KR101341932B1 - Gene encoding pepper transcription factor CaWRKY1 having resistance to frost damage and drought disaster, and use thereof - Google Patents

Abstract

The present invention relates to a method for improving the East Sea and drought resistance of plants by increasing the intracellular levels of East Sea and drought resistant polypeptides, wherein the plants transformed with the East Sea and drought resistant genes according to the present invention are East Sea and drought resistant It can be cultivated as a variety.

Description

Gene encoding pepper transcription factor CaWRKY1 having resistance to frost damage and drought disaster, and use approximately}

The present invention relates to the East Sea and drought resistant pepper transcription factor CaWRKY1 gene and its use. More particularly, the present invention relates to a method for regulating the intracellular level of a polypeptide having a specific amino acid sequence to enhance the East Sea and drought resistance of plants, in particular, the branched plants, and the plants produced according to the above methods and seeds or nutrients thereof.

Since plants are very mobile, their ability to avoid the many types of environmental stresses that face their survival process is significantly lower than that of animals, so most plants have a variety of defenses that are self-resistant to stresses such as low temperature, salt, and drying. Have a small Recently, research on the response to the stress or defense mechanism of the plant is being actively conducted, many genes related to the stress of the plant have been searched and its function is revealed. Genes that are resistant to plant stress, as found in these studies, can ultimately be used as a useful gene source for imparting resistance to environmental stresses on plants and crops.

In particular, changes in the expression of transcription factors have a significant effect on plants under stress. Arabidopsis has a variety of transcription factors with domains that can interact with DNA. These include proteins with EREBP / AP2, bZIP, Myb, WRKY, and Zinc finger domains, and studies are underway to determine whether they are induced under stress conditions. For example, tomatoes that overexpress Pti4, the tomato's ETHYLENE-RESPONSE-FACTOR, are plant fungal pathogens.Erysiphe orontiiThe bacterial pathogens of plantsPseudomonas syringae pv. Resistance to tomato was increased. Pti4 is thought to have a function of regulating the expression of genes with GCC box in the promoter. As another example, studies have reported that Arabidopsis overexpressing the Arabidopsis EREBP / AP2 type transcription factors, CBF1 / DREBP1 and DREBP1A, are resistant to environmental stresses such as drying, high concentrations of salt, and low temperature. These two transcription factors have been shown to interact with the CRT / DRE box, a promoter sequence involved in cold reactions, and to regulate the expression of target genes.

Regulators such as plant hormones are involved in different signaling systems. Salicylic acid, ethylene and jasmonic acid act as secondary signaling during disease-resistant signaling following pathogen invasion, inducing the expression of many defense-related genes. Dry and high concentrations of salts induce the production of large amounts of abscisic acid, while externally treated abscisic acid regulates the expression of several genes in response to drying and cold stress. These phenomena show different expression patterns of genes involved in biological and abiotic stress, which are the result of different regulation of different transcription factors and plant hormones.

On the other hand, food production is still required as the population grows, but agricultural water and farmland are decreasing. In addition, because crops have a fixed life, when the surrounding environment changes, a lot of stress occurs during the growing season, which brings a large difference in yield. Although Korea is one of temperate climates, unexpected climate change such as drought and East Sea occurs frequently due to global climate change. It is thousand ha, which is 8.87% of the total land area of 1,836 thousand ha. As such, deterioration of agricultural productivity due to natural disaster is a serious level, and it is absolutely necessary for food production to develop crops that withstand such poor environment.

Korean Patent Publication No. 10-2004-0096006

J. Korean Soc. Appl. Biol. Chem. 52 (5), 405-411 (2009)

Accordingly, the problem to be solved by the present invention is a method for improving the resistance of plants, particularly eggplant plants, using the East Sea and drought resistance genes, a method for producing plants having improved East Sea and drought resistance using such genes, and The present invention provides a transgenic plant having improved East Sea and drought resistance.

In order to achieve the above object, the present invention provides a method for enhancing the East Sea and drought resistance of plants by increasing the intracellular level of the CaWRKY1 polypeptide represented by SEQ ID NO: 2.

In order to achieve another object of the present invention, the present invention comprises the steps of (a) preparing an expression vector overexpressing the CaWRKY1 polypeptide represented by SEQ ID NO: 2; And (b) transforming the plant with the expression vector of step (a).

In order to achieve the other object of the present invention, the present invention provides a plant produced by the above production method.

In order to achieve the other object of the present invention, the present invention provides a plant seed (seed) or nutrients obtained from the plant.

That is, the present invention increases the intracellular level of the polypeptide having the amino acid sequence represented by SEQ ID NO: 2 to improve the East Sea and drought resistance of plants, more preferably having the amino acid sequence represented by SEQ ID NO: 2 Provided is a method of overexpressing a polypeptide to enhance plant's East Sea and drought resistance.

The present invention also provides a method for producing an East Sea and drought resistant plant, characterized by transforming a plant with a recombinant expression vector linking a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 to a promoter.

The present invention also provides a recombinant expression vector linking a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 to a promoter, and a transformant having improved East Sea and drought resistance by transforming with the recombinant expression vector and seeds thereof. Or provide nutrients.

Polypeptides having the amino acid sequence represented by SEQ ID NO: 2 (hereinafter also referred to as CaWRKY1 polypeptides) include functional equivalents thereof. The term "functional equivalent" is at least 70%, preferably 80% or more, more preferably 90% or more, and still more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution or deletion of the amino acid. Preferably, it refers to a polypeptide having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity with a polypeptide having an amino acid sequence represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" means activity that enhances the East Sea and drought resistance in plants. Substitution of the amino acid is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in the wild type are as follows; (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids is preferably located in portions not directly involved in the physiological activity of the CaWRKY1 polypeptide. The functional equivalent also includes polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic backbone of the CaWRKY1 polypeptide and its physiological activity. Examples include fusion polypeptides made by fusion with other polypeptides such as GFP while maintaining structural alterations and physiological activities to alter the stability, shelf life, volatility or solubility of the polypeptides of the invention. In addition, polypeptides having a bioactivity substantially equivalent to that of the wild type CaWRKY1 polypeptide as a fragment of the polypeptide having the amino acid sequence represented by SEQ ID NO: 2 form another preferred group of functional equivalents of the wild type CaWRKY1 polypeptide. The "fragment" refers to an amino acid sequence corresponding to a part of a polypeptide, which has a common element of origin, structure, and mechanism of action within the scope of the present invention, which is present in the wild type, is cleaved using a protease, or chemically. All cuts are included.

The base sequence of the gene encoding the CaWRKY1 polypeptide may include all gDNA, cDNA and RNA. The base sequence includes both base sequences encoding wild type polypeptides and their functional equivalents and sequences that hybridize with these sequences under high stringent conditions. High-tension conditions are as described in known literature (Molecular Cloning, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 1989). Preferably, CaWRKY1 according to the present invention Genes encoding polypeptides may include the nucleotide sequence represented by SEQ ID NO: 1 and sequence variants thereof. Specifically, the sequence variant is a base having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the base sequence represented by SEQ ID NO: 1 Sequences may be included.

As used herein, the term “homology” is intended to indicate a degree of similarity between the amino acid sequence of a wild type polypeptide or a nucleotide sequence encoding the same, and the amino acid sequence or the nucleotide sequence of the present invention and the percentage or more as described above. Include sequences with identical sequences. This homology comparison can be performed by using a comparison program that is easy to see with the naked eye. Commercially available computer programs can calculate homology between two or more sequences as a percentage, and homology (%) can be calculated for adjacent sequences.

The "intracellular level" refers to the amount present in a cell, which can be controlled by various methods known to those skilled in the art. For example, intracellular levels can be regulated through, but not limited to, regulation at the transcriptional stage or at the post-transcriptional stage. Modulation at the transcriptional stage is a method for enhancing the expression of a gene known to those skilled in the art, for example, a method for enhancing the expression of the gene by preparing a recombinant expression vector linking a target gene or a variant thereof to a promoter or the target gene. Or a method of inserting an expression control sequence such that the expression of the gene is enhanced around the variant thereof. Modulation at the post-transcriptional stage is a method for enhancing polypeptide expression known to those skilled in the art, for example, to enhance the stability of mRNA transcribed into a gene of interest or a variant thereof, to enhance the stability of the polypeptide of interest, or It can be carried out by a method for enhancing the activity of the polypeptide.

Preferably, in the present invention, the increase in the intracellular level of the polypeptide of SEQ ID NO: 2 or a functional equivalent thereof may be performed by a method of increasing the expression of the gene encoding the polypeptide. For this increase, a method known to those skilled in the art can be used. For example, a recombinant expression vector is prepared by linking a gene having a amino acid sequence represented by SEQ ID NO: 2 to a promoter or a gene encoding a functional equivalent thereof. By transformation, expression can be enhanced.

The term "recombinant" in the present invention refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a polypeptide encoded by a peptide, heterologous peptide or heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the wild-type form of the cells, either in sense or antisense form. Recombinant cells can also express genes found in wild-type cells, but the genes are modified and reintroduced into cells by artificial means. The term "vector" in the present invention is used to refer to a DNA fragment (s), a nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "recombinant expression vector" in the present invention means a recombinant DNA molecule comprising a desired coding sequence and a suitable base sequence necessary for expressing a coding sequence operably linked to a particular host organism. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. The recombinant vector is preferably a recombinant plant expression vector. A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Another type of Ti-plasmid vector (see EP 116,718 B1) is currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced which properly insert hybrid DNA into the genome of the plant. Preferred forms of Ti-plasmid vectors are so-called binary vectors. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly. Suitable recombinant expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers (promoter), and are prepared as desired. Can be. The recombinant expression vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable recombinant expression vector, a replication origin. The selection marker is a nucleotide sequence having properties that can be selected by a conventional chemical method, which corresponds to all genes that can distinguish the transformed cells from non-transformed cells. For example, there are herbicide resistance genes such as glyphosate or phosphinotricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol It is not limited to this.

In the recombinant expression vector according to the present invention, the promoter may be, for example, but not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. As used herein, the term "promoter" refers to a genome that regulates the expression of a nucleic acid sequence operably linked in a given host cell, wherein operably linked means that one nucleic acid fragment is different from another. Refers to a nucleic acid fragment that is affected by its function or expression by another nucleic acid fragment. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. A "plant promoter" is a promoter capable of initiating transcription in plant cells. The promoter may be a promoter (constitutive promoter) to induce the expression of the target gene at all times at all times or an inducible promoter (inducible promoter) to induce the expression of the target gene at a specific position, time, and conditions, Examples include RD29A promoter, SV40 promoter, CMV promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108: 193-199, 1991; Monahan et al., Gene Therapy, 7: 24-30, 2000), CaMV 35S promoter (Odell et al., Nature 313: 810-812, 1985), Rsyn7 promoter (US patent application Ser. No. 08 / 991,601), rice actin promoter (McElroy et al., Plant Cell, 2: 163-171 , 1990), ubiquitin promoters (Christensen et al., Plant Mol. Biol. 12: 619-632, 1989), ALS promoters (US Patent Application No. 08 / 409,297), and the like. In addition, U.S. Patent Nos. 5,608,149; 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463 and 5,608,142 can all be used. Preferably, East Sea and drought inducible promoters can be used. The terminator can use a conventional terminator, for example, nopaline synthase (NOS), potato α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) (Octopine) gene terminator, etc., but is not limited thereto. Terminator regions are known to increase the certainty and efficiency of transcription in plant cells.

Preferably, the recombinant expression vector provided by the present invention is operably linked to a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 or a gene having a nucleotide sequence represented by SEQ ID NO: 1 downstream of a promoter. , More preferably, a gene having a nucleotide sequence represented by SEQ ID NO: 1, or preferably the promoter is a CaMV (cauliflower mosaic virus) 35S promoter. That is, in the present invention, a recombinant vector pB7WG2D / CaRKY1 can be prepared by introducing the selected gene using pB7WG2D, which is a plant overexpression vector. Can be expressed throughout.

The present invention provides a transformant having improved East Sea and drought resistance transformed with the recombinant expression vector.

Transformation in the present invention can be carried out by a variety of transformation techniques known to those skilled in the art. Preferably microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion (PEG) mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method), Agrobacteria spraying method (Agrobacteria spraying method) can be used.

Also, bacteria that can be used for transformation include Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis ), Staphylococcus and Agrobacterium tumefaciens are possible, but are not limited to these.

In addition, the transformant may be a plant or a plant cell, but is not limited thereto. Plants include whole plants, parts of plants, callus, plant tissues, plant cells and plant seeds or nutrients. The plant is preferably an eggplant, and may be, for example, a plant such as potato, tomato, pepper, soybean, eggplant, tobacco or the like.

In the present invention, after transforming tobacco and potato for gene function analysis of CaWRKY1 pepper, after the transformation of tobacco and potato by producing a vector to be regulated by the 35S promoter under the gateway vector (Gateway vector) and selected as a basta, Transgene expression analysis was confirmed by Northern (Northern) analysis. As a result of the resistance test of tobacco and potato transformants, cell lines with high gene expression in CaWRKY1 transduced tobacco and potato showed increased tolerance and drought resistance.

In addition, the present invention provides a plant seed (seed) or nutrients obtained from the plant, as a cultivation method for obtaining plant seeds or nutrients well known to those skilled in the art to which the present invention belongs Method can be used.

Plants transformed with the East Sea and drought resistant genes according to the present invention can be grown into East Sea and drought resistant varieties, from which can significantly improve the productivity of plants, more specifically eggplant plants.

1 is a complete plant picture of differentiating callus.
Figure 2 shows the northern blot analysis of tobacco plants transformed with CaWRKY1 gene.
3 is a CaWRKY1 transformed tobacco plant subjected to cold stress treatment.
4 is CaWRKY1 Northern blot analysis of transformed potato plants.
5 is a pair of CaWRKY1 treatment Transformed potato plant.
6 is CaWRKY1 This is a photograph showing the external shape of the transformed potato plant.

Hereinafter, preferred examples and comparative examples are presented to aid in understanding the present invention. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1 Selection of Low Temperature Resistance Gene of Pepper

In order to select the genes used in this experiment, in the previous laboratory, cultivated the red pepper (capsicum annuum L., cultivar Cheongryong-cho) plants for 2 months in a laboratory until low temperature stress (4 ℃) ), RNA was isolated to prepare a control and cold library. Phage DNA of this library was transformed into pBluscript plasmid by mass excision and transformed to obtain colonies. About 11,000 plasmid DNA was isolated from the obtained colonies, and DNA chips were prepared from 3,100 gene clones.

DNA chip analysis and Northern blot analysis selected CaWRKY1 (AY789641) among a number of genes known to be involved in signaling systems among genes whose expression is induced or inhibited by cold stress.

The selected gene was introduced using pB7WG2D, a plant overexpression vector. The produced vector (pB7WG2D / CaWRKY1) has a CaMV (cauliflower mosaic virus) 35S promoter, so that the introduced gene is always expressed throughout the plant.

Example 2 Selection of Tobacco Transformant and Confirmation of Insertion

Construction and selection of tobacco transformants was performed according to the procedures described in Materials and Methods (Hwang, 2003). First, Agrobacteria liquid culture medium for transformation was prepared, tobacco leaves were cut and placed in PCM solid medium and pre-cultured for 24 hours. Callus was differentiated by infection with A. tumefaciens LBA4404 containing the overexpression vector pB7WG2D / CaRKY1, transferred to CIM medium, and co-cultured with cancer culture. Subcultured with fresh CIM solid medium every two to three weeks for callus induction. When the callus was differentiated for 2 to 3 months, an appropriate size callus was formed. The callus was transferred to SIM medium and shoots generated after 3-4 weeks were transferred to MS medium containing cefortaxime to differentiate into complete individuals.

1 is a complete plant picture of differentiating callus. Here, 1 is the disc of pre-cultured and co-cultured tobacco leaves, 2 represents callus induction, 3 represents chute induction, and 4 represents complete plant production.

CaWRKY1 Using Genomic DNA PCR Analysis of Transgenic Tobacco It was confirmed whether the gene was introduced into the plant. In this PCR analysis, primers complementary to the base sequence of each gene were prepared and used. As shown in Table 1, two primers were prepared for each of the transgenic plants, as sense and antisense (SEQ ID NOs: 3 and 4).

Genomic DNA PCR analysis of individual plants differentiated to CaWRKY1 transformed CaWRKY1 (75%) in 48 cell lines except for 12 cell lines.

Gene Primer Primer sequence  (5 '→ 3') CaWRKY1 CaWRKY1- att B1 5'-AAAAAGCAGGCTCCTTCATCTTATTTAAGGAGAAAAAACTG-3 ' CaWRKY1- att B2 5'-AGAAAGCTGGGTCAGACGCAAATCAAATTGGATATATTTC-3 '

Example 3 Expression Analysis of Transgenes

Based on the genomic DNA PCR analysis results performed in Example 2, a transformant was selected and Northern blot analysis was performed as follows, and the expression level of the introduced gene was examined.

First, total RNA was isolated, fractionated by electrophoresis and transferred to the membrane. The isolated RNAs were blotted and the blots hybridized with 32P- labeled CaWRKY1 cDNA.

2 shows Northern blot analysis of CaWRKY1 transformed tobacco plants. Here, numbers 1 to 36 are transgenic cell lines, and bottom panel represents RNA agarose gel stained with ethidium bromide. As shown in FIG. 2, high expression was observed in # 3, 4, 7, 8, 12, 26, 27, 30, 31, 32, 34, 35, 36 of CaWRKY1 transformant 36 cell lines, and # 2, 13, No expression was found in 15, 17, 18, 19, 20, 21, 22, 23, 24, and low expression in all other cell lines. As a selection criterion, a cell line whose expression was observed to be relatively high when compared with the wild type was selected and a cold stress resistance assay of the transformant was performed.

Example 4 Cold Stress Tolerance Analysis of Transgenic Tobacco

After confirming the insertion and expression of the genes by genome PCR and Northern blot analysis in Examples 2 and 3 and selecting the transgenic tobacco plants with high expression level of each gene of these genes and grown in MS medium for 3-4 weeks, Transfer to horticulture (Jiffy pot) and purified for 1 week.

For wild-type stress tolerance analysis After the selected tobacco transformants were treated at −6 ° C. for 16 hours, the cells were transferred to room temperature, and the survival rates of the wild type plants and the transformed plants were compared, and the results are shown in FIG. 3. 3 is a CaWRKY1 transformed tobacco plant subjected to cold stress treatment. Here, (A) is a plant before cold stress treatment, (B) is a plant 24 hours after cold stress treatment, WT is a wild type, and the number is a transformed cell line. As shown in FIG. 3, seven of the cell lines # 3, 4, 7, 8, 12, 26, 27, 30, 31, 32, 34, 35, 36 selected from CaWRKY1 transformants (# 3, 4, 7, 8, 31, 34, 35) showed strong resistance to low temperature stress.

Example 5 Preparation and Selection of Potato Transformant

A. tumefaciens by constructing the vector pB7WG2D / CaWRKY1 under the control of the 35S promoter to constantly express the CaWRKY1 gene It was infected with LBA4404 and transformed into potato varieties Sumi. Ten clones were transformed. As a template, genomic DNA isolated from 10 independently redivided plants was used as a template to confirm whether or not a structural gene site was inserted into the transformants obtained above. Reaction (PCR) was performed. Expression of the transformed clones was performed using the CaWRKY1 structural gene as a probe to isolate the total RNA, fractionated by electrophoresis, and transferred to the membrane. Separated RNAs were blotted and # 2, # 3, # 10 clones were selected as compared by Northern analysis by hybridizing the blots with 32P- labeled CaWRKY1 cDNA.

4 is CaWRKY1 Northern blot analysis of transformed potato plants.

Example 6 Drought Resistance Analysis of Transgenic Potatoes

CaWRKY1 overexpressing transgenic plant phenotypes for drought stress, one of the abiotic stresses, were investigated. The control group (wild type) and the transformants were incubated for 2 weeks in the plane, and then incubated for 2 weeks in hydroponic culture. After transfer to horticultural soil for 1 week to purify, the dry stress was treated for 4 weeks and observed for resistance. Soil moisture meters were used to maintain the same degree of drying in each pot, and experiments were carried out using 12 individuals for repeated experiments. The plants began to receive dry stress around 20 days, and the control group and CaWRKY1 overexpressing transformants showed a difference in resistance to one pair from around 25 days. After 28 days after the dry stress treatment, re-watering was carried out to observe the resilience of the plant.

Figure 5 is a one-shot CaWRKY1 transformed potato plant. As shown here, CaWRKY1 overexpressing transformants showed resistance to dry stress compared to the control.

Example 7 Agricultural Characterization of Transgenic Potatoes

The agricultural characteristics of CaWRKY1 overexpressing transformants were investigated in greenhouse conditions. As shown in Table 2, the apparent agricultural characteristics of the control group and the CaWRKY1 overexpressing transformants were examined to be almost similar.

Figure 6 is a photograph showing the external shape of the CaWRKY1 transformed potato plant. As shown here, the shape of the leaves and the size of the leaves could be observed to be almost similar, and the appearance of the potato was observed as a light brown round shape similar to the control.

According to the present invention, it is possible to easily and systematically breed useful plants having resistance to low temperature stress, and new transcription factor genes and proteins thereof for providing East Sea and drought resistance to plants used in the present invention are crops that have been edible for a long time. Since it is derived from the red pepper, it can be used to obtain safer transgenic plants and agricultural products, as well as to develop the East Sea and drought-resistant varieties, thereby improving productivity and supplying safe agricultural products.

Attach an electronic file to a sequence list

Claims (10)

delete A method of overexpressing a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 to enhance freezing and drought resistance of potatoes or tobacco. The method of claim 2, wherein the method of overexpressing the polypeptide comprises producing a recombinant expression vector linking a gene encoding the polypeptide to a promoter to transform the potato or tobacco, or to transform the gene encoding the polypeptide to potato or tobacco cells. A method characterized by the method of introduction. The method of claim 2, wherein the gene encoding the polypeptide has a nucleotide sequence represented by SEQ ID NO: 1. delete delete A method for producing East Sea and drought resistant potato or tobacco, characterized by transforming potato or tobacco into a recombinant expression vector linking a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 to a promoter. A recombinant expression vector linking a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 to a promoter. A transformant of potato or tobacco, which is transformed with the recombinant expression vector of claim 8 and has improved East Sea and drought resistance. Potato or tobacco seeds or nutrients obtained from the transformants of potato or tobacco of claim 9.

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