JP2007082557A - Gene encoding environmental stress-responding transcription factor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene encoding a stress-responding transcription factor. <P>SOLUTION: This gene comprises (a) a DNA composed of any base sequence selected from specific base sequences, (b) a DNA composed of a base sequence in which one or a plurality of bases are deleted, substituted or added in any base sequence selected from specific base sequences and encoding an environmental stress-responding transcription factor or (c) a DNA hybridizing under stringent conditions with DNA composed of any base sequence selected from specific base sequences and encoding the environmental stress-responding transcription factor. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a gene encoding an environmental stress responsive transcription factor.

  Gene sequencing projects have determined large amounts of genomic and cDNA sequences for several organisms, and in the plant model Arabidopsis thaliana, the complete genomic sequence of two chromosomes has been determined ( Lin, X. et al., (1999) Nature 402, 761-768 .; Mayer, K. et al., (1999) Nature 402, 769-777.).

  The expressed sequence tag (EST) project has also contributed greatly to the discovery of expressed genes (Hofte, H. et al., (1993) Plant J. 4, 1051-1061 .; Newman, T. et al., ( 1994) Plant Physiol. 106, 1241-1255 .; Cooke, R. et al., (1996) Plant J. 9, 1O1-124. Asamizu, E. et al., (2000) DNA Res. 7, 175- 180.). For example, dbEST (National Center for Biotechnology Information (NCBI) EST database) contains partial cDNA sequences, and more than half of all genes (i.e. about 28,000 genes) are reproduced (fully sequenced). Estimated from the gene content of chromosome 2 of Arabidopsis thaliana [Lin, X. et al., (1999) Nature 402, 761-768.]).

In recent years, microarray (DNA chip) technology has become a useful tool for analyzing genome-scale gene expression (Schena, M. et al., (1995) Science 270, 467-470 .; Eisen, MB and Brown, PO (1999) Methods Enzymol. 303, 179-205.). In this technique using a DNA chip, cDNA sequences are arranged on a slide glass at a density of 1,000 genes / cm ² or more. By directly hybridizing the thus-arranged cDNA sequences to two-color fluorescently labeled cDNA probe pairs prepared from RNA samples of different cell types or tissue types, gene expression can be directly and comparatively analyzed in large quantities. It becomes. This technique was first demonstrated by analyzing 48 Arabidopsis genes for differential expression in roots and shoots (Schena, M. et al., (1995) Science 270, 467-470.). In addition, microarrays have been used to investigate 1,000 clones randomly picked from human cDNA libraries to identify novel genes that respond to heat shock and protein kinase C activation (Schena, M et al., (1996) Proc. Natl. Acad. Sci. USA, 93, 10614-10619.).

  On the other hand, the expression profile of inflammatory disease-related genes under various induction conditions has been analyzed by the method using this DNA chip (Heller, RA et al., (1997) Proc. Natl. Acad. Sci. USA, 94, 2150-2155.). In addition, the dynamic expression of yeast genome consisting of more than 6,000 coding sequences has been analyzed using microarrays (DeRisi, JL et al., (1997) Science 278, 680-686 .; Wodicka, L. et al., (1997) Nature Biotechnol. 15, 1359-1367.).

  However, in the field of plants, only a few reports have been made on microarray analysis (Schena, M. et al., (1995) Science 270, 467-470 .; Ruan, Y. et al. , (1998) Plant J. 15, 821-833 .; Aharoni. A. et al., (2000) Plant Cell 12, 647-661 .; Reymond, P. et al., (2000) Plant Cell 12, 707 -719.)

  Plant growth is significantly affected by environmental stresses such as dryness, high salt concentration, and low temperature. Of these stresses, drought or water deficiency is the most severe limiting factor for plant growth and crop production. Drought stress causes various biochemical and physiological responses to plants.

  Plants acquire responsiveness and adaptability to stress in order to survive under these stress conditions. Recently, several genes that respond to desiccation at the transcriptional level have been described (Bohnert, HJ et al., (1995) Plant Cell 7, 1099-1111 .; Ingram, J., and Bartels, D. (1996 ) Plant Mol. Biol. 47, 377-403 .; Bray, EA (1997) Trends Plant Sci. 2, 48-54 .; Shinozaki. K., and Yamaguchi-Shinozaki, K. (1997) Plant Physiol. 115, 327-334.; Shinozaki, K., and Yamaguchi-Shinozaki, K. (1999). Molecular responses to drought stress. Molecular responses to cold, drought, heat and salt stress in higher plants. Edited by Shinozaki, K. and Yamaguchi -Shinozaki, KRG Landes Company .; Shinozaki, K., and Yamaguchi-Shinozaki, K. (2000) Curr. Opin. Plant Biol. 3, 217-223.).

  On the other hand, stress-inducible genes have been used to improve plant stress tolerance by gene transfer (Holmberg, N., and Bulow, L. (1998) Trends Plant Sci. 3, 61-66 .; Bajaj , S. et al., (1999) Mol. Breed. 5, 493-503.). It is important to analyze the function of stress-inducible genes not only to further elucidate the molecular mechanisms of stress tolerance and stress response in higher plants, but also to improve crop stress tolerance by genetic manipulation.

  DRE / CRT (Dry Responsive Element / C-Repetitive Sequence) is a dry, high salinity and low temperature stress responsive gene ABA (abscisic acid: a plant hormone that functions as a signaling factor for seed dormancy and environmental stress) Has been identified as an important cis-acting element in independent expression (Yamaguchi-Shinozaki, K., and Shinozaki, K. (1994) Plant Cell 6, 251-264 .; Thomashow, MF et al. (1999) Plant Mol. Biol. 50, 571-599 .; Shinozaki, K., and Yamaguchi-Shinozaki, K. (2000) Curr. Opin. Plant Biol. 3, 217-223.). In addition, a transcription factor (DREB / CBF) involved in DRE / CRT-responsive gene expression has been cloned (Stockinger. EJ et al., (1997) Proc. Natl. Acad. Sci. USA 94, 1035-1040. Liu, Q. et al., (1998) Plant Cell 10, 1391-1406 .; Shinwari, ZK et al., (1998) Biochem. Biophys. Res. Commun. 250, 161-170 .; Gilmour, SJet al., (1998) Plant J. 16, 433-443.). DREB1 / CBF is thought to function in cold-responsive gene expression, and DREB2 is involved in drought-responsive gene expression. Strong tolerance to freezing stress has been observed in transgenic Arabidopsis plants that overexpress CBF1 (DREB1B) cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter (Jaglo-Ottosen, KR et al., ( 1998) Science 280, 104-106.).

  We over-expressed DREB1A (CBF3) cDNA in transgenic plants under the control of the CaMV 35S promoter or the stress-inducible rd29A promoter, causing a strong constitutive expression of the stress-inducible DREB1A target gene, It has been reported that tolerance to freezing, drought and salt stress is improved (Liu, Q. et al., (1998) Plant Cell 10, 1391-1406 .; Kasuga, M. et al., (1999 ) Nature Biotechnol. 17, 287-291.). In addition, the present inventors have already identified six DREB1A target genes such as rd29A / lti78 / cor78, kin1, kin2 / cor6.6, cor15a, rd17 / cor47 and erd10 (Kasuga, M. et al (1999) Nature Biotechnol. 17, 287-291.). However, it has not been fully elucidated how overexpression of DREB1A cDNA in transgenic plants increases stress tolerance to freezing, drying and salinity. In order to study the molecular mechanism of drought and freeze resistance, it is important to identify and analyze more genes regulated by DREB1A.

  Meanwhile, DREB1A is known as a transcription factor showing responsiveness to environmental stress (Japanese Patent Laid-Open No. 2000-60558). Regarding DREB1A, by overexpressing the DREB1A gene in a plant body, the environmental stress resistance of the plant body can be increased. However, there are not many known transcription factors that can increase the environmental stress tolerance of plants.

  Accordingly, an object of the present invention is to provide a gene encoding an environmental stress responsive transcription factor.

  As a result of earnest research to solve the above-mentioned problems, the present inventor applied a cDNA microarray analysis to obtain a transcription factor that specifically responds to environmental stresses such as low temperature stress, drought stress, and salt stress. The present inventors have succeeded in identifying a gene to be encoded and have completed the present invention.

That is, the present invention includes the following.
(1) A gene encoding an environmental stress responsive transcription factor comprising the following amino acid sequence (a) or (b):
(a) any amino acid sequence selected from SEQ ID NO: 2n (n = integer from 1 to 36)
(b) It consists of an amino acid sequence in which one or more amino acids are deleted, substituted or added in any amino acid sequence selected from SEQ ID NO: 2n (n = integer from 1 to 36), and is responsive to environmental stress. And an amino acid sequence having transcription factor activity

  (2) The gene according to (1), which comprises the following DNA (a), (b) or (c):

(A) DNA comprising any nucleotide sequence selected from SEQ ID NO: 2n-1 (n = 1 to 36)
(B) An environmental stress responsive transcription comprising a base sequence in which one or more bases are deleted, substituted or added in any base sequence selected from SEQ ID NO: 2n-1 (integer of n = 1 to 36). DNA encoding the factor
(C) DNA that hybridizes under stringent conditions with DNA consisting of any one of the nucleotide sequences selected from SEQ ID NO: 2n-1 (n = 1 to 36), and that encodes an environmental stress-responsive transcription factor

(3) The gene according to (1) or (2), wherein the environmental stress is at least one selected from the group consisting of low temperature stress, drought stress and salt stress.
(4) An expression vector comprising the gene according to (1) or (2).
(5) A transformant comprising the expression vector according to (4).
(6) A transgenic plant comprising the expression vector according to (4).

  (7) The transgenic plant according to (6), wherein the plant is a plant body, plant organ, plant tissue, or plant cultured cell.

  (8) A method for producing a stress-tolerant plant, comprising culturing or cultivating the transgenic plant according to (6) or (7).

  According to the present invention, a gene encoding a stress-responsive transcription factor is provided. The gene of the present invention is useful in that it can be used for molecular breeding of environmental stress resistant plants.

Hereinafter, the present invention will be described in detail.
The inventors of the present invention have used Arabidopsis thaliana from plants with different conditions, such as dry-treated plants and low-temperature treated plants, by biotinylated CAP trapper method (Carninci. P. et al., (1996) Genomics, 37, 327-336.). A full-length cDNA library was constructed (Seki. M. et al., (1998) Plant J. 15, 707-720.) And about 7,000 full-length cDNAs containing stress-inducible genes were used to complete Arabidopsis thaliana. Each long cDNA microarray was prepared. In addition to these dry / cold-inducible full-length cDNAs, a cDNA microarray was prepared using a gene targeted by DREB1A, a transcriptional regulator that controls the expression of stress-responsive genes. Then, the gene expression pattern under drought stress and low temperature stress was monitored, and the stress responsive genes were comprehensively analyzed. As a result, 277 drying-inducible genes, 53 cold-inducible genes, and 194 high-salt stress-inducible genes were isolated from a cDNA microarray containing about 7,000 full-length cDNAs.

Then, by encoding the nucleobase sequence of these environmental stress responsive genes into amino acid sequences, a blast X search is performed on the amino acid sequences Data registered in the GenBank Database, so that transcription factors are encoded (known transcriptions). It has succeeded in identifying the gene encoding the factor and E-value (which shows homology with a score higher than e- ¹⁰ ). In the following description, the transcription factor identified in the present invention may be referred to as an environmental stress responsive transcription factor.

As described above, the full-length cDNA microarray is an effective tool for analyzing the expression pattern of a dry and low-temperature stress-inducible gene in Arabidopsis and the target gene of a stress-related transcriptional regulatory factor.
1. Isolation of transcription factor A transcription factor has a function of binding to a cis element existing upstream of a gene and activating transcription of the downstream gene. The transcription factor isolated in the present invention is induced by environmental stress such as low temperature, drying, and high salt concentration.

  Environmental stress responsive transcription factors are those belonging to the DREB family, those belonging to the ERF family, those belonging to the zinc finger family, those belonging to the WRKY family, those belonging to the MYB family, those belonging to the bHLH family, those belonging to the NAC family These are broadly classified into those belonging to the homeodomain family and those belonging to the bZIP family.

  In isolating a transcription factor, first, a stress responsive gene is isolated using a microarray. Microarrays include genes isolated from the Arabidopsis full-length cDNA library, RD (Responsive to Dehydration) gene, ERD (Early Responsive to Dehydration) gene, and λ control template DNA fragment (TX803, Takara Shuzo) as an internal standard. As a negative control, a total of about 7000 cDNA consisting of mouse nicotinic acetylcholine receptor epsilon subunit (nAChRE) gene and mouse glucocorticoid receptor homologous gene should be used as negative controls. Can do.

  Plasmid DNA extracted using a Kurabo plasmid preparation apparatus is used for sequence analysis, and the sequence is determined by a DNA sequencer (ABI PRISM 3700. PE Applied Biosystems, CA, USA). Based on the GenBank / EMBL database, a sequence homology search is performed using the BLAST program.

  Next, after poly A selection, reverse transcription is performed to synthesize double-stranded DNA, and the cDNA is inserted into a vector.

  The cDNA inserted into the cDNA library creation vector is amplified by PCR using primers complementary to the vector sequences on both sides of the cDNA. Examples of the vector include λZAPII and λPS.

  The microarray can be produced according to a usual method, and is not particularly limited. For example, using the gene tip microarray stamp machine GTMASS SYSTEM (manufactured by Nippon Laser & Electronics Lab.), The PCR product is loaded from a microtiter plate and spotted on a micro slide glass at predetermined intervals. Thereafter, the slide is immersed in a blocking solution to prevent the expression of non-specific signals.

  Plant materials include wild-type strains and strains of specific genes, but transgenic plants into which DREB1A cDNA has been introduced can be used. Examples of the plant species include Arabidopsis thaliana, tobacco, rice and the like, but Arabidopsis thaliana is preferable.

  The drying and low-temperature stress treatment can be performed by a known method (Yamaguchi-Shinozaki, K., and Shinozaki, K. (1994) Plant Cell 6, 251-264.).

  After exposure to stress treatment, plant bodies (wild type and DREB1A overexpression transformants) are sampled and stored frozen using liquid nitrogen. Wild type and DREB1A overexpressing transformants are used in experiments to identify target genes for DREB1A. MRNA is isolated and purified from the plant using a known method or kit.

  Each mRNA sample is reverse-transcribed in the presence of Cy3 dUTP or Cy5 dUTP (Amersham Pharmacia) for labeling and used for hybridization.

  After hybridization, the microarray is scanned using a scanning laser microscope or the like. As a microarray data analysis program, Imagene Ver 2.0 (BioDiscovery), QuantArray (GSI Lumonics), or the like can be used.

  After scanning, the gene is isolated by preparing a plasmid having the target gene.

  The transcription factor is determined by analyzing the base sequence of the isolated gene and using a gene analysis program based on the genome information in the database (GenBank / EMBL, ABRC). The isolated genes can be classified into those having both drought stress-inducible and low-temperature stress-inducible properties, those specific to drought stress inducibility, and those specific to low-temperature stress inducibility. According to the gene analysis program, genes encoding 36 types of transcription factors are identified from the above genes. The nucleotide sequences of the genes encoding these 36 types of transcription factors are shown in SEQ ID NO: 2n-1 (n is an integer of 1 to 36), and the amino acid sequences of transcription factors are shown in SEQ ID NO: 2n (n is an integer of 1 to 36). Table 1 shows the sequence number and the gene name of the gene encoding the transcription factor.

  However, as long as the transcription factor of the present invention functions as an environmental stress responsive transcription factor, one or more, preferably 1 in any base sequence selected from SEQ ID NO: 2n-1 (n is an integer of 1 to 36). Alternatively, it may have a base sequence in which several (eg, 1 to 10, 1 to 5) bases are deleted, substituted, or added. Further, a DNA that hybridizes under stringent conditions with a DNA comprising any one of the nucleotide sequences selected from SEQ ID NO: 2n-1 (n is an integer of 1 to 36) and encodes an environmental stress responsive transcription factor And included in the transcription factor of the present invention. Here, the stringent conditions are a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68 ° C., preferably 42 to 65 ° C. More specifically, 5 × SSC (83 mM NaCl, 83 mM sodium citrate), temperature 42 ° C.

The 36 types of transcription factors isolated in the present invention can be classified as follows.
1) DREB family: RAFL05-11-M11, RAFL06-11-K21, RAFL05-16-H23, RAFL08-16-D16
2) ERF family: RAFL08-16-G17, RAFL06-08-H20
3) Zinc finger family: RAFL07-10-G04, RAFL04-17-D16, RAFL05-19-M20, RAFL08-11-M13, RAFL04-15-K19, RAFL05-11-L01, RAFL05-14-C11, RAFL05- 19-G24, RAFL05-20-N02
4) WRKY family: RAFL05-18-H12, RAFL05-19-E19, RAFL06-10-D22, RAFL06-12-M01
5) MYB family: RAFL05-14-D24, RAFL05-20-N17, RAFL04-17-F21
6) bHLH family: RAFL09-12-N16
7) NAC Family: RAFL05-19-I05, RAFL05-21-I22, RAFL08-11-H20, RAFL05-21-C17, RAFL05-08-D06
8) Homeodomain family: RAFL05-20-M16, RAFL11-01-J18, RAFL11-09-C20
9) bZIP family: RAFL05-18-N16, RAFL11-10-D10, RAFL04-17-N22, RAFL05-09-G15
RAFL05-21-L12 cannot be classified into the above 1) to 9).

  Once the base sequence of the gene encoding the transcription factor of the present invention has been determined, it is then hybridized by chemical synthesis, by PCR using the cloned probe as a template, or by using a DNA fragment having the base sequence as a probe. By doing so, a gene encoding the transcription factor of the present invention can be obtained. Furthermore, a mutant form of the gene encoding the transcription factor of the present invention having a function equivalent to that of the gene encoding the transcription factor before mutation can also be synthesized by site-directed mutagenesis.

  In order to introduce a mutation into the base sequence of a gene encoding a transcription factor, a known method such as the Kunkel method or the Gapped duplex method or a method equivalent thereto can be employed. For example, using a mutagenesis kit (for example, Mutant-K (TAKARA) or Mutant-G (TAKARA)) using site-directed mutagenesis, or TAKARA LA PCR in vitro Mutagenesis Mutation is introduced using a series kit.

  Here, “environmental stress” generally means abiotic stress, such as drought stress, low temperature stress, high salt concentration stress, intense light stress, and the like. “Dry” means a state deficient in moisture, and “low temperature” means a state exposed to a temperature lower than the optimum temperature of each species (for example, a temperature of -20 to + 21 ° C. in Arabidopsis thaliana). In addition, “high salt concentration” means a state in which NaCl at a concentration of 50 mM to 600 mM is continuously treated for 0.5 hour to several weeks. “Intense light stress” means a state in which a plant is irradiated with strong light exceeding the photosynthetic ability, and for example, it is applied when light of 5,000 to 10,000 Lx or more is applied. May be loaded, or a plurality of types may be loaded.

2. Construction of Expression Vector The expression vector of the present invention can be obtained by linking (inserting) a gene encoding the transcription factor of the present invention to an appropriate vector. The vector for inserting the gene encoding the transcription factor of the present invention is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, shuttle vectors, helper plasmids and the like.

  As plasmid DNA, plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YCp50, etc.) And λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.

  In order to insert a gene encoding the transcription factor of the present invention into a vector, first, the purified DNA is cleaved with an appropriate restriction enzyme and inserted into a restriction enzyme site or a multicloning site of an appropriate vector DNA. The method of connecting is adopted.

  When the gene encoding the transcription factor of the present invention is used by linking a reporter gene, for example, a GUS gene widely used in plants, to its 3 ′ end, the intensity of expression of the gene can be determined by examining GUS activity. Can be easily evaluated. In addition to the GUS gene, luciferase, green fluorescein protein, and the like can be used as the reporter gene.

3. Production of transformant The transformant of the present invention can be obtained by introducing the expression vector of the present invention into a host. Here, the host is not particularly limited as long as it can express the gene encoding the transcription factor of the present invention, but a plant is preferable. When the host is a plant, a transformed plant (transgenic plant) can be obtained as follows.

Plants to be transformed in the present invention include whole plants, plant organs (e.g. leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g. epidermis, phloem, soft tissue, xylem, vascular bundles, etc.) ) Or plant cultured cells. Examples of plants used for transformation include plants belonging to the Brassicaceae, Gramineae, Solanum, Legumes, etc. (see below), but are not limited to these plants.
Brassicaceae: Arabidopsis thaliana
Solanum: Tobacco (Nicotiana tabacum)
Gramineae: maize (Zea mays), rice (Oryza sativa)
Legumes: Soybean (Glycine max)

  The recombinant vector can be introduced into a plant by a conventional transformation method such as electroporation (electroporation), Agrobacterium method, particle gun method, PEG method and the like.

  For example, when the electroporation method is used, the gene is introduced into the host by treatment with an electroporation apparatus equipped with a pulse controller under conditions of a voltage of 500 to 1600 V, 25 to 1000 μF, and 20 to 30 msec.

  When the particle gun method is used, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The sample thus prepared can be processed using a gene introduction apparatus (for example, PDS-1000 / He manufactured by Bio-Rad). The treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 1000-1800 psi and a distance of about 5-6 cm.

  Moreover, a target gene can be introduced into a plant body by using a plant virus as a vector. Examples of plant viruses that can be used include cauliflower mosaic virus. That is, first, a virus genome is inserted into a vector derived from E. coli to prepare a recombinant, and then these target genes are inserted into the virus genome. The gene of interest can be introduced into a plant host by excising the viral genome thus modified from the recombinant with a restriction enzyme and inoculating the plant host.

  In the method using the Agrobacterium Ti plasmid, when a bacterium belonging to the genus Agrobacterium infects a plant, it takes advantage of the property of transferring a part of the plasmid DNA that it has into the plant genome. The target gene is introduced into the plant host. Among the bacteria belonging to the genus Agrobacterium, Agrobacterium tumefaciens infects plants to form a tumor called crown gall, and Agrobacterium u rhizogenes infects plants. To generate hairy roots. These are because the region called T-DNA region (Transferred DNA) on the plasmid that is present in each bacterium called Ti plasmid or Ri plasmid at the time of infection is transferred into the plant and integrated into the genome of the plant. This is due to

  If the DNA to be incorporated into the plant genome is inserted into the T-DNA region on the Ti or Ri plasmid, the target DNA is transferred into the plant genome when Agrobacterium bacteria infect the plant host. Can be incorporated.

  Tumor tissue, shoots, hairy roots, and the like obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be used at an appropriate concentration using a conventionally known plant tissue culture method. Of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).

  The vector of the present invention is not only introduced into the plant host, but also Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, or Pseudomonas putida such as Pseudomonas putida. Transformants are introduced into yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, animal cells such as COS cells and CHO cells, or insect cells such as Sf9. It can also be obtained. When a bacterium such as E. coli or yeast is used as a host, the recombinant vector of the present invention can autonomously replicate in the bacterium, and at the same time, a gene encoding the transcription factor of the present invention, a ribosome located upstream of the gene It is preferably composed of a binding sequence, a promoter sequence, and a transcription termination sequence located downstream of the gene.

  The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

  When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used. 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. Examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

  When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.

  When insect cells are used as hosts, Sf9 cells and the like are used. Examples of the method for introducing a recombinant vector into insect cells include the calcium phosphate method, lipofection method, electroporation method and the like.

  Whether or not the gene has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization or the like. For example, DNA is prepared from the transformant, PCR is performed by designing a DNA-specific primer. PCR is performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, Confirm that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence or enzyme reaction may be employed.

4). Manufacture of a plant In this invention, it can reproduce | regenerate to a transformed plant body from the said transformed plant cell. As a regeneration method, a method is employed in which callus-like transformed cells are transferred to a medium with different hormone types and concentrations and cultured to form somatic embryos to obtain complete plants. Examples of the medium to be used include LS medium and MS medium.

  The “method for producing a plant body” of the present invention comprises introducing a plant expression vector into which a gene encoding the environmental stress responsive transcription factor is inserted into a host cell to obtain a transformed plant cell, and the transformed plant cell. From which the plant is regenerated, plant seeds are obtained from the obtained transformed plants, and the plant is produced from the plant seeds.

  In order to obtain plant seeds from transformed plants, for example, the transformed plants are collected from the rooting medium, transplanted to pots containing water-containing soil, and grown at a constant temperature to form flowers. And finally seeds are formed. In order to produce a plant from seeds, for example, when the seed formed on the transformed plant has matured, it is isolated, sown in water-containing soil, and grown under constant temperature and illuminance. As a result, a plant body is produced. The plant bred in this way becomes an environmental stress resistant plant according to the stress responsiveness of the gene encoding the introduced environmental stress responsive transcription factor.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Isolation of a gene encoding an environmental stress responsive transcription factor Materials and methods
(1) Arabidopsis cDNA clone
In addition to genes isolated from the full-length cDNA library of Arabidopsis, RD (Responsive to Dehydration) gene, ERD (Early Responsive to Dehydration) gene, kin1 gene, kin2 gene, cor15a gene, and λ control template DNA as internal standard As a negative control, a total of about 7000 cDNAs of a mouse nicotinic acetylcholine receptor epsilon subunit (nAChRE) gene and a mouse glucocorticoid receptor homologous gene were used for microarray preparation.
Positive control: desiccation-inducible gene (dehydration responsiveness: rd and initial dehydration responsive gene: erd)
Internal standard: Fragment obtained by PCR amplification of λ control template DNA (TX803, manufactured by Takara Shuzo Co., Ltd., hereinafter referred to as “control fragment”)
Negative control: nicotinic acetylcholine receptor ε subunit (nAChRE) gene and mouse glucocorticoid receptor homolog that has virtually no homology to any sequence in the Arabidopsis database to assess non-specific hybridization gene

(2) Arabidopsis full-length cDNA microarray Using the biotinylated CAP trapper method, the present inventor was able to analyze Arabidopsis plants from different conditions (for example, dry treatment, low temperature treatment and untreatment at various growth stages from germination to mature seeds). ) To construct a full-length cDNA library. From the full-length cDNA library, the present inventors isolated about 7000 independent Arabidopsis full-length cDNAs, respectively. According to a known method (Eisen and Brown, 1999), cDNA fragments amplified by PCR were aligned on a slide glass. The inventor prepared a full-length cDNA microarray containing about 7000 Arabidopsis full-length cDNAs containing the following genes.

(3) Dryness-inducible gene, low-temperature-inducible gene, and high salt concentration-inducible gene using cDNA microarray In this example, a dryness-inducible gene using a full-length cDNA microarray containing about 7000 full-length Arabidopsis cDNAs A low temperature inducible gene and a high salt concentration inducible gene were isolated.

Cy3 and Cy5 fluorescently labeled probes of the above-mentioned various stressed plants and non-stressed plants were mixed and hybridized with a full-length cDNA microarray containing about 7000 full-length Arabidopsis cDNAs. Double labeling of a pair of cDNA probes that label one mRNA sample with Cy3-dUTP and the other mRNA sample with Cy5-dUTP allows for simultaneous hybridization to DNA elements on the microarray. Direct quantitative measurement of gene expression between conditions (ie stressed or unstressed) is facilitated. The hybridized microarray was scanned by two separate laser channels for Cy3 and Cy5 emission from each DNA element. Subsequently, the intensity ratio of the two fluorescent signals of each DNA element was measured as a relative value, and the change in differential expression of the gene represented by the cDNA spot on the microarray was determined. In this example, an α-tubulin gene whose expression level was almost equivalent under the two experimental conditions to be analyzed was used as an internal control gene.
The identification procedure of a drought-inducible gene, a low-temperature-inducible gene and a high salt concentration-inducible gene in a full-length cDNA microarray containing about 7000 Arabidopsis full-length cDNAs is shown.

  Among the stresses described above, mRNA derived from a plant loaded with one kind of stress and mRNA derived from an unstressed wild type plant were used for the preparation of a Cy3-labeled cDNA probe and a Cy5-labeled cDNA probe, respectively. These cDNA probes were mixed and hybridized with a cDNA microarray. In this example, a control fragment having almost the same expression level under the two conditions was used as an internal control gene. Genes whose expression ratio (dry / no stress, low temperature / no stress, or high salt concentration / no stress) exceeds 5 times that of the control fragment were determined to be genes induced by the applied stress.

(4) Sequence analysis Plasmid DNA extracted using a DNA extractor (model Biomek, manufactured by Beckman Coulter) and purified using a multi-screen 96-well filter plate (manufactured by Millipore) for homology search of gene sequences Was used for sequence analysis. The sequence analysis was determined by the dye terminator cycle sequencing method using a DNA sequencer (ABI PRISM 3700. PE Applied Biosystems, CA, USA). Based on the GenBank / EMBL database, sequence homology searches were performed using the BLAST program.

(5) cDNA amplification
λZAP and λFLC-1 were used as cDNA library production vectors. The cDNA inserted into the library vector was amplified by PCR using primers complementary to the vector sequences on both sides of the cDNA.
Primer sequences are as follows.
FL forward 1224: 5'-CGCCAGGGTTTTCCCAGTCACGA (SEQ ID NO: 73)
FL reverse 1233: 5'-AGCGGATAACAATTTCACACAGGA (SEQ ID NO: 74)

  Plasmid (1-2 ng) was added as a template to 100 μl of PCR mixture (0.25 mM dNTP, 0.2 μM PCR primer, 1 × Ex Taq buffer, 1.25 U Ex Taq polymerase (Takara Shuzo)). PCR was first reacted at 94 ° C for 3 minutes, followed by 35 cycles of 95 ° C for 1 minute, 60 ° C for 30 seconds and 72 ° C for 3 minutes, and finally 72 ° C for 3 minutes. went. The PCR product was ethanol precipitated and then dissolved in 25 μl of 3 × SSC. The quality of the obtained DNA and the amplification efficiency of PCR were confirmed by electrophoresis using 0.7% agarose gel.

(6) Preparation of cDNA microarray
Using a gene tip microarray stamp machine GTMASS SYSTEM (Nippon Laser & Electronics Lab.), 0.5 μl of PCR product (500-1000 ng / ml) was loaded from a 384-well microtiter plate, and 48 microslide glasses 0.5 nl each was spotted on a model Super Aldehyde substrate (manufactured by Telechem International) at intervals of 300 μm. The slide after spotting was dried in an atmosphere with a relative humidity of 30% or less and subjected to ultraviolet irradiation for a crosslinking reaction.

  Thereafter, the slide was shaken in 0.2% SDS for 2 minutes three times, and then treated to penetrate in distilled water twice. Thereafter, the slide was placed on a slide rack, and the slide rack was placed in a chamber containing hot water and left for 2 minutes. The blocking solution (containing 1 g borohydride, 300 ml PBS and 90 ml 100% ethanol) was then poured into the chamber. After gently shaking the glass chamber containing the slide rack, the slide rack was transferred to a chamber containing 0.2% SDS and gently shaken for 1 minute three times. Thereafter, the slide rack was transferred to a glass chamber containing distilled water, gently shaken for 1 minute, and then dried by centrifuging for 20 minutes.

(7) Isolation of plant material and RNA Plant material was seeded on agar medium and cultivated for 3 weeks (Yamaguchi-Shinozaki and Shinozaki, 1994). The wild type and cauliflower mosaic virus 35S promoter contains DREB1A cDNA (Kasuga et al. 1999) was used to introduce Arabidopsis plants (Colombia). Drying and low-temperature stress treatments were performed by the method of Yamaguchi-Shinozaki and Shinozaki (1994). That is, the plant body extracted from the agar medium was placed on a filter paper and dried under conditions of 22 ° C. and a relative humidity of 60%. The plant grown at 22 ° C was transferred to 4 ° C for low temperature treatment. The high salt concentration stress treatment was carried out by hydroponics in an aqueous solution containing 250 mM NaCl.

  Wild-type plants were subjected to stress treatment for 2 hours or 10 hours, sampled, and stored frozen using liquid nitrogen. In addition, wild-type and DREB1A overexpressing transformants cultivated on an agar medium not added with kanamycin were used in experiments for identifying a target gene of DREB1A. The DREB1A overexpression type transformant was not subjected to stress treatment. From the plant body, total RNA was isolated using ISOGEN (Nippon gene, Tokyo, Japan), and mRNA was isolated and purified using Oligotex-dT30 mRNA purification kit (Takara, Tokyo, Japan).

(8) Probe fluorescent labeling
Each mRNA sample was reverse transcribed in the presence of Cy3 dUTP or Cy5 dUTP (Amersham Pharmacia). Specifically, the reverse transcription reaction was performed using 1 μg of denatured poly (A) ⁺ containing 1 ng of λ poly A ⁺ RNA-A (TX802, Takara Shuzo) as an internal standard, 50 ng / μl of 12-18mer oligo dT primer (life Technology 1), each containing 0.5 mM dATP, dGDP and dCTP, 0.2 mM dTTP, 0.1 mM Cy3 dUTP or Cy5 dUTP, 100 U Rnase inhibitor, 10 mM DTT and 200 U Superscript II reverse transcriptase Superscript first-strand buffer was performed to a total volume of 20μl (50mM Tris-HCl, pH8.3 , 75mM KCl, the 3mM MgCl _2, 20mM DTT-containing. Life technologies, Inc.) in.

  The reaction solution having the above composition was incubated at 42 ° C. for 35 minutes, 200 U of Superscript II reverse transcriptase was added, and further incubated at 42 ° C. for 35 minutes. Thereafter, 5 μl of 0.5M EDTA, 10 μl of 1N sodium hydroxide and 20 μl of distilled water were added to the reaction solution to stop the enzymatic reaction in the reaction solution and to decompose the template. Thereafter, the reaction solution was incubated at 65 ° C. for 1 hour. Thereafter, the reaction solution was neutralized with 1M Tris-HCl (pH 7.5).

  The reaction solution was transferred to a Microcon 30 micro concentrator (Amicon). The process of adding 250 μl of TE buffer, centrifuging until the buffer volume reached 10 μl, and discarding the effluent was repeated 4 times. The probe contained in the reaction solution was recovered by centrifugation, and several μl of distilled water was added. To the obtained probe, 5.1 μl of 20 × SSC, 2 μg / μl of yeast tRNA, and 4.8 μl of 2% SDS were added. Further, the sample was denatured at 100 ° C. for 2 minutes, placed at room temperature for 5 minutes, and then used for hybridization.

(9) Microarray hybridization and scanning The probe was subjected to high-speed centrifugation for 1 minute using a benchtop micro centrifuge. To avoid bubble formation, the probe was placed in the center of the array and a cover slip was placed over it. Four drops of 5 μl of 3 × SSC were placed on the glass slide to keep the chamber at an appropriate humidity to prevent the probe from drying out during hybridization. The slide glass was sealed in a hybridization cassette (THC-1, BM instrument), and then treated at 65 ° C. for 12 to 16 hours. Remove the glass slide and place it on a slide rack. Carefully remove the coverslip in Solution 1 (2X SSC, 0.03% SDS), wash the rack by shaking, and move the rack into Solution 2 (1X SSC). Washed for minutes. Further, the rack was transferred to Solution 3 (0.05X SSC), allowed to stand for 2 minutes, and dried by centrifugation (2500 g, 1 min).

  The microarray was scanned at a resolution of 10 μm per pixel using a scanning laser microscope (ScanArray4000; GSI Lumonics, Watertown, Mass.). QuantArray version 2.0 (GSI Lumonics) was used as a microarray data analysis program. Background fluorescence was calculated based on the fluorescence signals of the negative control genes (nicotinic acetylcholine receptor ε subunit (nAChRE) gene and mouse glucocorticoid receptor homolog gene). Samples with a fluorescence signal value of less than 1000 (this value means less than twice the background fluorescence signal value) were not analyzed. Gene clustering analysis was performed using Genespring (Silicon Genetics).

(10) Northern analysis Northern analysis was performed using total RNA (Yamaguchi-Shinozaki and Shinozaki, 1994). A DNA fragment isolated by PCR from a full-length cDNA library of Arabidopsis thaliana was used as a probe for Northern hybridization.

(11) Determination of gene encoding transcription factor Based on the genome information of Arabidopsis thaliana in the database (GenBank / EMBL, ABRC), the gene encoding the transcription factor was analyzed using the gene analysis program BLAST.

2. result
(1) Identification of stress-inducible gene From mRNA isolated from an unstressed Arabidopsis plant, reverse transcription was carried out in the presence of Cy5-dUTP to prepare fluorescently labeled cDNA. A second probe labeled with Cy3-dUTP was prepared from a plant that had been subjected to a drying treatment, a low temperature treatment (2 hours), or a high salt concentration treatment. Both probes were simultaneously hybridized to a cDNA microarray containing about 7000 Arabidopsis cDNA clones, and a pseudo color image was prepared.

  Genes induced and repressed by stress are represented by red and green signals, respectively. Genes that are expressed at approximately the same level in both treatments give a yellow signal. The intensity of each spot corresponds to the absolute value of the expression level of each gene. The cold-inducible gene (rd29A) has a red signal and the control fragment (internal control) has a yellow signal.

  As a result of scanning the microarray, 277 genes specifically induced by drying treatment were identified, 53 genes specifically induced by low-temperature treatment were identified, and specifically induced by high salt concentration treatment 194 genes were identified. In addition, the gene induced | guided | derived by the expression ratio 5 times or more compared with the expression ratio of the control fragment was made into the gene induced | guided | derived by various stress.

As a result of analysis using a database, 35 types of transcription factors that can be classified into the following families were identified. RAFL05-21-L12 could not be classified into the following families, but a blast X search was performed on the amino acid sequence data registered in the GenBank Database for the nucleobase sequence converted to the amino acid sequence. However, it was identified as a transcription factor because it showed homology with a score of a known transcription factor (heat shock transcription factor-like protein) and E-value: e- ¹⁰⁰ . That is, according to the present example, 36 types of transcription factors could be identified.
1) DREB family: RAFL05-11-M11, RAFL06-11-K21, RAFL05-16-H23, RAFL08-16-D06
2) ERF family: RAFL08-16-G17, RAFL06-08-H20
3) Zinc finger family: RAFL07-10-G04, RAFL04-17-D16, RAFL05-19-M20, RAFL08-11-M13, RAFL04-15-K19, RAFL05-11-L01, RAFL05-14-C11, RAFL05- 19-G24, RAFL05-20-N02
4) WRKY family: RAFL05-18-H12, RAFL05-19-E19, RAFL06-10-D22, RAFL06-12-M01
5) MYB family: RAFL05-14-D24, RAFL05-20-N17, RAFL04-17-F21
6) bHLH family: RAFL09-12-N16
7) NAC Family: RAFL05-19-I05, RAFL05-21-I22, RAFL08-11-H20, RAFL05-21-C17, RAFL05-08-D06
8) Homeodomain family: RAFL05-20-M16, RAFL11-01-J18, RAFL11-09-C20
9) bZIP family: RAFL05-18-N16, RAFL11-10-D10, RAFL04-17-N22, RAFL05-09-G15

(2) Relationship between various stress treatment times and expression ratios For the genes encoding 36 types of stress-responsive transcription factors isolated as described above, the relationship between various stress treatment times and expression ratios is as follows. The results of studying the relationship are shown in FIGS. Table 2 shows the correspondence between the gene names shown in FIGS.

1 to 57, the vertical axis represents the gene expression ratio, which is calculated as follows. In the following formula, FI represents fluorescence intensity.
Expression ratio = [(FI of each cDNA under stress load condition) / (FI of each cDNA under no stress condition)] ÷ [(FI of control fragment under stress load condition) / (Condition without stress application) Control fragment in FI)]

  As shown in FIGS. 1 to 57, the genes encoding the stress-responsive transcription factors isolated by this method have different profiles, but it can be seen that expression is induced by the addition of various stresses.

It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL08-16-G17. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-11-M11. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL05-11-M11. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL06-11-K21. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL06-11-K21. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL06-08-H20. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL06-08-H20. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL05-16-H23. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-16-H23. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL08-16-D06. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL07-10-G04. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL04-17-D16. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-19-M20. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL08-11-M13. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL04-15-K19. It is a characteristic view which shows the relationship between low temperature stress and an expression ratio about RAFL04-15-K19. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-11-L01. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL05-11-L01. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-14-C11. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL05-19-G24. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-19-G24. It is a characteristic view which shows the relationship between low temperature stress and expression ratio about RAFL05-19-G24. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-20-N02. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-18-H12. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL05-18-H12. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL05-19-E19. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL06-10-D22. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL06-12-M01. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL06-12-M01. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-14-D24. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL05-14-D24. It is a characteristic view which shows the relationship between low temperature stress and expression ratio about RAFL05-20-N17. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-20-N17. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL04-17-F21. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL09-12-N16. It is a characteristic view which shows the relationship between a drought stress and an expression ratio about AFL05-19-I05. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL05-19-I05. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL05-21-I22. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL08-11-H20. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL08-11-H20. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL05-21-C17. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-21-C17. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL05-08-D06. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-20-M16. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL05-20-M16. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL11-01-J18. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL11-01-J18. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL11-09-C20. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL05-18-N16. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL11-10-D10. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL11-10-D10. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL04-17-N22. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL04-17-N22. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-09-G15. It is a characteristic view which shows the relationship between high salt concentration stress and expression ratio about RAFL05-09-G15. It is a characteristic view which shows the relationship between drought stress and expression ratio about RAFL05-21-L12. It is a characteristic view which shows the relationship between high salt concentration stress and an expression ratio about RAFL05-21-L12.

SEQ ID NOs: 73 and 74 are synthetic primers.

Claims (8)

A gene encoding a drought stress responsive transcription factor comprising the following amino acid sequence (a) or (b):
(a) the amino acid sequence of SEQ ID NO: 8
(b) An amino acid sequence consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 8, which is responsive to drought stress and has transcription factor activity The gene according to claim 1, comprising the following DNA (a), (b) or (c):
(a) DNA consisting of the base sequence of SEQ ID NO: 7
(b) DNA comprising a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 7, and encoding a drought stress responsive transcription factor
(c) DNA that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 7 under stringent conditions and encodes a drought stress responsive transcription factor   An expression vector comprising the gene according to claim 1 or 2.   A transformant comprising the expression vector according to claim 3.   A transgenic plant comprising the expression vector according to claim 3.   The transgenic plant according to claim 5, wherein the plant is a plant body, a plant organ, a plant tissue or a plant cultured cell.   A method for producing a drought stress-tolerant plant, wherein the transgenic plant according to claim 5 or 6 is cultured or cultivated. A gene encoding a drought stress responsive transcription factor comprising the following amino acid sequence (a) or (b):
(a) the amino acid sequence of SEQ ID NO: 10
(b) an amino acid sequence consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 10, which is responsive to drought stress and has transcription factor activity

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