KR20130033033A - Salt and drought stress tolerant gene, ossdt1 and use thereof - Google Patents

Abstract

PURPOSE: A transgenic plant with salt and/or cold stress resistance is provided to grow salt resistant and cold resistant variety, and to stably supply agricultural products without the influence by sudden climate change. CONSTITUTION: A method for improving salt resistance or cold resistance in a plant comprises a step of enhancing the polypeptide level with an amino acid sequence in sequence number 2. The method also comprises a step of over-expressing a polypeptide with an amino acid sequence in sequence number 2. The over-expression of the polypeptide comprises: a step of preparing a recombinant expression vector and transforming, or a step of transducing a gene encoding the polypeptide into a plant cell. [Reference numerals] (AA) Moisture loss rate(%); (BB) Time; (CC,EE) Average survived leaf number per plant; (DD,FF) Dongjin-rice;

Description

Salt and drought stress tolerant gene, OsSDT1 and use approximately}

The present invention relates to a salt-tolerant or cold-tolerant gene and a use thereof, and more particularly, to a method for enhancing the salt-tolerant or cold-tolerant resistance of a plant by regulating intracellular levels of a polypeptide having a specific amino acid sequence, a plant prepared according to the method, and It's about seeds.

Agriculture has its own risks and instabilities that are not seen in other industries. Agriculture is not only uncertain because of the large losses caused by disasters, but also the high price of agricultural commodities and the relatively low price of agricultural commodities relative to the purchase price of production materials. Agriculture is a natural danger that is more prone to damage than any other industry due to drought, flooding, frost or hail. This is because it is closely connected to the land of agricultural production and the land which cannot be moved, and because the object of production is flora and fauna, it is largely dominated by natural conditions due to the limitations of life.

A drought causes crop loss due to lack of precipitation, or there is no constant precipitation period, and crops are cultivated depending on irrigation water. Drought can occur at any time during the growing season and may occur year round depending on the region. In the early stage of growth, delayed seeding time or seeding, and the mid-term drought lasting, have a fatal effect on the yield due to the delay of flowering time. Korea has abundant annual rainfall of 1,250 mm, but is concentrated in July-August, so it has a semi-dry climate in spring and autumn. Therefore, spring and autumn usually occur in pairs, and sometimes even in July-August, there is a shortage of rainfall, which leads to a year.

The salt damage of crops occurs when growing crops in salt and sodium soils. It may also appear as a result of splashes of sea water caused by breezes or blue waves. In Korea, three sides are surrounded by the sea, and most of them consist of coasts, and there are many salt spots. Salt salt is a land where plants are impeded to grow because salt concentration increases due to brine intrusion or evaporation of soil moisture. Some areas have been used as salt fields, and since 1960, a large part has been converted into reclaimed land. Many of these areas have been matured to increase the yield of rice, but in the case of new land reclamation projects that have been recently reclaimed, they are often damaged by salt.

With the development of plant transformation technology, it is possible to express foreign genes that are far from flexible, and thus, it is possible to develop a tolerant crop by introducing genes tolerant to poor environments. Therefore, the isolation and development of genes to withstand cold and drought and salt is an essential requirement for the development of such crops.

In particular, salt and drought cause water deprivation due to osmotic pressure of crops, resulting in morphological and metabolic changes in plants, and the detailed mechanisms thereof have not been fully understood. This lack of water causes not only morphological changes in plants, but also pore closure, decreased photosynthetic and photorespiratory rates, increased small molecules and plant hormones, and altered gene expression.

As the population grows, food production continues to increase, 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 weather events such as droughts and high temperatures are occurring 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.

Accordingly, the problem to be solved by the present invention is a method of improving the abiotic stress resistance of plants using salt and / or drought resistance genes, and plants having improved salt and drought resistance using such genes. To provide a method for producing a, and a transgenic plant with improved salt and drought resistance prepared by such a method.

In order to achieve the above object, the present invention provides a method for enhancing the flame resistance of plants by increasing the intracellular level of the OsSDT1 polypeptide represented by SEQ ID NO: 2.

In order to achieve another object of the present invention, the present invention provides a method for enhancing the cold resistance of plants by increasing the intracellular level of the OsSDT1 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 OsSDT1 polypeptide represented by SEQ ID NO: 2; And (b) it provides a method for producing a flame resistance enhanced plant comprising the step of transforming the plant with the expression vector of step (a).

In order to achieve another object of the present invention, the present invention comprises the steps of (a) preparing an expression vector overexpressing the OsSDT1 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 another object of the present invention, the present invention provides a plant seed (seed) obtained from the plant.

That is, the present invention is a method of improving the salt resistance or cold resistance of plants by increasing the intracellular level of the polypeptide having the amino acid sequence represented by SEQ ID NO: 2, more preferably having the amino acid sequence represented by SEQ ID NO: 2 Provided is a method of overexpressing a polypeptide to enhance the tolerability or cold tolerance of a plant.

The present invention also provides a method for producing a salt tolerant or cold-resistant plant, characterized in that the plant is transformed with a recombinant expression vector in which a gene encoding a polypeptide having a amino acid sequence represented by SEQ ID NO: 2 is linked 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 salt resistance or cold resistance by transforming with the recombinant expression vector and seeds thereof. To provide.

Polypeptides having the amino acid sequence represented by SEQ ID NO: 2 (hereinafter also referred to as OsSDT1 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 resistance to salt or drought in a plant. 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; Aliphatic amino acids (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). The deletion of the amino acid is preferably located at a portion not directly involved in the physiological activity of the OsSDT1 polypeptide. The functional equivalent range also includes polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic backbone of OsSDT1 polypeptide and its physiological activity. For example, fusion polypeptides made by fusion with other polypeptides, such as GFP, while maintaining structural alterations and physiological activities to alter the stability, storage, volatility or solubility of the polypeptides of the present invention. In addition, polypeptides having a bioactivity substantially equivalent to that of the wild type OsSDT1 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 OsSDT1 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 OsSDT1 polypeptide may include all gDNA, cDNA and RNA. The base sequence includes both base sequences encoding wild type polypeptides and functional equivalents thereof 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, OsSDT1 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 the percent homology between two or more sequences, and the percent 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.

As used herein, the term “recombinant” 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, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in the wild-type form of the cell in either the 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 when referring to DNA fragment (s), nucleic acid molecules that are delivered into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. As used herein, the term “recombinant expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable base sequence necessary for expressing the coding sequence operably linked in 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. Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. 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 can be advantageous, especially when it is difficult to properly transform a plant host. 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 a Maize Ubiquitine promoter for monocotyledonous plants, but may be, but is 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, in which one nucleic acid fragment is operably linked to 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; The promoters disclosed in 5,608,144 5,604,121 5,569,597 5,466,785, 5,399,680 5,268,463, 5,608,142, and the like can all be used. Preferably, environmental stress inducible promoters, more preferably salt or drone stress inducible promoters can be used. The terminator may use a conventional terminator, for example nopalin synthase (NOS), potato α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacteriumtumefaciens) Octopine) terminators, 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 may be capable of linking 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. will be.

The present invention provides a transformant having improved salt tolerant and / or cold tolerance 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.

In addition, transformants include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Staphylococcus and Agrobacterium tumefaciens is possible, but is not limited to this.

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. The plant may be a dicotyledonous plant such as beans, potatoes, cabbages, radishes, peppers, but more preferably monocotyledonous plants such as rice, wheat, barley, corn, and the like. In one embodiment of the present invention, the recombinant expression vector was transformed into Agrobacterium tumefaciens, and the rice callus and the recombinant expression vector were co-cultured with Agrobacterium tumefaciens to prepare a transformant.

The present inventors prepared a recombinant expression vector comprising the OsSDT1 gene having the nucleotide sequence represented by SEQ ID NO: 1 and transformed it into rice to increase the intracellular expression level of OsSDT1. As a result of examining the characteristics of the overexpressing transformant of OsSDT1, it was confirmed that the overexpression of OsSDT1 improved the resistance to salt and drought.

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

Plants transformed with the genes resistant to salt and / or drought stress according to the present invention can be grown into flame-resistant and / or cold-resistant varieties, thereby providing stable agricultural products without being affected by productivity even under sudden climate change such as global warming. This is possible.

1 is a schematic diagram of a recombinant expression vector overexpressing the OsSDT1 gene according to Example 1 of the present invention.
Figure 2 is a result of overexpression assay electrophoresis using Genomic PCR in the assay of the rice transformant according to Example 3 of the present invention.
3 is a result of overexpression assay electrophoresis using RT-PCR in the assay of the rice transformant according to an embodiment of the present invention.
4 is a result of evaluating the effect on the growth of Dongjin rice (middle of white pot) and transformants (left and right side of white pot), which are varieties before salt treatment or drought treatment. 4A to 4A are untreated controls, and FIG. 4B is a representative graph of quantification and water loss rate of the medicated leaves and the number of surviving leaves during drought treatment, and FIG. Ratio.
5 is a result of evaluating the effect of the high osmotic stress treatment on the transformant of the present invention.

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 Construction of OsSDT1 Overexpressing Transformation Vector and Agro Bacteria Containing the Same

The rice cultivar used in this experiment was Dongjin rice, and the sterilized brown rice seeds were grown for 2 weeks in solid medium (pH5.8) added 1% Sucrose and 0.4% phytagel at 1 / 2MS for 2 weeks. Total RNA was isolated from these plants using Trizol, and then 30 cDNAs were synthesized from 5 RNAs using reverse transcriptase. The coding region of A30 gene from these cDNA was amplified by PCR using Forward primer-CACC ATG GTG GAG GTG GGA GGA GGA G, Reverse primer-CCG GTC GAG GGG CTC GGT. An entry clone was prepared as a PCR product by using the pENTR / D-Topo kit. For overexpression of the gene, tetracycline was used as a bacterial marker gene and hygromycin as a plant marker gene. It was cloned into the pGA2897 vector, which is a binary vector. PGA2897-OsSDT1, whose gene insertion and frame were identified through sequencing, was first transformed into Agrobacterium tumefaciens-LBA4404 (strain with rifampicin and streptomycin as a marker) for rice transformation. 2μl of pGA2897-OsSDT1 DNA is added to 100μl of Agrobacterium and placed on ice. After putting it in a cuvette, immediately after applying 1.25kV electric shock, 1ml of YEP medium was transferred to a 15ml tube and incubated at 30 ° C. for 3 hours. 300 µl of the culture solution was plated on YEP medium containing tetracycline, rifampicin and streptomycin, and grown at 30 ° C for one day. One of these colonies was selected to confirm gene insertion through colony PCR and stored in stock.

A schematic diagram of the vector prepared in Example 1 is shown in FIG. 1.

Example 2 Preparation of Rice Transformant

Rice seed used for rice transformation was made from brown rice, peeled. Soybean seeds were soaked in 70% ethanol for 1 minute and then sterilized for 40 minutes in 50% solution. At this time, it is more preferable to use 10uL Tween 20 dropped per 30mL of lacx.

After disinfecting the disinfectant solution, wash the rice seed 5 times with sterile water. When the disinfectant solution was washed well, water was removed from the rice seed using filter paper and the rice seed was soaked in 2N6 medium. At this time, rice seed is embedded in the medium so that the contact with the medium is good, and the seed is directed upward. 2N6 medium is made of some modified medium in the medium described in the literature (Hiel et al., 1994). 4g / L and 30g / L sucrose, 0.5g / L proline, 0.5g / g of N6 medium (Chu et al., Scin Sin (1975) 18: 659-668) L glutamine, 0.3 g / L casamino acids, 0.01 g / L myo-inositol, 2 mg / L 2,4-D, 4 g / L phytagel, solid medium (pH5.8). Incubation temperature is 28 ℃, dark treatment.

Incubated for 5 days at 2N6, the embryo was removed when callus is induced. The drainage was carefully removed using tweezers to prevent the callus from being damaged, and the cotyledons and roots were left intact to prevent the callus from being damaged.

Agrobacterium was prepared by inserting the gene of interest, incubated in AB solid medium for 3 days, and then collected in AAM liquid medium to make a suspension. The concentration of the suspension was measured using an absorbance machine. First, the suspension was made to be OD ₅₉₅ = 0.1, and then diluted 10-fold with AAM to make OD ₅₉₅ = 0.01.

It is preferable to inoculate Agrobacterium by immersing the callus from which the oil is removed in the suspension for 2 minutes and gently shaking the solution for 2 minutes. At the end of the inoculation, only the suspension was poured from the callus and the filtrate was removed using filter paper. This is a rapid process and care should be taken not to allow the callus to dry too long on filter paper. Callus was co-cultured by transferring to 2N6-AS medium. 2N6-AS medium was adjusted to pH5.2 by adding 10g / L glucose and 100uM acetosyringone to 2N6 medium. Place filter paper one by one on the medium in advance so that the medium is sufficiently infiltrated, and then place the inoculated callus on the filter paper. The filter paper is intended to prevent excessive growth of Agrobacterium during the co-culture and the callus receding. Co-culture was incubated for 7 days at 25 ℃.

At the end of the coculture, the Agrobacterium, which was responsible for inserting useful genes into the callus, is no longer needed. The washing was performed with a solution containing 500 mg of antibiotic carbenicillin in 1 L of sterile distilled water. After washing the callus about three times, all of the filtrate was removed using a filter paper. When using the Agrobacterium LBA4404 strain, it is more preferable not to perform the above washing operation because no excessive growth of Agrobacterium was achieved during the coculture period. Callus is transferred to 2N6-CH medium in which antibiotics cefotaxime 200mg / L and Hygromycin 40mg / L are added to 2N6 medium. At this time, the cotyledons and roots are removed using tweezers, and the callus is divided into 2-3 pieces so that the contact area with the medium is maximized and cultured. The culture temperature of 28 ℃ was appropriate and incubated for 14 days in the cancer incubator to induce transformed callus.

After 14 days, when the transformed callus was split and grown in 2N6-CH medium to which the antibiotic hygromycin was added, it was passaged to MSR medium, which is a regeneration medium capable of inducing chute and root. The distinction of transformed callus was judged by the presence of newly differentiated callus mass, and the browned and dead callus was discarded.

Example 3 Rice Transformant Assay and Overexpression Assay Using RT-PCR

To confirm that the transformants were lyophilized rice cultivated for one month, and then genomic DNA was isolated from the DNeasy Plant Mini Kit (QIAGEN). The following hygromycin gene primers were identified using genomic DNA. By confirming the insertion was confirmed whether the insertion of the OsSDT1 gene. In addition, the total RNA of the identified rice transformant was isolated using RNeasy Plant Mini Kit (QIAGEN), and cDNA was synthesized from the RNA using SuperScript III Reverse Transcriptase (Invitrogen). Using the synthesized cDNA, the following OsSDT gene primers Forward primer- CCG TCC ACC TGG TTT GGT CT, Reverse primer- GGC TCG GTT TGG TCC TTG AC Actin gene primer Forward primer- TGC TCT GTA CGT CGC CAT CCA G, Reverse primer AAT GAG TAA CCA CGC TCC GTC A overexpression of OsSDT1 gene was confirmed by RT-PCR. The results of the rice transformant assay and the overexpression assay using RT-PCR are shown in FIGS. 2 and 3, respectively.

Example 4 Evaluation of Cold Resistance and Flame Resistance

For the cold-resistance test of overexpressing transformants, the rice grown in the greenhouse for 4 weeks was stopped for 4 days, and the plant growth was examined 3 weeks after the water supply was resumed. For 4 weeks, the plant was tested for flame resistance of the overexpressing transformants. Plant growth after 8 days was assayed by treating with 250 mM NaCl in rice grown at. The results are collectively shown in FIG.

As shown in Figure 4, under the conditions of drought and high salt conditions, the overexpression growth was better or higher than that of the control rice Dongjin rice. In the water loss assay, the rice seeds selected from the hygromycin medium were transferred to the soil, grown for 5 weeks, and the top leaves of each overexpression were cut and placed on a filter paper. . The graph shows the percentage of water loss relative to initial weight. In the salt treatment, the transformants selected from the hygromycin medium were transferred to the soil in the white square pot along with the control of Dongjin rice and grown for 4 weeks, and then treated with 250 mM salt for 8 days. The graph was then shown by examining the number of dead leaves against the number of leaves in one individual for each transformant. Droght treatment also showed the graph by growing the rice by drought treatment in the same way as the salt treatment, drought it for 4 days, watering again and growing for 23 days.

Example 5 Evaluation of High Osmotic Resistance

Rice germinated at 1/2 MS for 3 days was transferred to 1 / 2MS media to which 400 mM mannitol was added for high osmotic stress treatment, and growth state was observed after 7 days, and the results are shown in FIG. 5.

As shown in Figure 5, Dongjin can be seen that the shoot site is dying, this was not found in the overexpressing transformant according to the present invention. In addition, the growth of overexpressing transformants in the high osmotic medium was not seen, but rather suppressed.

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Claims (11)

A method of improving the tolerability or cold tolerance of a plant by increasing the intracellular level of a polypeptide having the amino acid sequence represented by SEQ ID NO: 2. The method according to claim 1, wherein the polypeptide having the amino acid sequence represented by SEQ ID NO: 2 is overexpressed to improve flame resistance or cold resistance of plants. The method of claim 2, wherein the method of overexpressing the polypeptide is by producing a recombinant expression vector linking a gene encoding the polypeptide to a promoter and transforming the plant, or by introducing a gene encoding the polypeptide into a plant cell. How is characterized by the thing. The method of claim 1, wherein the gene encoding the polypeptide has a nucleotide sequence represented by SEQ ID NO: 1. The method of claim 1 wherein the plant is a monocotyledonous plant. 6. The method of claim 5 wherein the plant is rice, wheat, barley or corn. 7. The method of claim 6, wherein the plant is rice. A method for producing a salt tolerant or cold resistant plant comprising transforming a plant into a recombinant expression vector in which a promoter is linked to a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 2. 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 having improved salt toleration or cold resistance by transformation with the recombinant expression vector of claim 9. Plant seeds obtained from the transformant of claim 10.

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