KR100990333B1 - Hordeum vulgare Na+/H+ antiporter HvNHX gene, method for improving salt tolerance in plants by expressing HvNHX gene and transgenic plants with improved salt tolerance developed by the application of the method - Google Patents

Abstract

The present invention plays a role of sequestering the cytosolic sodium ions of plant cells into vacuoles to improve flame resistance ( Hordeum vulgare ) sodium ion / hydrogen reverse carrier (Na + / H + antiporter) ( HvNHX1 ) cDNA sequence, recombinant vector comprising the gene, yeast and plant transformed with the recombinant vector, and flame resistance of the plant using the gene It relates to a method for enhancing the flame resistance of the plant and its seeds, and the plant containing the gene and the salt resistance enhanced using the method.

The method according to the present invention can be grown in high salted reclaimed land by using genes that sequester the sodium ions of plants with vacuoles in the cytoplasm and by using the genes to increase the salt resistance of cells and plant individuals. In addition, there is an advantage that can produce and develop excellent plants, it can be usefully used for production conditions, increasing the utilization efficiency and plant seed industry.

Barley, Sodium Ion / Hydrogen Reverse Carrier, HvNHX1, Mutant, Rice, Salt Tolerant, Yeast, Transgenic Plant

Description

NHH gene derived from barley, a method for improving the flame resistance of plants using the same, and a transgenic plant having improved salt resistance using the method (Hordeum vulgare Na + / H + antiporter (HvNHX) gene, method for improving salt tolerance in plants by expressing HvNHX gene and transgenic plants with improved salt tolerance developed by the application of the method}

The present invention relates to a method for enabling flame resistance enhancement of plants, and more particularly barley ( Hordeum) that serves to sequester sodium ions of the cytoplasm of plant cells with vacuoles to enhance flame resistance. vulgare ) sodium ion / hydrogen reverse carrier (Na + / H + antiporter) ( HvNHX1 ) cDNA sequence, recombinant vector comprising the gene, yeast and plant transformed with the recombinant vector, and flame resistance of the plant using the gene It relates to a method for enhancing the flame resistance of the plant and its seeds, and the plant containing the gene and the salt resistance enhanced using the method.

Sodium ion / hydrogen ion carriers catalyze the transport of sodium ions through the vacuole membrane from the cytoplasm to the vacuole and the exchange of hydrogen ions in the reverse direction with sodium ions to regulate the pH of the cytoplasm, the levels of sodium ions, and the size of the cells (Aronson, PS, Annu. Rev. Physiol. 1985. 47: 545-560; Orlowski, J., Grinstein, S., J. Biol. Chem. 1997. 272: 22373-22376). In particular, the vesicle-type sodium ion / hydrogen ion reverse carriers present in the vacuole membranes of plant cells sequester high concentrations of sodium ions in the cytoplasm of the plant cells with vacuoles under high salt conditions, thereby lowering the sodium ions in the cytoplasm to enhance salt resistance. (Zhang, HX and E Blumwald. 2001. Nat. Biotechnol. 19: 765-768).

In addition, yeast complementation studies on the complementary ability of vacuole sodium ion / hydrogen ion reverse carriers to inhibit sodium ion and hygromycin susceptibility of yeast mutants lacking sodium ion / hydrogen ion reverse carriers have already been performed by AtNHX1 (Gaxiola et al. Proc. Natl. Acad. Sci. USA. 96: 1480-1485 ), OsNHX1 (Fukuda et al. 2004. Journal of Experimental Botany, 55: 585-594) and TaNHX1 (Brini et al. 2004. Plant Physiology and Biochemistry 43: 347-354), and studies have shown that NHX inhibits salt susceptibility by partitioning sodium ions entering the yeast cytoplasm into vacuoles and inhibits hygromycin by modulating NHX for toxic cations in yeast vacuoles. Specific evidence suggests that it also reduces sensitivity.

In recent years, the sodium ion / hydrogen reverse carrier gene (AgNHX1) of transgenic plants has been enhanced to increase the salt resistance of rice (Ohta M. et al. 2002. FEBS Letters 26785: 1-4). The usefulness of the sodium ion / hydrogen ion reverse carrier genes presented in the study results in enhancing the salt resistance was also found in monocotyledonous plants.

The present invention has been made in accordance with the above requirements, and in the present invention, the sodium ion / hydrogen reverse carrier gene ( HvNHX1 ) was isolated and identified from barley, and the gene was deficient in the sodium vesicle- type sodium ion / hydrogen ion reverse carrier gene. Susceptible Mutant Yeast ( Sacchromyces) cerevisiae ) lineage R100 (Δnhx1) was tested for the function of the salt of HvNHX1 by heterologous expression. In addition, the gene was recombined into a plant gene expression carrier and inserted into rice cells using Agrobacterium tumefaciens , and T1 has improved salt resistance through differentiation and generation progression from the gene-transmitted cell to the complete rice plant. Generation rice system was obtained. The rice line obtained using the above-described method and the above method differs from the conventional hybridization and selection method and the cultivars in which a plurality of genes obtained are combined. In other words, the rice varieties obtained by the present invention have only improved flame resistance while maintaining the excellent traits of the material varieties used in the present invention, and have improved salt resistance in rice.

The present invention has the advantage of producing and developing an environmentally superior plant as a useful gene for improving adaptability by separating the NHX gene, which is the most effective for improving flame resistance among the known genes, from barley, and salts such as reclaimed land. It can increase the use efficiency of the production conditions, including a lot, can be usefully used in plant seed industry.

In order to solve the above problems, the present invention serves to sequester sodium ions of the cytoplasm of plant cells into vacuoles to enhance flame resistance, and is a sodium ion / hydrogen ion reverse carrier derived from barley ( Hordeum vulgare ) (Na + / H + antiporter). A recombinant vector comprising the ( HvNHX1 ) gene is provided.

The present invention also provides yeast and plants transformed with the recombinant vector.

The present invention also provides a method for enhancing the flame resistance of plants using the gene.

The present invention also provides plants and seeds thereof having improved flame resistance using the above method.

The present invention also provides a method of cultivating the plant with enhanced salt resistance in reclaimed land or production conditions known as high salt.

The present invention also provides a composition for enhancing salt resistance of a plant comprising the gene.

According to the present invention, the cDNA sequence isolated in the present invention is a sodium ion / hydrogen reverse carrier ( HvNHX1 ) cDNA derived from barley, and the HvNHX1 cDNA is a salt of a mutant of a yeast lacking a vesicular sodium ion / hydrogen ion reverse carrier. It has been confirmed that the resistance to the disease can be restored, and the salt resistance of the plant at the seedling stage can be enhanced under salt stress conditions. Therefore, HvNHX1 cDNA, a reverse carrier of sodium ion / hydrogen ion derived from barley, is a gene that has a high effect on enhancing flame resistance and is useful for improving adaptability to plant production conditions. It is useful for plant seed industry by producing and developing environmentally superior plants. Can be used.

In order to achieve the object of the present invention, the present invention is barley ( Hordeum) vulgare ) NHX (Na + / H + antiporter) gene HvNHX1 And it provides a recombinant vector comprising the same.

The present invention relates to cDNA encoding barley sodium ion / hydrogen ion reverse carriers that isolate high concentrations of sodium ions in the cytoplasm of plant cells under high salt conditions, thereby reducing cytosolic sodium ions. NHX function by heterologous expression The assay utilizes yeast mutant yeast R100 (Δnhx1) lacking a vesicular sodium ion / hydrogen ion reverse carrier and control line K601 (Dr. R. Rao, Johns Hopkins University, USA) to restore the mutant properties of the yeast HvNHX1 cDNA. It is about contents which confirmed whether or not.

Preferably the HvNHX1 The gene may consist of the nucleotide sequence represented by SEQ ID NO: 1. HvNHX1 The protein sequence deduced by the gene is the amino acid sequence shown in SEQ ID NO: 2. In addition, variants of such sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in base sequence but has similar functional properties to that of SEQ ID NO: 1. Specifically, HvNHX1 The gene may comprise a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1.

The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

Recombinant vector according to an embodiment of the present invention may be a yeast expression vector, more preferably the vector may be a pMG515 vector described in FIG. The yeast expression vector pMG515 was isolated by identifying and identifying a plant sodium ion / hydrogen ion reverse carrier ( HvNHX1 ) cDNA derived from barley, and was constructed by recombination into pRG410 using uracil as the selection marker. However, the recombinant vector of the present invention is not limited to the vector described in FIG. 2, and any vector useful for yeast transformation can be used.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment (s), 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 "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.

The present invention also provides yeast transformed with the recombinant vector of the present invention. More specifically, HvNHX1 in yeast seedling R100 lacking the sodium ion / hydrogen ion reverse carrier gene By introducing a recombinant yeast expression vector pMG515, the cDNA is to provide a Y.PMG515 yeast that has been restored to the salt resistance.

The host cell that can be used in the present invention is preferably Saccharomyces cerevisiae strain, but is not limited thereto, Pichia pastoris; Hansenula polymorpha; Yarrowia; Schizosaccharomyces pombe; Kluyveromyces hosts, eg K. K. lactis, K. Fragilis (K. fragilis), K. Bulgaricus (K. bulgaricus), K. K. wickeramii, K. K. waltii, K. K. drosophilarum, K. Thermotelerans (K. thermotolerans) and K. K. marxianus is available.

As a method for transforming a host cell with the recombinant vector of the present invention, a method commonly used in the art such as electroporation can be used.

The recombinant vector of the present invention may be a 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 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the HvNHX1 gene according to the invention into a plant host are viral vectors such as those derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc., For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous, especially when it is difficult to properly transform a plant host.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, which corresponds to all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. It is not limited. The plant expression vector according to the embodiment of the present invention used a hygromycin resistance gene.

In the plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

The terminator may use a conventional terminator, and examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Ogrobacterium tumefaciens ) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

The recombinant plant expression vector according to one embodiment of the present invention may preferably be the pMG-HvNHX1 vector described in FIG. 5. The pMG-HvNHX1 vector is a binary vector for plant gene expression prepared by recombining HvNHX1 cDNA derived from barley into pGA1611. However, the recombinant vector of the present invention is not limited to the vector described in FIG. 5, and any vector useful for plant transformation can be used.

The present invention also provides a plant having enhanced flame resistance transformed with the recombinant vector of the present invention. More specifically, by using the plant transformation vector pMG-HvNHX1 to express HvNHX1 to maintain a homeostasis to sodium ions to provide a transgenic plant having increased flame resistance.

Plants according to the present invention is rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum food crops selected from the group consisting of; Vegetable crops selected from the group consisting of Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Special crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Fruit trees selected from the group consisting of apple trees, pear trees, jujube trees, peaches, lambs, grapes, citrus fruits, persimmons, plums, apricots and bananas; Flowers selected from the group consisting of roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops selected from the group consisting of lysis, redclover, orchardgrass, alphaalpha, tolsquescue and perennial lygragrass. Preferably, the plant may be monocotyledonous plants, such as rice, barley, corn, wheat, rye, oats, grass, forage, millet, sugar cane, rice, orchardgrass, and most preferably, the plant is rice to be.

The present invention also provides seed of the plant. Preferably, said seed is a seed of rice.

The invention also comprises the steps of transforming a plant cell with a recombinant vector according to the invention; And

It provides a method for producing a tolerant transgenic plant comprising the step of re-differentiating the transformed plant from the transformed plant cells.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74), electroporation of protoplasts (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102 ), Microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), particle bombardment methods (DNA or RNA-coated) of various plant elements (Klein TM et al., 1987, Nature 327, 70), infection with (incomplete) virus in agrobacterium tumerfaciens mediated gene transfer by invasion of plants or transformation of mature pollen or vesicles, and the like. have. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell. Preferably, the plant is rice.

"Plant tissue" refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. Plant tissue may be in planta or in organ culture, tissue culture or cell culture.

The method of the present invention comprises the step of transforming plant cells with the recombinant vector according to the present invention, wherein the transformation can be mediated by Agrobacterium tumefaciens . The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

In the present invention, a gene recombinant (pMG-HvNHX1) prepared by recombining barley-derived HvNHX1 cDNA into pGA1611, a carrier for plant gene expression, is introduced into Agrobacterium tumefaciens and Agrobacterium into which the gene recombinant is introduced. Tumefaciens was co-cultured with a 1-2 mm diameter callus of rice to develop a transformed rice strain that increased sodium ion homeostasis from high concentrations of sodium ion toxicity entering the cytoplasm in the plant. Since the transformed rice is a high-efficiency environmentally superior rice plant that can be used in high salt concentration and reclaimed land and production conditions, there is an excellent practical effect of maximizing the utilization efficiency of reclaimed land and production conditions. Plants other than rice may also be useful for the development of the plant seed industry by providing a way to develop varieties with increased flame resistance.

The present invention also provides a method for enhancing the salt resistance of a plant, comprising the step of transforming plant cells with the recombinant vector according to the present invention, overexpressing the HvNHX1 gene. Plant transformation methods, recombinant vectors used for transformation and plants that can be used as host cells are as described above.

The present invention also provides a plant having improved flame resistance prepared by the above method. Plants having improved flame resistance may preferably be monocotyledonous plants such as rice, barley, corn, wheat, rye, oats, grass, forage, millet, sugarcane, ligras, orchardgrass, and most preferably, the plants Is rice.

The present invention also provides a method of cultivating the plant with enhanced salt resistance in reclaimed land or production conditions known as high salt. General plants can not grow normally in the high salt land reclaimed land or production site called, but since the flame resistance enhanced plant of the present invention can grow normally in the region, it is possible to maximize the utilization efficiency of the reclaimed land and production site called.

The invention also relates to barley ( Hordeum) vulgare ) NHX (Na + / H + antiporter) gene HvNHX1 It provides a composition for improving salt resistance of plants comprising a. In the composition of the present invention, the HvNHX1 The gene may be preferably composed of the nucleotide sequence represented by SEQ ID NO: 1. The composition for enhancing flame resistance of plants of the present invention is barley ( Hordeum) as an active ingredient vulgare ) NHX (Na + / H + antiporter) gene HvNHX1 Including, the gene HvNHX1 It is possible to enhance the flame resistance of the plant by transforming the plant.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> barley cDNA  Barley (through library screening) Hordeum vulgare Origin HvNHX1 cDNA  Securing sequence

Basically, cDNA library was prepared using mRNA extracted from barley (dongbo) using uni-ZAP cDNA library biosynthesis kit (Stratagene, California, USA). Barley cDNA library (4x10 ⁶ pfu ) was obtained from HvNHX1 of barley. Partial gene probe was used for searching. Lambda phages presumed to have inserted the sodium ion / hydrogen ion reverse carrier gene of barley were isolated and converted to plasmid vectors (pBluscript SK (+)) clones utilizing the inherent properties of lambda Zap II vectors. The obtained nucleotide sequence was obtained by using both T7 (5'-AATACGACTCACTATAG-3 '; SEQ ID NO: 3) and T3 (5'-AATTAACCCTCACTAAAGGG-3'; SEQ ID NO: 4) sequencing primers present in the vector. The nucleotide sequence was determined by a sequencing autonomic analyzer (model ABI 3730xl, Macrogen).

As a result of determining the sequencing, the clone identified as cDNA of the sodium ion / hydrogen ion reverse carrier gene was named HvNHX1 , which is a sodium ion / hydrogen ion reverse carrier (Na + / H + antiporter) of barley . The amino acid sequence that fits the base sequence and reading frame of the determined gene is shown in FIG. 1.

Sequence homology was performed using GenBank program BLAST [Altschul et al., Nucleic Acid Res, 25: 3389-3402 (1997)] based on DNA sequence in GenBank, and sequencing and amino acid sequence analysis were performed by DNASIS (Hitachi, Yokohama). , Japan) program. SIGFIND [Reczko et al., Version 2.10, Leture Note in Computer Science, 2452: 60-67 (2002)], SignalP 3.0 [Bendtsen et al., In order to predict hydrophobic polypeptides, post-translational mutation sites, protein secondary structure, and the like. , Proteomics, 4: 1633-1649 (2004)], Net Phos 2.0 [Chen et al., J. Mol. Biol. 40: 247-260 (2007), NetNGlyc 1.0 [Blom et al., Proteomics, 4: 1633-1649 (2004)], WoLFPSORT (http://wolfpsort.seq.cbrc.jp), PREDATOR [Frishman and Argos , Protein Engineering, 9: 133-142 (1996), and SOPMA [Geourjon and Deleage, Comput. Appl. Biosci., 11: 681-684 (1995). Homology analysis between several NHX sequences was performed using the AliBee (Brodsky et al., Biosystems, 30: 65-79 (1993)) program.

< Example  2> barley ( Hordeum vulgare Origin HvNHX1 cDNA Recombinant vector for yeast expression pMG515 Introduced yeast Y. PMG515 Of NHX Function by Production and Heterologous Expression

(Step 1) Barley ( Hordeum vulgare Origin HvNHX1 cDNA For yeast expression, including Recombinant  Filling and yeast Intracellularly  Introduction

To express HvNHX1 cDNA in yeast, an open reading frame (ORF) sequence of HvNHX1 cDNA was inserted into pRG410, a gene expression carrier for yeast (provided by Roberto Gaxiola, University of Connecticut, USA). First, the ORF region was amplified by PCR using Extaq (Takara, Shiga, Japan) using HvNHX1 as a template to set the restriction enzyme site. The sequences of the primers used to amplify the gene are 5'-TTCGGGCGGCCGCAAGGAAGAA-3 '(SEQ ID NO: 5) and 5'-CATCTTCAGTCGACACTCTGAA-3' (SEQ ID NO: 6). The amplified PCR products were cloned into pGEM T-Easy vectors (Promega, Madison, USA) and sequenced by conventional methods. The identified recombinant HvNHX1 cDNAs were inserted into the pRG410 vector as NotI-SalI restriction enzyme sites, respectively, and HvNHX1 PMG515, a recombinant carrier of genes, was generated (FIG. 2).

HvNHX1 The gene inserted pMG515 was introduced into the yeast mutant strain R100 (Δnhx1) strain lacking the vesicular sodium ion / hydrogen ion reverse carrier using electroporation to develop the Y.PMG515 yeast strain. It was analyzed whether HvNHX1 produced by Y.PMG515 yeast restores the mutant characteristics of R100 yeast, and the selection medium for yeast used at this time was SD medium (0.17% Yeast nitrogen base without amino acid and ammonium sulfate (AS), 5). Uracil in% (NH ₄ ) ₂ SO ₄ 0.01% L-leucine, 0.02% L-tryptophan, 0.02% L-histidine, 0.02% uracil, 5 ml 20x Drop-out medium mix, 2% Glucose) Was used to remove.

(Step 2) HvNHX1 cDNA Introduced yeast Y. PMG515 To produce NHX  Activity Hygromycin  Confirmation using added medium

It was confirmed whether the HvNHX1 cDNA produced by the yeast Y.PMG515 obtained by introducing pMG515 restored the mutant characteristics of the yeast.

Yeast Y.PMG515 transformed with wild type control yeast K601, mutant yeast R100, and HvNHX1 cDNA were inoculated in 5 ml YPAD medium (containing 1% Bacto yeast extract, 2% Bacto peptone, 0.0075% Adenine hemisulfate, 2% Glucose), respectively. Incubation was carried out at 250 rpm for 24 hours at 28 ° C. so that the measured value of OD ₆₀₀ was 1.0 to 1.8. Hygromycin is a toxic, high molecular weight alkaline cation that accumulates in the cytoplasm in response to an electrochemical proton gradient, and the nhx1 yeast mutant R100 is supersensitive to hygromycin (McCusker et al., (1987) Mol. Cell. Biol. 11, 4082-4088). OD ₆₀₀ in YPAD solid medium with or without 0.05 mg / ml hygromycin After inoculating 2.5 μl each of the bacteria that had a value of 0.3, the cells were cultured at 28 ° C. for 48 hours. HvNHX1 was analyzed for the growth of yeast on a selection medium containing 0.05 mg / ml hygromycin after incubation. It was confirmed whether cDNA complements the hypersensitivity of hygromycin in yeast.

The results of Example 2 were shown in Figure 3, all strains, including hygromycin supersensitive nhx1 yeast mutant R100, were grown in YPAD medium without hygromycin, but 0.05 mg / ml hygromycin was added. In the YPAD medium, only yeast Y.PMG515 transformed with wild type control yeast K601 and HvNHX1 cDNA was grown. These results indicate that the expression of HvNHX1 cDNA can inhibit the hygromycin supersensitivity of mutant yeasts with a sodium ion / hydrogen ion reverse carrier defect.

(Step 3) HvNHX1  Yeast Y with the gene introduced. PMG515 Resistance to salts was determined using a medium containing NaCl

Transformed yeast Y.PMG515, NHX mutant yeast R100, and wild type yeast K601 strains were inoculated in 5 ml YPAD medium and incubated for 24 hours at 28 ° C. so that the value of OD ₆₀₀ was 1.0 to 1.8.

Inoculate 100μl of yeast cultures with OD ₆₀₀ value 0.12 in YPAD medium containing 0, 300, 600, 1200mM NaCl, incubate for 48 hours at 28 ℃, and measure the absorbance at OD ₆₀₀ , respectively, to measure HvNHX1 cDNA. Recovery of resistance to salts of yeast Y.PMG515 by expression of was confirmed.

In order to further solidify the results, spotting bacteria having a value of OD ₆₀₀ of 0.3 in the YPAD solid medium to which 0, 300, and 600 mM NaCl was added was 0.3 μl, and incubated for 48 hours at 28 ° C. to express HvNHX1 cDNA. The recovery of resistance to the salt of yeast Y.PMG515 was confirmed.

All strains were grown in YPAD medium containing 0 and 300 mM NaCl, but only wild type yeast strain K601 strain and transformed yeast Y.PMG515 were grown in 600 and 1200 mM NaCl (FIG. 4A).

Yeast strains of all strains were grown in YPAD medium without NaCl, but salt-sensitive yeast mutant R100 was inhibited in YPAD solid medium containing 300 and 600 mM NaCl, whereas transformed yeast Y.PMG515 and wild-type yeast K601 all grown tolerant (FIG. 4B).

The result of this increased yeast Y.PMG515 increased susceptibility inhibition to hygromycin and high salt is that the HvNHX1 cDNA isolated from barley compensates for the deficiency of hygromycin in yeast mutants lacking sodium ion / hydrogen ion reverse carriers. It is evidence that it can and is a gene that confers resistance to salts.

< Example  3> barley ( Hordeum vulgare Origin HvNHX1 cDNA Rice Intracellular  Insertion and transformation

(Step 1) is a carrier for expressing plant genes pGA1611 on HvNHX1 cDNA For plant transformation of Recombinant  write

The plant gene expression carrier, pGA1611, was distributed by Professor Ahn Jin-heung from Pohang University of Science and Technology (Pohang, Korea). HvNHX1 cDNA nucleotide sequence and restriction enzyme sites were analyzed to generate fragments containing the entire reading frame so that genes were normally expressed, and recombined into pGA1611 to generate a transgenic gene recombinant (pMG-HvNHX1) (FIG. 5).

(Step 2) rice plant Intracellularly  Introduction of recombinant gene carriers and selection of cells into which the carriers have been introduced

Freezing-thawing, An G, Ebert PR, Mitra A, Ha SB, 1988, Plant Molecular Biology Manual A3: 1-19. (Gelvin., SB, Schilperoort, R., and Verma, DP, Eds .), Kluwer Academic Pub., The Netherlands) using the Agrobacterium tumefaciens pMG-HvNHX1 prepared in the first step The strain was introduced into LBA 4404 and the recombinant gene carrier was introduced in the hygromycin medium, and the selected strain was cultured to isolate pMG-HvNHX1, followed by HvNHX1 through restriction enzyme treatment. It was confirmed that the gene was introduced into the strain.

Agrobacterium tumefaciens with pMG-HvNHX1 cDNA recombinant LBA 4404 was inoculated into a YEP (1% yeast extract, 1% peptone, 0.5% NaCl) liquid medium containing 50 mg / L hygromycin, followed by incubation at 250 rpm for at least 48 hours at 28 ° C. in the dark. When the OD ₆₀₀ of the Agrobacterium culture was 0.6, the culture was diluted 10-fold in AAM liquid medium and inoculated with a callus having a diameter of 1-2 mm using the dilution for 10 minutes. Callus inoculated with Agrobacterium was selected first from N6 base medium supplemented with 2,4-D (2 mg / L), cefotaxime ( 250 mg / L), and hygromycin (40 mg / L). One callus was again added with 2,4-D (2 mg / L), cefotaxime ( 250 mg / L), hygromycin (40 mg / L), BA (0.5 mg / L) Secondary selection in N6 base medium (Fig. 6).

(Step 3) Regeneration of Rice Plants from Cells into Which Carriers Are Introduced

The seedlings induced in rooting were transplanted by transplanting the differentiated shoots selected in step 2 into the MS basal medium to which NAA (0.1 mg / L), kinetin (2.0 mg / L), and cetactac (125 mg / L) were added. After transplanting into the soil, normal growth and development were grown as flowering plants of depleted rice (Fig. 6), and seeded to secure T0 generation seeds of 121 strains. In order to select T0 generation seeds as hygromycin, germinated experiments were carried out under 50 mg / ml hygromycin conditions for non-transformed rice and transformed rice seeds, and 50 strains of hygromycin-resistant T0 generation and 9 strains of high Gromycin sensitive T0 generation was selected. The selected T0 generation seedlings were transplanted into the soil to grow normal rice plants, and then the transfer genes were confirmed by polymerase chain reaction (PCR) method and Southern blot analysis method (FIG. 7). Both the strains with confirmed insertion of the transgene and the hygromycin selection strain were seeded in the transgenic plant sequestration water test packaging to harvest T1 generation seeds of 231 strains.

(Step 4) In the salt stress condition of the transformed rice plants confirmed the insertion of the recombinant gene Seedling  Monitor growth and recovery rates to identify salt tolerance and select lineages with unique properties

To determine if salt resistance is given to replanted rice plants by the introduction of recombinant genes, the T1 generation seeds of the transformed lines and the seed of the untransformed rice are surface sterilized in 70% ethanol for 5 minutes, and then 0.1%. Seed surface disinfection for 20 minutes in% sodium hypochlorite was washed three times or more with tap water and distilled water. Nontransformed rice (Dongjin rice) and transformed strains (44-1, 44-3, 44-4, 44-6, 90-1, 90-2, 90-8, 90-10, 90-11, 96 -1, 96-2, 96-4, 100-3) rice seeds 3 repetitions of 3 grains each for 3 days in 25 ℃ dark conditions 5 days after 2 days (2 days in dark conditions and 3 days in light conditions Treatment was observed by treatment with 100 mM NaCl. After 15 minutes of salt treatment, normal plant irrigation was observed to increase the recovery rate. The recovery rate was measured in terms of percentage based on non-transformed control rice without NaCl treatment.

After 5 days of salt stress treatment, the growth of rice seedlings of the transgenic systems was better than that of non-transformed rice plants (FIG. 8). After 5 days of salt stress treatment, the growth of rice seedlings of the transgenic systems was also good after 15 days of normal irrigation, and the increase in recovery after stress was also high (FIG. 9). In particular, the ground parts of 44-1, 90-1, 90-2, 90-8, and 90-11 strains, which were stressed by salt, showed significantly faster growth than other seedlings after 15 days of normal irrigation. 47.3, 85.5, 92.4, 91.5, and 83.1% increase, respectively. The recovery rate of total transgenic rice increased by 45.8% on average compared to non-transgenic control rice without NaCl (Table 1).

Table 1.Land length and recovery rate of seedling growth after 15 days of normal irrigation after 100 mM NaCl stress treatment (5 days)

system Length of the ground part (cm) % Increase in recovery H ₂ O H ₂ O + NaCl H ₂ O H ₂ O + NaCl Nontransformer
(Dongjin Rice) 4.52 ± 0.84 3.95 ± 1.45 0.00 0.00 44-1 4.36 ± 2.48 5.82 ± 0.65 3.57 47.27 44-3 6.39 ± 0.76 4.89 ± 1.27 41.29 23.73 44-4 6.61 ± 1.83 5.37 ± 1.00 46.07 35.74 44-6 5.48 ± 0.97 4.78 ± 1.38 21.14 20.99 90-1 6.65 ± 1.57 7.33 ± 0.52 47.05 85.50 90-2 6.83 ± 1.72 7.61 ± 1.11 51.04 92.44 90-8 5.62 ± 1.24 7.57 ± 0.15 24.26 91.49 90-10 5.49 ± 0.34 5.73 ± 0.13 21.50 44.88 90-11 6.11 ± 0.25 7.24 ± 0.76 35.01 83.10 96-1 5.07 ± 0.35 4.85 ± 0.09 12.19 22.68 96-2 6.54 ± 3.52 4.40 ± 0.72 44.57 11.30 96-4 5.24 ± 0.55 4.40 ± 0.25 15.91 11.29 100-3 6.02 ± 0.20 4.92 ± 1.29 33.12 24.57 Transformation system
Average 5.88 ± 1.21 5.76 ±± 72 29.97 45.77

The results indicate that resistance to salt stress is actively achieved through the expression of HvNHX1 cDNA, and proves that salt resistance can be increased even in all plants transformed using HvNHX1 cDNA.

1 is a barley obtained by screening barley cDNA library (cDNA library) ( Hordeum vulgare ) shows the nucleotide sequence and amino acid sequence of HvNHX1 cDNA. arrow; NHX1 Protein Signal Peptide Ten Positions

2 is a block diagram of a yeast expression vector (pMG515) including the expression structure of HvNHX1 cDNA. PMA1, promoter from pRS699: HvNHX1, Hordeum vulgare Na + / H + antiporter: CYC1, terminator: URA3, yeast selection marker (uracil): Amp, ampicillin.

3 shows HvNHX1 in control strain K601 of yeast resistant to salt and yeast mutant strain R100, yeast R100 that is sensitive to salt. The yeast Y.PMG515 strain in which the carrier pMG515 in which the cDNA was recombinant was introduced was cultured for 3 days in YPAD medium with or without 50 ug / ml hygromycin.

4 shows HvNHX1 in control strain K601 of yeast resistant to salt and yeast mutant strain R100, yeast R100 that is sensitive to salt. The yeast Y.PMG515 strains in which the carrier pMG515 recombinant cDNA was introduced were measured at an absorbance of 600 nm at the immobilized NaCl concentration (A) and the strains were immobilized for 24 hours in solid YPAD medium at the immobilized NaCl concentration. It is the result of culture (B).

5 is a partial configuration diagram of the plant gene expression binary vector (pMG-HvNHX1) including the expression structure of HvNHX1 cDNA. pUbi, maize ubiquitin promoter: HvNHX1, Hordeum vulgare Na + / H + antiporter: Tnos, polyadenylation signal of the nopaline synthase gene: p35S, 35S promoter: Hyg, Hygromycin phosphotransferase: 7 ', polyadenylation signal of the gene 7 of pTiA6: RB -DNA right border: LB, T-DNA left border.

FIG. 6 shows the selection (A) of cells incorporating genes in a medium containing antibiotic hygromycin after co-culture of sterile germinated embryonic fragments with Agrobacterium containing HvNHX1 recombinant gene vector, shoots (B) from selected cells and Regeneration of roots (C), nutrient solution (D), regeneration of soil plants and greenhouses (E), and transformants of transformed T1 generation (F), and growth processes in sequestering water G).

A: Antibiotic Selection

B: regeneration

C: rooting

D: Port Purification

E: Greenhouse Purification

F, G: cultivated transgenic sequestration water

Figure 7 is non-transformed rice ( dongjin rice) and HvNHX1 HvNHX1 in DNA extracted from T1 generation rice plants of cDNA transformed lines This is the result of Southern blot analysis for the presence or absence of the transgene.

Lane 1: untransformed rice.

Lane 2: 1-1, lane 3: 11-1, lane 4: 44-1, lane 5: 46-1, lane 6: 47-1,

Lane 7: 49-1, Lane 8: 62-1, Lane 9: 64-1

Lane 10: nontransformed rice

Lane 11: 66-1, Lane 12: 70-1, Lane 13: 76-1, Lane 14: 77-1, Lane 15: 81-1, Lane 16: 82-1, Lane 17: 87-1, Lane 18: 90-1, lane 19: 96-1, lane 20: 97-1, lane 21: 99-1, lane 22: 100-1

FIG. 8 shows that after transgenic T1 generation seeds of non-transformed rice (Dongjin rice) and transformed lines (transformed lines 44-1, 90-1, 90-2, 90-8, 90-10, 90-11) The growth of plants for the salt of seedlings was observed for 5 days under 100 mM NaCl.

Figure 9 after minimizing T1 generation seeds of non-transformed rice (Dongjin rice) and transformed lines (transformed lines 44-1, 90-1, 90-2, 90-8, 90-10, 90-11) After growing for 5 days in 100mM NaCl conditions and normal irrigation for 15 days, the growth of the plants and the length of the ground of the recovered plants was measured and converted into percentage.

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Hordeum vulgare Na + / H + antiporter (HvNHX) gene, method for          improving salt tolerance in plants by expressing HvNHX gene and          transgenic plants with improved salt tolerance developed by the          application of the method <130> PN08071 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 2559 <212> DNA Hordeum vulgare <400> 1 aacggaacct tctccagata ccccgcccgc gcgaaaagaa tagaggagaa tcccgacctc 60 cccgcccgcg cggctgcgca tctgcccccc cctccttttc cctcctcgct ccccaccccg 120 ggtttcccgt gccattcttt ccctccccac cccggccccg ggcacgaagc nncggcggag 180 acggggccag gaggaggagg agctcggctg ttcttcgtct ccccgtcgat tcgtctccgg 240 attagcgccg ccggccgttc cccgagggct ccgtccgggt tgattcgatc tgattgaaaa 300 agcccgcgtc tttccccgag ggcgcgcgct cgctcgccgg agctagctgt gtctcgttcg 360 gccgggctca aggaagaaga gtaacgggcg ggatggcgtt cgaagtggtg gcggcgcagc 420 tggcgcggct gagcgacgcg ctggccacct cggaccacgc ctccgtggtc tccatcaacc 480 tcttcgtcgc gctgctctgc gcctgcatcg tcctcggcca cctcctcgag gagaaccgct 540 ggctcaacga gtccatcacc gccctcatca tcgggctgtg caccggcgtg gtgatcctga 600 tgaccaccaa ggggaagagc tcgcacgtgc tcgtcttcag cgaggacctc ttcttcatat 660 acctcctccc tcccatcatc ttcaacgccg gtttccaggt gaggaagaag cagttcttcc 720 ggaatttcat gacaatcaca tcattcggcg ctgtcgggac gatgatttca ttcttcacaa 780 tctctcttgc tgccattgcg atattcagca agatgaacat tgggacactg gatgtatcag 840 attttctcgc aattggagcc atcttttccg cgacagattc ggtctgcact ttacaggttc 900 tcaatcagga cgagacgccc tttctgtaca gtctagtttt cggggaaggt gttgtgaacg 960 atgccacatc agtcgtgctt ttcaacgcgc tccagaactt cgatcctaac caaatcgatg 1020 caatcgtcat tctgaagttc ttgggaaact tctgctactt atccgtgtca agcaccttcc 1080 ttggagtatt ttctggattg ctcagtgcat acataatcaa gaagttatac ataggaaggc 1140 attctactga ccgtgaggtt gtgcttatga tgctcatggc ctacctctca tatatgctag 1200 ctgagctgct tgatttgagt ggcatcctca ccgtgttctt ctgtggtatt gtgatgtcgc 1260 attatacttg gcataatgtg acagagagct caagagttac aacaaagcat gcttttgcaa 1320 ccttgtcttt cattgttgag acctttctct tcctttatgt tgggatggat gcactggata 1380 tcgagaagtg gaaatttgct agtgacagcc ctggcaaatc catcggaata agctcaattt 1440 tgctaggatt agttctggtt ggaagagctg cttttgtctt cccgctttca ttcttatcca 1500 acctgacaaa gaagacggag ctcgaaaaaa taagctggag gcagcaaatc gtaatatggt 1560 gggctgggct gatgagagga gctgtgtcga tcgctcttgc ttacaataag tttacaagat 1620 ctggccacac acagctacac ggcaacgcga taatgatcac cagcaccatc actgtcgttc 1680 tgtttagcac tatgctgttt ggcatattga caaagcctct gatccggttc ctgctgcccg 1740 cgtcgagcaa tggcgacccc tcggagccct cgtcaccgaa gtccctgcac tctcctctcc 1800 tcacaagcat gctaggctcg gacatggagg cgcctctccc catcgtcagg ccctccagcc 1860 tccggatgct catcaccaag ccgacccaca ccatccacta ctactggcgc aagttcgacg 1920 acgcgctgat gcgccctatg ttcggcgggc gcgggttcgt gccctactcc cctggatcac 1980 ccaccgatcc aaacgtaatc gtggcatgaa cgttgtggag agaagagaaa agccattaca 2040 tcttcaggag acactctgaa ctgttgtaac tggaagagaa ggaggtgcta cagcttcgga 2100 agaaggcaaa gtctccatta ctattatagt gtttggctga ctcggagggc cgaagaaggc 2160 gcccctctga tgatggttca gatgaacggt tggttgcggc accaacagga agatgaaccc 2220 tagtaacggt gatgcgagta ccatcgcctt atcggttacg acaagcctgt acatttttgt 2280 atgtagatta acaagccaat tgtaccctat gagatgagat ctcctctggc aggcaggcag 2340 gccatttcct tgctccttgg ctaggagtct ctggcctcct gcatatctac cagtgcttat 2400 taatctcctc ccccactttc tagtggattg gtgtaatggt gtgtacttta ccaagttgtg 2460 tgagatgagt gatgatcttg tggcctggtt gctacaaaga actcatctca aagttatcta 2520 tctattttct atattgaatt gaaaaaaaaa aaaaaaaaa 2559 <210> 2 <211> 538 <212> PRT Hordeum vulgare <400> 2 Met Ala Phe Glu Val Val Ala Ala Gln Leu Ala Arg Leu Ser Asp Ala   1 5 10 15 Leu Ala Thr Ser Asp His Ala Ser Val Val Ser Ile Asn Leu Phe Val              20 25 30 Ala Leu Leu Cys Ala Cys Ile Val Leu Gly His Leu Leu Glu Glu Asn          35 40 45 Arg Trp Leu Asn Glu Ser Ile Thr Ala Leu Ile Ile Gly Leu Cys Thr      50 55 60 Gly Val Val Ile Leu Met Thr Thr Lys Gly Lys Ser Ser His Val Leu  65 70 75 80 Val Phe Ser Glu Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile                  85 90 95 Phe Asn Ala Gly Phe Gln Val Arg Lys Lys Gln Phe Phe Arg Asn Phe             100 105 110 Met Thr Ile Thr Ser Phe Gly Ala Val Gly Thr Met Ile Ser Phe Phe         115 120 125 Thr Ile Ser Leu Ala Ala Ile Ala Ile Phe Ser Lys Met Asn Ile Gly     130 135 140 Thr Leu Asp Val Ser Asp Phe Leu Ala Ile Gly Ala Ile Phe Ser Ala 145 150 155 160 Thr Asp Ser Val Cys Thr Leu Gln Val Leu Asn Gln Asp Glu Thr Pro                 165 170 175 Phe Leu Tyr Ser Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr             180 185 190 Ser Val Val Leu Phe Asn Ala Leu Gln Asn Phe Asp Pro Asn Gln Ile         195 200 205 Asp Ala Ile Val Ile Leu Lys Phe Leu Gly Asn Phe Cys Tyr Leu Ser     210 215 220 Val Ser Ser Thr Phe Leu Gly Val Phe Ser Gly Leu Leu Ser Ala Tyr 225 230 235 240 Ile Ile Lys Lys Leu Tyr Ile Gly Arg His Ser Thr Asp Arg Glu Val                 245 250 255 Val Leu Met Met Leu Met Ala Tyr Leu Ser Tyr Met Leu Ala Glu Leu             260 265 270 Leu Asp Leu Ser Gly Ile Leu Thr Val Phe Phe Cys Gly Ile Val Met         275 280 285 Ser His Tyr Thr Trp His Asn Val Thr Glu Ser Ser Arg Val Thr Thr     290 295 300 Lys His Ala Phe Ala Thr Leu Ser Phe Ile Val Glu Thr Phe Leu Phe 305 310 315 320 Leu Tyr Val Gly Met Asp Ala Leu Asp Ile Glu Lys Trp Lys Phe Ala                 325 330 335 Ser Asp Ser Pro Gly Lys Ser Ile Gly Ile Ser Ser Ile Leu Leu Gly             340 345 350 Leu Val Leu Val Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu         355 360 365 Ser Asn Leu Thr Lys Lys Thr Glu Leu Glu Lys Ile Ser Trp Arg Gln     370 375 380 Gln Ile Val Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Ile 385 390 395 400 Ala Leu Ala Tyr Asn Lys Phe Thr Arg Ser Gly His Thr Gln Leu His                 405 410 415 Gly Asn Ala Ile Met Ile Thr Ser Thr Ile Thr Val Val Leu Phe Ser             420 425 430 Thr Met Leu Phe Gly Ile Leu Thr Lys Pro Leu Ile Arg Phe Leu Leu         435 440 445 Pro Ala Ser Ser Asn Gly Asp Pro Ser Glu Pro Ser Ser Pro Lys Ser     450 455 460 Leu His Ser Pro Leu Leu Thr Ser Met Leu Gly Ser Asp Met Glu Ala 465 470 475 480 Pro Leu Pro Ile Val Arg Pro Ser Ser Leu Arg Met Leu Ile Thr Lys                 485 490 495 Pro Thr His Thr Ile His Tyr Tyr Trp Arg Lys Phe Asp Asp Ala Leu             500 505 510 Met Arg Pro Met Phe Gly Gly Arg Gly Phe Val Pro Tyr Ser Pro Gly         515 520 525 Ser Pro Thr Asp Pro Asn Val Ile Val Ala     530 535 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> T7 primer <400> 3 aatacgactc actatag 17 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> T223 primer <400> 4 aattaaccct cactaaaggg 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 5 ttcgggcggc cgcaaggaag aa 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 6 catcttcagt cgacactctg aa 22  

Claims (19)

delete delete delete delete delete delete delete Plants with enhanced salt resistance which are transformed with a recombinant vector comprising a NHX (Na + / H + antiporter) gene HvNHX1 derived from barley ( Hordeum vulgare ) consisting of the nucleotide sequence represented by SEQ ID NO: 1. The plant of claim 8, wherein the plant is a monocotyledonous plant. 10. The plant of claim 9, wherein the monocotyledonous plant is rice. Seeds of plants according to claim 8. Transforming a plant cell with a recombinant vector comprising an NHX (Na + / H + antiporter) gene HvNHX1 derived from barley ( Hordeum vulgare ) consisting of the nucleotide sequence represented by SEQ ID NO: 1; And Regenerating a transgenic plant from the transformed plant cell comprising the method of producing a salt tolerant transgenic plant. Flame retardant of plants comprising overexpressing HvNHX1 gene by transforming plant cells with a recombinant vector comprising NHX (Na + / H + antiporter) gene HvNHX1 derived from barley ( Hordeum vulgare ) consisting of the nucleotide sequence represented by SEQ ID NO: 1 How to promote. The method of claim 13, wherein the plant is a monocotyledonous plant. The method of claim 14, wherein the monocotyledonous plant is rice. 16. Flameproof enhanced plant produced by the method of any one of claims 13-15. The method of growing a salt tolerant plant of claim 16 in a high salt reclaimed land. Composition for enhancing salt resistance of plants comprising NHX (Na + / H + antiporter) gene HvNHX1 derived from barley ( Hordeum vulgare ) consisting of the nucleotide sequence represented by SEQ ID NO: 1. delete

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