA8ox2在植物光形态建成和根发育中的应用
CN114540407A (zh) * 2022-01-13 2022-05-27 安庆市长三角未来产业研究院 SlCYP707A基因作为负调控因子在促进番茄亚低温抗性中的应用
CN114540407B (zh) * 2022-01-13 2023-11-28 安庆市长三角未来产业研究院 SlCYP707A基因作为负调控因子在促进番茄亚低温抗性中的应用

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