KR101905267B1 - Abiotic Stress-Inducible OsSta2 Gene, Protein and Transformant Enhancing Tolerance to Salt Stress - Google Patents

Abstract

The present invention relates to a nucleic acid construct comprising a salt stress-inducible OsSta2 protein comprising an amino acid sequence of SEQ ID No. 2 and enhancing tolerance to salt stress in the plant when overexpressed in a plant, and a nucleic acid comprising a nucleotide sequence encoding the OsSta2 protein A method for producing a transgenic plant transformed with said vector, a method for producing a transgenic plant having enhanced salt stress tolerance, and a method for enhancing tolerance to salt stress produced by said method Providing a transgenic plant. The nucleotide sequence used in the present invention is involved in increasing the resistance of the plant to abiotic stress (for example, salt stress) and seed production, and when the plant is transformed using the nucleotide sequence, the plant resistance to the stress and the grain yield Can be increased at the same time. Therefore, it is possible to overcome the limit of the cultivation area of the plant, and when applied to rice, it is possible to maximize productivity by being able to produce rice with high yield and strong against abiotic stress.

Description

(Abiotic Stress-Inducible OsSta2 Gene, Protein and Transformant Enhancing Tolerance to Salt Stress), which promotes osteosarcoma resistance,

The present invention relates to an abiotic stress-inducible OsSta2 gene, a protein and an OsSta2-expressing resistant transformant which promote salt stress tolerance.

More than half of the world's population is using rice as a major edible crop, and the global demand for rice will continue to increase with population growth. A variety of environmental stresses react at the molecular level by altering the expression levels of genes that play a key role in regulatory mechanisms or signaling leading to stress tolerant plants and genes that encode stress tolerant proteins. Drying and salt stress are environmental factors limiting rice productivity. Rice productivity in areas affected by salinity is significantly reduced by 30-50% per year. Significant progress has been made in recent decades to understand and achieve salt stress tolerance in many plant species, including rice. To protect cells from stress under salt stress and maintain normal growth, many cellular responses occur, such as cytosolic calcium release, ionic imbalance in vacuoles, stress signaling, and gene regulation.

AP2 / ERF, bZIP. Transcription factors such as MYB, NAC, zinc-finger, MYC and WRKY are known to be important for the stress response and stress tolerance of plants because they can regulate the expression of many stress-responsive genes. Transforming transcription factors to produce plants with enhanced resistance to abiotic stress has proven to be an important approach. Among the transcription factors, AP2 / ERFs play a variety of roles such as developmental processes such as plant growth, flower development, fruit ripening, epidermal cell identification of leaves and leaflet generation. The AP2 / ERF protein is also associated with plant responses to biological stress. For example, the ERF protein binds to the GCC box (AGCCGCC) and regulates the expression of the oncogenic gene. Medicoga The AP2 / ERF proteins, such as ERN1, 2, 3 and EFD from truncatula , co-occur with nitrogen-fixing bacteria to regulate the development of root nodules in legumes.

The AP2 / ERF protein plays a role in response to abiotic stresses such as dryness, salt and low temperature in addition to its role in response to biological stress. The AP2 / ERF protein contains a conserved AP2 / ERF domain. The most studied AP2 / ERF protein is CBF / DREB, which activates expression of many stress-related proteins and improves resistance to drying, salt and cold.

Rice ( Oryza sativa ssp. Japonica ) has at least 139 AP2 / EFR family genes. Various environmental stresses induce the expression of rice AP2 / ERF gene related to stress tolerance. For example, the AP2 / ERF proteins SNORKEL1 and SNORKEL2 (SK1 and SK2) rapidly accelerate accumulation and stem elongation of gibberellic acid with avoidance strategies in inundation classics (Hattori et al., 2009). On the other hand, the AP2 / ERF protein, SUB1A-1, has a resting strategy that inhibits shoot elongation and increases survival to immersion-resistant (Xu et al., 2006). In addition, continuous expression of AP2 / ERF genes such as DREB1A, HARDY ( arabidopsis ), HvCBF4 (barley) and TERF1 (tomato) increases abiotic stress tolerance (Oh et al., 2005, 2007; , 2007; Gao et al., 2008). Overexpression of rice AP2 / ERF gene AP37 confers dry tolerance at the growth stage with high grain yield (Oh et al., 2009). Overexpression of the AP2 / ERF protein TSRF1 improves rice osmosis and dry tolerance (Quan et al., 2010). Recently, overexpression of OsEREB1 in rice has been reported to increase resistance to biological and abiotic stress (Jisha et al., 2015).

Functional genomes including rice reverse genetic and forward genetic methods are important research areas for identifying novel genes involved in plant stress response and tolerance. These genes could be a new target for genetic engineering to improve the tolerance of rice and other crops.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

 Hattori, Y. et al. (2009) The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water. Nature. 460, 1026-1030.  Xu, K. et al. (2006) Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice. Nature. 442, 705-708.  Oh, S. J. et al. (2005) Arabidopsis CBF3 DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth. Plant Physiol. 138, 341-351.  Oh, S. J. et al. (2007) Expression of barley HvCBF4 enhances tolerance to abiotic stress in transgenic rice. Plant Biotechnol. J. 5, 646-656.  Karaba, A. et al. (2007) Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene. Proc. Natl Acad. Sci. 104, 15270-15275.  Gao, S. et al. (2008) Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity. Plant Cell Rep. 27, 1787-1795.  Oh, S. J. et al. (2009) Overexpression of the transcription factor AP37 in rice improves grain yield under drought conditions. Plant Physiol. 150, 1368-1379.  Quan, R. et al. (2010) Overexpression of an ERF transcription factor TSRF1 improves rice drought tolerance. Plant Biotechnol. J. 8: 476-488.  Jisha, V. et al. (2015) Overexpression of an AP2 / ERF type transcription factor OsereBP1 confers biotic and abiotic stress tolerance in rice. PLoS one. 10, 6-e0127831.

As a result of intensive efforts to discover genes capable of promoting resistance to abiotic stress, the present inventors have found that OsSta2 gene induced by salt stress from rice ( Oryza sativa ) Of the present invention exhibits enhanced resistance to salt stress, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a salt stress-inducible OsSta2 protein.

It is another object of the present invention to provide a nucleic acid molecule comprising a nucleotide sequence encoding a salt stress-inducible OsSta2 protein.

It is another object of the present invention to provide a vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a salt stress-inducible OsSta2 protein.

It is another object of the present invention to provide a transformant transformed by the vector of the present invention.

It is still another object of the present invention to provide a method for producing transgenic plants with enhanced salt stress tolerance.

It is another object of the present invention to provide a transgenic plant having enhanced resistance to salt stress.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing an abiotic stress-inducible OsSta2 protein, which comprises the amino acid sequence of the second sequence of the Sequence Listing and enhances tolerance to salt stress in the plant when overexpressed in the plant .

As a result of intensive efforts to discover genes capable of promoting resistance to abiotic stress, the present inventors have found that OsSta2 gene induced by salt stress from rice ( Oryza sativa ) Showed enhanced tolerance to salt stress.

The abiotic stress-inducible OsSta2 protein of the present invention has substantial identity with the above-mentioned amino acid sequence and is also interpreted to include an amino acid sequence within a range capable of enhancing tolerance to salt stress . The above-mentioned substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence as much as possible, and analyzing the aligned sequence using a program commonly used in the art (for example, ClustalX and PROSIS) Means an amino acid sequence that exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology.

As used herein, the term " abiotic stress " is defined as the negative effect of a microbial element on a living being in a particular environment and includes salt stress, drought stress, cold stress and osmotic stress do.

According to one embodiment of the present invention, the abiotic stress is salt stress.

According to another embodiment of the present invention, the abiotic stress-inducible OsSta2 protein exhibits a resistance to salt stress of 1.5-5.0, 1.5-4.5 or 1.5-4.0 times compared to the wild type.

According to another embodiment of the present invention, the salt stress-inducible OsSta2 protein exhibits an increase of 2-15%, 3-15% or 4-13% relative to the grain yield compared to the wild type .

According to another aspect of the invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding said abiotic stress-inducible OsSta2 protein.

As used herein, the term " nucleic acid molecule " has the meaning inclusive of DNA (gDNA and cDNA) and RNA molecules. In the nucleic acid molecule, the nucleotide which is a basic constituent unit is not only a natural nucleotide, Also included are analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

According to an embodiment of the present invention, the nucleotide sequence is a sequence of SEQ ID NO: 1. The nucleic acid molecule of the present invention encoding an abiotic stress-inducible OsSta2 protein is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The above-mentioned substantial identity can be determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible, and analyzing the aligned sequence using a program commonly used in the art (for example, ClustalX and DNASIS) Means a nucleotide sequence which, in one case, exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology.

According to another aspect of the present invention, the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned abiotic stress-inducible OsSta2 protein.

According to one embodiment of the present invention, the vector of the present invention comprises (a) a nucleotide sequence encoding a salt-inducible OsSta2 protein, and (b) a vector comprising a promoter operably linked to said nucleotide to provide.

As used herein, the term " operably linked " refers to a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The sequence will control the transcription and / or translation of the different nucleic acid sequences.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) This document is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pL? Promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) It is common to include a ribosome binding site and a transcription / translation termination sequence for initiation of translation. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) and the left promoter of phage l Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)).

Meanwhile, vectors that can be used in the present invention include plasmids such as pGA series (Kim et al., 2009), ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19 ), A phage (e.g., λgt4λB, λ-Charon, λΔz1, and M13), or a virus (eg, SV40 or the like).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell (for example, a metallothionein promoter) or a mammalian virus Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The vectors of the present invention may be fused with other sequences to facilitate purification of the salt stress-inducible OsSta2 protein expressed therefrom. The fusion sequences include, for example, maltose binding protein (NEB, USA), glutathione S-transferase (Pharmacia, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA). Because of the additional sequence for such purification, proteins expressed in the host are rapidly and easily purified through affinity chromatography.

The vector of the present invention may be a selection marker and may include an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The gene of the present invention has been isolated from plants and has the most favorable utility for plants since it can act to enhance plant tolerance to abiotic stress. Thus, the vector of the present invention comprises (i) a nucleotide sequence encoding a salt-inducible OsSta2 protein; (Ii) a promoter operatively linked to the nucleotide sequence of (i) above and acting on plant cells to form RNA molecules; And (iii) a plant expression vector comprising a 3'-non-detoxified site that acts in plant cells to cause polyadenylation of the 3'-end of the RNA molecule.

According to one embodiment of the present invention, the promoter suitable for the present invention may be any of those conventionally used in the art for transgenic introduction of a plant, for example, a ubiquitin promoter of corn, a Cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase (nos) promoter, Piguet mosaic virus 35S promoter, water crab basil lipid viral promoter, combellinella yellow mothball virus promoter, ribulose-1, 5-bis- (TPI) promoter, adenine phosphoribosyltransferase (APRT) promoter of Arabidopsis, and the tryptophan synthase promoter of ssRUBISCO, the cytosolic triphosphate isomerase (TPI) promoter of Arabidopsis thaliana, .

According to one embodiment of the present invention, the 3'-non-detoxified site causing polyadenylation in accordance with the present invention is derived from nos 3 'end (Bevan et al., Nucleic Acids Research, 11 (2): 369-385 (1983)), derived from the Octopane synthase gene of Agrobacterium tumefaciens, protease inhibitor I of tomato or potato End < / RTI > portion and a CaMV 35S terminator.

Alternatively, the vector additionally carries a gene encoding a reporter molecule (e.g., GFP (Green Fluorescence Protein)). In addition, the vector of the present invention can be used as a selection marker for a gene resistant to antibiotics (e.g. neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin etc.) (for example, neomycin phosphotransferase (nptII) Mycophosphotransferase (hpt), etc.).

According to another aspect of the present invention, there is provided a transformant transformed by the above-mentioned vector of the present invention.

Host cells capable of continuously cloning and expressing the vector of the present invention in a stable manner can be used in any host cell known in the art, such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli . such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus strains, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas spp. Enterobacteriaceae and strains.

When the vector of the present invention is transformed into eukaryotic cells, Saccharomyce cerevisiae , insect cells, human cells (e.g., Chinese hamster ovary, W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells.

Methods of delivering a vector of the present invention into a host cell include the CaCl2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)) when the host cell is a prokaryotic cell , Hanahan, D., J. MoI. Biol., 166: 557-580 (1977)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 1983) and electroporation method (Dower, WJ et al., Nucleic Acids Res., 16: 6127-6145 (1988)). When the host cell is a eukaryotic cell, the microinjection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (Wong, TK et al., Gene, 10: 87 (1980)), DEAE- (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 ) Or the like into the host cell.

The vector injected into the host cell is expressed in host cells, in which case a large amount of salt stress-inducible OsSta2 protein is obtained.

According to one embodiment of the present invention, the transformant shows improved bounce rate.

According to another aspect of the present invention, the present invention provides a method for producing a plant cell, comprising: (a) transforming a plant cell or a plant tissue with the vector of the present invention; (b) screening the transformed plant cell or plant tissue; And (c) regenerating the plant from the transformed plant cell or plant tissue to obtain a transformed plant. The present invention also provides a method for producing a transformed plant having enhanced salt stress tolerance.

According to another aspect of the present invention, the present invention provides a transgenic plant having a salt stress tolerance.

The explant used in the present invention is a plant cell or a plant tissue, and when plant tissue is used, a callus is preferably used.

In the method of the present invention, transformation of plant cells can be carried out according to conventional methods known in the art, including electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982) ), Particle bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and Agrobacterium-mediated transformation (U.S. Patent Nos. 5,004,863, 5,349,124 and 5,416,011) . Of these, Agrobacterium-mediated transformation is most preferred.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent such as a metabolic inhibitor, an antibiotic and a herbicide. Plant cells that stably contain a marker gene that is transformed and conferring selectative resistance are grown and divided in the above cultures. Exemplary labels include, but are not limited to, hygromycin phosphotransferase gene, glycophosphate tolerance gene and neomycin phosphotransferase (nptII) system.

Methods for the development or regeneration of plants from plant protoplasts or from various expansions are well known in the art. The development or regeneration of plants containing foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (U.S. Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

Although the method of the present invention is applied to various plants, according to one embodiment of the present invention, crop plants such as rice, barley, wheat and corn are applied, and more preferably, rice is applied.

The features and advantages of the present invention are summarized as follows:

(a) the present invention includes a salt stress-inducible OsSta2 protein comprising the amino acid sequence of SEQ ID NO: 2 and enhancing tolerance to salt stress in the plant when overexpressed in the plant, and a nucleotide sequence encoding the OsSta2 protein A method for producing a transgenic plant having enhanced salt stress tolerance, and a method for producing a transgenic plant having resistance to salt stress produced by the method, Thereby providing an enhanced transgenic plant.

(b) The nucleotide sequence used in the present invention is involved in increasing the resistance of the plant to abiotic stress (for example, salt stress) and seed production, and when the plant is transformed using the nucleotide sequence, the plant resistance to the above- And grain yield can be increased at the same time, so that the limit of the cultivation area of the plant can be overcome, and when applied to rice, it is possible to maximize productivity by being able to produce rice with high yield and strong against abiotic stress.

FIGS. 1A to 1C show the result of confirming the salt stress tolerance of OsSta2 tagging mutation. FIG. 1A shows the expression pattern of salt tolerance of seedlings after a 6-day recovery period after salt treatment for 7 days or salt treatment for 48 hours at 250 mM NaCl condition at 100 mM NaCl condition on germination on day 9 of germination. Figure 1b shows a schematic view, and RT-PCR results of the T-DNA inserted between the OsSta2 and LOC _ Os02g43830. Figure 1c shows the results of qRT-PCR for the expression of OsSta2 in the tagged mutant and wild-type.
Figures 2a and 2e show the expression patterns of OsSta2 in the tissues under stress. Figure 2a shows the qRT-PCR result of OsSta2 expression over the developmental stage. Ca = callus, Ss = 7-DAG shoot, Sr = 7 days old root, Lf = mature leaf, Ls = flag leaf sheaths ), Ih = mature seeds at 3 days after pollination, RAc1 was used as a reference gene. Figures 2b to 2e show the results of qRT-PCR of OsSta2 expression in seedlings treated with salt, dried, ABA and cold stress for 0, 1, 3, 6 and 24 hours respectively .
Figures 3a-3d show the OsSta2 overexpression (Ox) transfection construct. Figure 3a schematically depicts the p Ubi :: OsSta2 construct used for transformation. P ubi , Maze Ubiquitin promoter; P 35S , CaMV 35S promoter; Termination sequence of the nos T furnace sold synthase gene; T7, the terminator sequence of transcript 7; hph , hygromycin phosphotransferase gene; RB (right border) and LB (left border) are the right border and left border sequences of the Ti plasmid derived from Agrobacterium tumefaciens , respectively. Figure 3b shows the results of qRT-PCR analysis of OsSta2- Ox transformation. Actin is a loading control. Figure 3c shows the phenotype on day 8 of germination with OsSta2- Ox transfection. Figure 3d shows the phenotype of the seed germination on day 14 in 1 / 2MS.
Figures 4a-4d show improved salt stress tolerance in the OsSta2- Ox transgenic growth step. Figure 4a shows transgenic and wild-type phenotypes having a 6 day recovery period with 100 mM NaCl treated for 7 days in seedlings at 8 days germination. Figure 4b shows the fresh weight after recovery. Figure 4c shows the dry weight after drying for 2 days. Figure 4d shows the stress-tolerance as judged by the survival ratio of OsSta2- Ox transformation. On the 8th day after germination, the seedlings were subjected to stress (single treatment for 32 hours), salt treatment (treatment with 100 mM NaCl for 7 days and treatment with 250 mM NaCl for 72 hours) a is the number of surviving graves / total number of grains, b is the percentage of surviving seedlings in total grains, and * indicates the significant difference between the transgenic and wild type ( t- test and χ ² test P <0.05).
Figures 5a to 5d show the photosynthetic rate of OsSta2- Ox transfection under stress conditions. Figure 5a shows leaf disc analysis in which homozygous T4 OsSta2-Ox transgenic strains (Ox13, Ox19 and Ox20), wild type and T-DNA mutant strains were stress treated with salt (100, 200 and 250 mM NaCl). Figures 5b and 5c show the homozygous T4 OsSta2- Ox transformation chlorophyll evaluation (mg per g fresh weight). FIG. 5d shows the fluorescence intensity ( Fv / g ) of the homozygous T4 OsSta2- Ox transfection reaction at high salt (500 mM NaCl 48 hr), dry (3 hr anhydrous in Petri dish) Fm ). Three homozygotes T4 and wild type of OsSta2- Ox transformants were cultured in soil for 12 days. Fv / Fm values were measured using a pulse-regulated fluorescent light system (mini-PAM; Walz) after various stresses on the leaf discs. All plants were stressed under a continuous light source of 150 μmol m ^-2 s ^-1 . * Values indicate significant differences between OsSta2- Ox transgenic and wild-type ( t- test, P <0.05).
Figures 6a-6k show the agronomic characteristics of OsSta2- Ox transfectants under normal conditions. Fig. 6 (a) shows the number of small water droplets per hill, Fig. 6 (c) shows the number of small water droplets per hill, Fig. 6 Figure 6h shows the length of the cones, Figure 6h shows the weight of 1000 grains, Figure 6i shows the morphology of the mature rice cones, Figure 6j shows the morphology of the mature cultured rice, Figure 6k shows the OsSta2- And a spider-type graph of agricultural characteristics of the wild type. Each measurement point represents a percentage of the median value (n = 8) listed in Figures 7A through 7H. The median value of the wild type was set as a reference value of 100%. CL is the length of the stem (Culm length), NFG is the number of filled grains, NSH is the number of spikelets per hill (number of spikelets per hill), NSP is the number of small pieces per cone spikelets per panicle, NP is the number of panicles per hill per hill, NT is the number of tillers per hill per hill, and 1,000 GW is 1,000 grain weights. The error bars represent the standard error values of the 8 experiments. * Values indicate significant differences between OsSta2- Ox transgenic and wild-type (t-test, P <0.05).
Figures 7a and 7b show an analysis of the OsSta2- Ox transgenic ABA susceptibility. OsSta2- Ox transformants exhibit an ABA-sensitive phenotype during early seedling growth. Two kinds of OsSta2- Ox (Ox19 and Ox20) on the 5th day of germination were transferred to half-strengh MS medium supplemented with different concentrations of ABA. 7A shows the inhibition of OsSta2- Ox growth by ABA treatment. Seven days after transplantation, it is a seedling's grave. The scale bar represents 5 cm. Figure 7b shows the length of the relative shoots of OsSta2- Ox and wild type. Results are mean ± number of seedlings per experiment).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Plant material and abiotic stress conditions

A seed pool of T-DNA insertion mutant and overexpressed rice ( Oryza sativa ssp. Japonica cv. Dongjin) was surface-sterilized and germinated in a wet paper towel for 2 days. Plants were cultivated in pots in a work-in growth chamber in a temperature cycle ranging from 22-30 ° C and a 12-hour photoperiod condition. Yoshida solutions of dried (water removed), salt (300 mM NaCl), low temperature (4 ° C) and ABA (Abscisic acid, 100 μM) were incubated for 0, 1, 3, 6 , 12 and 24 hours after germination on day 8, and cultured by hydroponics. T4 seeds were analyzed for survival rate of abiotic stress tolerance. Germinated seedless germinated seeds on day 8 of germination were cultured in a box containing 100 g soil. Dry stress was induced by not sprinkling for 30-40 hours until the leaves were dry. The seedlings were transferred to 100 mM NaCl and 250 mM NaCl solutions and incubated for 7 and 72 hours, respectively, to induce salt stress. The salt solution was changed twice to keep the concentration until the leaves were free. The seedlings were poured with 2 liters of cold water and incubated at 4 ° C for 48-72 hours to induce cold stress. The survival rate of the plants after the recovery period was calculated for 6 days after the stress treatment. Photographs representing the stress tolerance of the plants were taken and the fresh weight was measured. The dry weight of the plant was measured after drying at 80 DEG C for 2 days.

The T5 generation of the OsSta2- Ox transformants was subjected to the ABA sensitivity test. Surface-sterilized and peeled seeds were germinated on half-strength MS medium containing hygromycin (50 mg / L) and the parental transformants were determined after germination. Five days after germination, a similar transgenic tobacco seedlings were seeded at 6.125 sq. G. Containing half-strength MS medium supplemented with 0, 5, 10 [mu] M ABA. Inch &lt; / RTI &gt; and 3 &quot; sized cultivated area. The seedlings were grown vertically. Growth of the seedlings was measured 7 days after implantation.

Active tagging screening for salt stress tolerance

T-DNA transfection To screen for salt tolerance, germinated 8-day old seedlings were treated with 100 mM NaCl for 7 days or 250 mM NaCl for 48 hours. After treatment, the seedlings were restored for 6 days and the survival rate was determined. The expression of T-DNA tagged genes was confirmed by RT-PCR and qRT-PCT.

Analysis of gene expression using RT-PCR and qRT-PCR

RNeasy mini plant kit (QIAGEN, Germany) was used to isolate total RNA and cDNA was synthesized according to the manufacturer's instructions using RT Complete kit (BIOFACT, Korea). For amplification of PCR products using Gene Runner software ( http://www.generunner.net/ ) and NCBI primer blast ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) The primer was designed. The primer sequences are shown in Table 1. The final concentrations of the primers were 10 pM for RT-PCR and 5 pM for qRT-PCR, and 3 μl cDNA (6 ng total RNA) was used as the template.

- order Analysis method Sequence List OsSta2-F AGATGCAATTGCGCGAAC RT-PCR Fifth sequence OsSta2-R CACCGTTGAGGTTGTCGTTG RT-PCR 6th sequence Pubi-F TTGATATACTTGGATGATGGCATA gD-PCR Seventh sequence Tnos-R GCGGGACTCTAATCATAAAAACC gD-PCR Eighth sequence RAc1-F CGCAGTCCAAGAGGGGTATC RT-PCR Ninth sequence RAc1-R TCCCTCACAATTTCCCGCTC RT-PCR Tenth sequence RAc1-qF GACTCTGGTGATGGTGTCAGCCACAC qRT-PCR Eleventh sequence RAc1-qR CGCACTTCATGATGGAGTTGTAT qRT-PCR 12th sequence

PCR was carried out at 95 ° C for 5 minutes, followed by 25-35 cycles at 95 ° C for 30 seconds, 58 ° C for 30-60 seconds, and 72 ° C for 30-60 seconds. The amplified product was resolved in 0.8% agarose gel. The RT-PCR results were validated with the total RNA prepared independently by repeating the experiment one or more times. For qRT-PCR analysis, KAPA SYBER FAST Universal PCR Kit and LightCycler 96 (ROCHE LIFE SCIENCE, Germany) were used. The PCR cycle was carried out at 95 ° C for 3 minutes, followed by amplification at 40 cycles of 95 ° C for 3 seconds, 60 ° C for 20 seconds, and 72 ° C for 20 seconds, followed by reaction at 72 ° C for 20 seconds. The melting curve was obtained under the conditions of 95 占 폚 for 5 seconds, 65 占 폚 for 1 minute, and 97 占 폚 for 1 minute. And then cooled at 40 DEG C for 10 minutes. Relative expression level was calculated using the 2 ^- △ ΔCt method as the internal gene RAc1 .

Inositolysis of the OsSta2 promoter

The 2 kb upstream upstream promoter sequence of the ATG initiation codon was analyzed by Oryzabase (Kurata and Yamazaki, 2006), and the cis -elements in the promoter were analyzed using the PLACE database (http://www.dna.affrc.go.jp/PLACE/) Respectively.

Manufacture of OsSta2 overexpression

For construction of the OsSta2-overexpression vector, OsSta2 cDNA was purchased from KOME (http://cdna01.dna.affrc.go.jp/cDNA/). It placed the cDNA under maize (maize) ubiquitin 1 promoter regulatory having BamH1 and SacI site and a selling furnace (nopaline) synthase terminator of the pGA3426 binary vector. The calyx-derived blastocyst of the Japanese rice variety, 'Dongjin', was transformed with Agrobacterium-mediated co-culture. Transformants were transferred to designated paddies for further growth.

Transformational Isolation Analysis

Genes of OsSta2 gene in T2-T5 generation were analyzed. The offspring were evaluated for resistance to hygromycin. More than 50 T2-T3 seeds of independent transformants of Japanese rice varieties were selected with hygromycin-containing medium (50 mg / L). After 5 days of incubation at 30 ° C, the number of viable seedlings was counted and all viable seedlings were judged to be homozygous.

Analysis of abiotic stress-tolerance through chlorophyll fluorescence measurement

OsSta2- OX transformants and wild-type control rice seeds were germinated and grown in a soil box in a work-in growth chamber (12 h photoperiod [150 μmol m ^-2 s ^-1 ] 12 h cancer cycle 28 ° C.) for 12 days . Prior to stress treatment with Invit, the 5th green leaf of 10 seeds was cut with scissors. High salt stress was induced by incubation at 28 ° C for 48 hours in a continuous light source of 110 μmol m-2 s-1 in 500 mM NaCl. Drying stress was induced by air drying at 28 ° C under a continuous light source of 110 μmol m ^-2 s ^-1 for 3 hours. Cold stress was induced by immersing seedlings in 4 ° C deionized water for 48 hours under a continuous light source of 110 μmol m ^-2 s ^-1 . Fv / Fm values were measured using a MINI-PAM-II Photosynthesis Yield Analyzer (WALZ, Germany).

Salt tolerance analysis of leaf disc

For leaf disc analysis, 30 ml of NaCl solution (100, 200 and 250 mM, 24h) was prepared by washing the transformed (maturing), wild type, healthy, fully extended leaves with deionized water and cutting into 1 cm diameter leaf discs. . Phenotypic changes were observed and chlorophyll content was measured to quantify the effect of salt stress treatment on leaf discs.

Survey of agricultural characteristics

Dongjinbyeo (Oriza sativa ssp japonica cv Dongjin..), Wild-type (OsSta2 -Ox attention non-transformant) and the independent homozygous in OsSta2 -Ox note seeds surface-sterilized and were germinated at 30 ℃ dark conditions. Two days after germination, the seedlings were grown in a soil box in a growth chamber (28 ° C, 60% RH, 12-h light period / 12-h dark period, light source 150 μmol m ^-2 s ^-1 ). One week old tomb moved to the greenhouse. After 4 weeks, the plant was fed into the rice LMO field and harvested at the end of October, 2012, 2013, 2014 and 2015 every year. The test nursery is the National University of Korea, Gunwi County, Kyungbuk, and Kyunghee University, Suwon. In order to analyze the agricultural characteristics of OsSta2 -Ox T4 and T5 homozygotes and wild type rice, the number of ginseng per plant, number of panicle per plant, number of spikelets panicle, number of filled grain, length of cone, length of stem, and grain weight were analyzed. To culture in agar medium, the seedlings were incubated for 5 days at 30 ° C under dark conditions and transferred to continuous light conditions.

Statistical analysis

The median value ± standard deviation was calculated from the results of three repeated experiments. The differences between treatments were determined by Student t-test and x2 test (P < 0.05).

Experiment result

Isolation of Salt Stress-Resistant Active Tagging Mutagenesis

PFG T-DNA tagging mutants The pools of T2 generations were screened to obtain PFG_3A-05272.R having both salt stress tolerance at 100 mM and 250 mM NaCl (FIG. 1A and Table 2). LOC _ Os02g43820 and reveals a T-DNA tagging between LOC _ Os02g43830 using the chair in the reverse (inverse) PCR analysis of molecular PFG_3A-05272.R. The results confirmed the two, RT-PCR or qRT-PCR of the gene LOC _ Os02g43820 only was induced compared with the wild-type strings charged 8-fold (Fig. 1c). This gene was named Oryza sativa Salt tolerance activation 2-Dominant ( OsSta2 , LOC_Os0g43820 ).

Salt stress Wild type OsSta2-D 100 mM NaCl 9/48 ^a
(18.8) ^b 16/43 ^*
(37.2) 250 mM NaCl 0/48
(0.0) 5/48 ^*
(10.4)

Table 2 above shows the salt-tolerance of wild type and OsSta2 tagging mutants determined by the survival ratio. Seedlings on the 8th day of germination were treated with salt stress (treatment with 100 mM NaCl for 7 days and treatment with 250 mM NaCl for 48 hours) and survival rate was measured after 6 days of recovery. a is the number of surviving graves / total graves, and b is the percentage of graves surviving in total graves. * Significance means significant difference from wild type (χ ² test, P <0.05 ).

Isolation and structural analysis of OsSta2

The putative amino acid composed of the 1035 bp ORF (open reading frame) of the OsSta2 gene is a 344 amino acid protein whose molecular weight and pI are expected to be 36.3 kDa and 5.63, respectively (SEQ ID No. 3 and 4). The presumed secondary structure of the OsSta2 protein is one sheet, two β-hairpin, one strand, one helix and one spiral-spiral interaction . It is estimated that the putative OsSta2 protein has three amino acid sites (A24HAAAGEAEAVYRAAAAAEPVMIRFGGEVSPVSDPRRPPLTISLPPTSAWAAAEAVHPAALLQAQTAAAAAD, P172PPPPPPP and S205SKSVKTETYTSPASSSLA-STTTSTVTSSSTSPSPSS) rich in AAV, Pro and Ser conserved DNA-binding domain (AP2 / ERF domain) of 58 amino acids. The analysis of the OsAt2 alignment and phylogenetic tree showed that Zea mays ZmEREBP (KJ727501, 62%), Setaria italic SiERF5 (XP_004953326, 58%), Brachypodium distachyon BdERF6 (XP_003572768, 53%), Sorghum bicolor Sb06g024360 (XP_00446882, 43%), Arabidopsis and at amino acid level with thaliana AtERF5 (At5g47230, 42%).

The OsSta2 cDNA used to prepare OsSta2- Ox transgenic strains consists of 775 bp and 56 amino acids in truncated form (Sequence Listing 1 and Sequence 2).

Expression analysis of OsSta2

Expression of OsSta2 in other tissues was analyzed by RT-PCR and validated by qRT-PCR. It was expressed in all tissues, and was abundant in 3-8 cm long cones, mature cones, callus, roots of 7 days after germination, leaflet of leaflets, and the best (top) Germination on day 7 of germination, and mature seed on day 3 of water (Fig. 2a). OsSta2 expression decreased for 3 hours after 12 hours of salt stress induction (FIG. 2b). Expression was induced after 24 hr dry stress (FIG. 2C). During ABA treatment, OsSta2 expression decreased for 3 hours and then increased (Fig. 2d). OsSta2 was not induced under cold stress (Fig. 2E).

Preparation of OsSta2-Ox Transformational Assay

To prepare an OsSta2 over-expression transformant, the cDNA sequence (J065129D08) obtained from KOME (http://cdna01.dna.affrc.go.jp/cDNA/) was inserted under the maze ubiquitin promoter in the pGA3426 vector (Fig. 3A) . The cassette was replaced with O. sativa L. cv. And transformed to Dong Jinbin. A total of 21 independent transgenic strains were produced. Genomic DNA PCR analysis confirmed the insertion of OsSta2 . Among the first transgenic strains, five strains expressing normal seed production were selected for T1 generation. The seeds were harvested and selected with hygromycin-containing medium (50 mg / L). The salt stress test using T2 homozygous seeds confirmed the strong salt stress tolerance of three selected T2 generation OsSta2 overexpressing strains selected for further analysis. The above-noted OsSta2 overexpression was confirmed by RT-PCR and validated by qRT-PCR (Fig. 3B). Three independent strains increased expression of OsSta2 over wild type and did not show any morphological differences (Fig. 3c and 3d).

OsSta2-Ox Transformational Salts Stress Resistance

In order to investigate whether overexpression of OsSta2 confers salt stress tolerance, salt stress was given to rice seedlings in a work-in growth chamber. The OsSta2- Ox transgenic line survived 10.1-22, 7% and the wild type survived 7.2% after treatment with 100 mM NaCl for 7 days. Similar results were obtained under the condition of treatment with 250 mM NaCl for 72 h. Transgenic lines survived 25.8-34.5% but only wild type 11.6% survived (FIG. 4). The fresh weight and dry weight of the OsSta2- Ox transformed strain were increased after restoration of the stress than the wild type (Figs. 4B and 4C).

OsSta2- Ox transgenic lines were treated with various concentrations of NaCl and leaf disc analysis to further confirm salt stress tolerance. Wild-type and T4 OsSta2- Ox Transformational leaf disks were floated in 100, 200 and 250 mM NaCl solution for 4 hours. Salinity-induced loss of chlorophyll was less in OsSta2- Ox transgenic strains as compared to wild type (Figure 5a). After 24 hours, the degree of blistering of the leaf tissue, which indicates damage due to stress, was observed. Measurements of the chlorophyll content of stressed leaf discs indicate that the OsSta2- Ox transgenic strain and the salt stress are present in the cytosol (FIG. 5c).

To further confirm the stress-resistant phenotype, the OsSta2- Ox transgenic strain and the wild-type Fv / Fm value of the growth stage were measured (Fig. 5d). Fv represents the variable fluorescence, Fm represents the maximum fluorescence, and Fv / Fm represents the maximum photochemical efficiency of PSII in the cancer adaptation state. The Fv / Fm value was about 6% higher in the OsSta2- Ox transgenic strain than in the wild type at high salt conditions. In contrast, the Fv / Fm values of wild-type and OsSta2- Ox transgenic strains were almost similar under dry and low temperature conditions. Therefore, this result implies that overexpression of OsSta2 in the transgenic strain increases the resistance to high salt at the growth stage.

OsSta2-Ox Transformational ABA Sensitivity

The OsSta2- Ox transgenic strain showed an ABA-sensitive phenotype in early seedling growth (Fig. 7). Two weeks of germinated OsSta2- Ox transgenic (Ox) were transferred to half-strength medium supplemented with different concentrations of ABA. OsSta2- Ox transgenic shoot growth was inhibited by ABA treatment. At 5 μM and 10 μM ABA, shoots were significantly inhibited by 63% and 74%, respectively, than wild type. These results suggest that OsSta2- Ox transgenic strain is ABA sensitive and OsSta2 The gene is associated with the ABA signaling pathway.

Increased Breadth Gain by Overexpression of OsSta2

To investigate the role of OsSta2 on yield of grain under packaging conditions, 3 week OsSta2 -Ox transgenic T4 and T5 homozygous strains were grown in rice fields with wild type and analyzed for eight agricultural characteristics. The mature OsSta2- Ox transgenic strain did not show any difference up to the 4 leaf stage but showed anti-dwarfism. Overall, the adult OsSta2- Ox transgenic trunks were 7-9% shorter than the wild type (Figure 6). The OsSta2- Ox transgenic strain also showed more buds than the wild type. Investigation of the harvesting parameters under normal paddy conditions showed that the yield of OsSta2 -Ox transgenic paddy was higher than wild type. Statistical analysis of the harvest parameters indicates that the yield of OsSta2- OX transgenic bark is significantly increased over wild type. In particular, the filling rates of Ox13 and Ox19 were 17% and 23% higher than wild type and 5% and 8% higher total grain weight, respectively. Ox20 , which expresses less OsSta2 than other overexpressing strains , did not show any significant difference compared to wild type strains. Ox20 showed a 8% increase in the number of small cone cornflowers and a 10% increase in total grain weight.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> SOGANG UNIVERSITY RESEARCH FOUNDATION <120> Abiotic Stress-Inducible OsSta2 Gene, Protein and Transformant          Enhancing Tolerance to Salt Stress <130> PN160387 <160> 12 <170> KoPatentin 3.0 <210> 1 <211> 775 <212> DNA <213> Oryza sativa <400> 1 ggcagatgca attgcgcgaa cacctccaaa ctcccgagtc ccctctcctc tctcgtcttc 60 ctctccacga cgacctctcc gcttcaattc tgcgccgccc ccgccgccga cgacgtcgac 120 ccacggcaaa cggaagcggc acgagacggc ggcggccgac ccggacgtcg aggtgatcgg 180 agaatcatcc aaatccgtca agaccgagac gtacacatcg ccggcgtcgt cgtcgctggc 240 ctcaacgaca acctcaacgg tgacatcatc atccacgtcg ccttccccct cgtcggaggc 300 agctgcctgc ggcggcggcg gcggcgagct gttcgtccca ccgatgccgt cgagctggtc 360 gtgggatcag ttggaggggt tcttcggcat cctctcgccg ctctcgccgc acccgcaaat 420 gggattcccg gaggtcgccg taaactaatt aagcgcgcta atgatctctc taagcatgat 480 taattaggcg caataatcgg caattaatta gtggcttcta gttctcgacg gccttcgctt 540 tggacacatc tcaggagatt tggcttggag cagtgtacta tactgtacag aggagagaag 600 atagtatatt agcgcatgca tgtgacgggt actgaatgtg tgcatcgaca taggtgttgg 660 agatacactc tagatatctt gaaaataata agaggaaata atagtatagg atgggtgata 720 ataacattgt aaattaatta attaaatgaa gagtaaatgt gtactagttt gtttt 775 <210> 2 <211> 56 <212> PRT <213> oryza sativa <400> 2 Met Gln Leu Arg Glu His Leu Gln Thr Pro Glu Ser Pro Leu Leu Ser   1 5 10 15 Arg Leu Pro Leu His Asp Asp Leu Ser Ala Ser Ile Leu Arg Arg Pro              20 25 30 Arg Arg Arg Arg Arg Arg Pro Thr Ala Asn Gly Ser Gly Thr Arg Arg          35 40 45 Arg Arg Pro Thr Arg Thr Ser Arg      50 55 <210> 3 <211> 1035 <212> DNA <213> oryza sativa <400> 3 atggacttca gcggcgacat cgatgacctg atctccctcc agctcctcca cgatcagctg 60 ctcggggtcg aggccgacgc gtgcctcccg gtggcagttc atcacgatgg cgtcgccgcg 120 ttcgccgagc atcagcaggg tttccatccc gcggcgttct tgccgcagca gccgatgacg 180 atgacaccgg ctgggtacgt ggacatggcg aatgatcagt atttgggtgc gcatgccgcc 240 gctggcgagg cggaggcggt ctaccgggcg gcggcggcgg agccggtgat gatacggttc 300 ggcggcgagg tgtccccggt gtcggacccc cggaggccgc ccctgacgat ctcgctgcct 360 ccgacgtcgc acgcgtgggc ggcggcggag gcggtgcacc cggccgctct gctgcaggcg 420 cagaccgcgg ccgcggccgc cgacccgaac gacttccgga agtaccgcgg cgtgcggcag 480 cggccgtggg gcaagttcgc ggcggagatc cgcgacccca agaagcgggg ctcgcgcgtg 540 tggctcggca cgtacgacac ggccatcgag gcggcgcgcg cgtacgaccg cgccgccttc 600 cgcatgcgcg gcgccaaggc catcctcaac ttccccaacg aggtcggctc ccgcggcgcc 660 gacttcctgg caccgccgcc gccgcccccg ccgccgacga cgtcgaccca cggcaaacgg 720 aagcggcacg agacggcggc ggccgacccg gacgtcgagg tgatcggaga atcatccaaa 780 tccgtcaaga ccgagacgta cacatcgccg gcgtcgtcgt cgctggcctc aacgacaacc 840 tcaacggtga catcatcatc cacgtcgcct tccccctcgt cggaggcagc tgcctgcggc 900 ggcggcggcg gcgagctgtt cgtcccaccg atgccgtcga gctggtcgtg ggatcagttg 960 gaggggttct tcggcatcct ctcgccgctc tcgccgcacc cgcaaatggg attcccggag 1020 gtcgccgtaa actaa 1035 <210> 4 <211> 345 <212> PRT <213> oryza sativa <400> 4 Met Asp Phe Ser Gly Asp Ile Asp Asp Leu Ile Ser Leu Gln Leu Leu   1 5 10 15 His Asp Gln Leu Leu Gly Val Glu Ala Asp Ala Cys Leu Pro Val Ala              20 25 30 Val His His Asp Gly Val Ala Ala Phe Ala Glu His Gln Gln Gly Phe          35 40 45 His Pro Ala Ala Phe Leu Pro Gln Gln Pro Met Thr Met Thr Pro Ala      50 55 60 Gly Tyr Val Asp Met Ala Asn Asp Gln Tyr Leu Gly Ala His Ala Ala  65 70 75 80 Ala Gly Ala Gla Ala Gla Ala Ala Ala Gla Pro Val                  85 90 95 Met Ile Arg Phe Gly Gly Glu Val Ser Ser Val Ser Asp Pro Arg Arg             100 105 110 Pro Pro Leu Thr Ile Ser Leu Pro Pro Thr Ser His Ala Trp Ala Ala         115 120 125 Ala Glu Ala Val His Pro Ala Ala Leu Leu Gln Ala Gln Thr Ala Ala     130 135 140 Ala Ala Ala Asp Pro Asn Asp Phe Arg Lys Tyr Arg Gly Val Arg Gln 145 150 155 160 Arg Pro Trp Gly Lys Phe Ala Ala Glu Ile Arg Asp Pro Lys Lys Arg                 165 170 175 Gly Ser Arg Val Trp Leu Gly Thr Tyr Asp Thr Ala Ile Glu Ala Ala             180 185 190 Arg Ala Tyr Asp Arg Ala Ala Phe Arg Met Arg Gly Ala Lys Ala Ile         195 200 205 Leu Asn Phe Pro Asn Glu Val Gly Ser Arg Gly Ala Asp Phe Leu Ala     210 215 220 Pro Pro Pro Pro Pro Pro Pro Thr Thr Ser Thr His Gly Lys Arg 225 230 235 240 Lys Arg His Glu Thr Ala Ala Ala Asp Pro Asp Val Glu Val Ile Gly                 245 250 255 Glu Ser Ser Lys Ser Val Lys Thr Glu Thr Tyr Thr Ser Pro Ala Ser             260 265 270 Ser Ser Leu Ala Ser Thr Thr Thr Ser Thr Ser Thr Ser Ser Thr         275 280 285 Ser Pro Ser Ser Ser Glu Ala Ala Ala Cys Gly Gly Gly Gly Gly     290 295 300 Glu Leu Phe Val Pro Pro Met Pro Ser Ser Trp Ser Trp Asp Gln Leu 305 310 315 320 Glu Gly Phe Phe Gly Ile Leu Ser Pro Leu Ser Pro His Pro Gln Met                 325 330 335 Gly Phe Pro Glu Val Ala Val Asn ***             340 345 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> OsSta2-F <400> 5 agatgcaatt gcgcgaac 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OsSta2-R <400> 6 caccgttgag gttgtcgttg 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Pubi-F <400> 7 ttgatatact tggatgatgg cata 24 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Tnos-R <400> 8 gcgggactct aatcataaaa acc 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RAc1-F <400> 9 cgcagtccaa gaggggtatc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RAc1-R <400> 10 tccctcacaa tttcccgctc 20 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> RAc1-qF <400> 11 gactctggtg atggtgtcag ccacac 26 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RAc1-qR <400> 12 cgcacttcat gatggagttg tat 23

Claims (10)

A composition for improving salt-stress resistance comprising a gene carrier comprising a nucleic acid molecule consisting of a nucleotide sequence of the first sequence of the sequence listing.
The composition according to claim 1, wherein the salt-stress resistance improving composition increases the grain yield.
delete delete delete A transformant transformed by the composition for improving the salt-stress resistance of claim 1.
7. The transformant of claim 6, wherein the transformant exhibits an improved grain yield.
A method for producing a transgenic plant having enhanced salt stress tolerance comprising the steps of:
(a) transforming a plant cell or a plant tissue with the salt-stress resistance-enhancing composition of claim 1;
(b) screening the transformed plant cell or plant tissue; And
(c) regenerating the plant from the transformed plant cell or plant tissue to obtain a transformed plant.
9. The method according to claim 8, wherein the plant is rice, barley, wheat, or corn.
9. A transgenic plant having enhanced tolerance to salt stress produced by the method of claim 8 or 9.

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