AU2009201381A1 - Hordeum vulgare NA+/H+antiporter (HVNHX1) gene, method for improving salt tolerance in plants by expressing HVNHX1 gene and transgenic plants with improved salt tolerance developed by using the method - Google Patents

Description

AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant/s: Industrial Cooperation Foundation Chonbuk National University Actual Inventor/s: Song Joong Yun and Hee-Sun Kook and Myoung-Ryoul Park Address for Service is: SHELSTON IP 60 Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: HORDEUM VULGARE NA+/H+ ANTIPORTER (HVNHX1) GENE, METHOD FOR IMPROVING SALT TOLERANCE IN PLANTS BY EXPRESSING HVNHX1 GENE AND TRANSGENIC PLANTS WITH IMPROVED SALT TOLERANCE DEVELOPED BY USING THE METHOD The following statement is a full description of this invention, including the best method of performing it known to me/us: File: 62055AUP00 - Ia HORDEUM VULGARE NA+/H+ ANTIPORTER (HVNHX 1) GENE, METHOD FOR IMPROVING SALT TOLERANCE IN PLANTS BY EXPRESSING HVNHXI GENE AND TRANSGENIC PLANTS WITH IMPROVED SALT TOLERANCE DEVELOPED BY USING THE METHOD 5 TITLE OF THE INVENTION Hordeum vulgare Na+/H+ antiporter (HvNHXJ) gene, method for improving salt tolerance in plants by expressing HvNHXJ gene and transgenic plants with improved salt tolerance developed by using the method 10 FIELD OF THE INVENTION The present invention relates to a method for increasing salt tolerance of a plant. More specifically, the present invention relates to cDNA of Hordeum vulgare Na+/H+ antiporter gene (HvNHXI), which contributes to the improvement of salt 15 tolerance of a plant cell by compartmentalizing sodium ions of the plant cell cytosol into a vacuole; a recombinant vector comprising the same; a yeast and a plant transformed with the said recombinant vector; a method for increasing salt tolerance of a plant by using the said gene; a plant having high salt tolerance prepared based on the said method; seeds of the plant; and a composition for increasing salt tolerance of a plant 20 comprising the said gene. BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common 25 general knowledge in the field. Na+/H+ antiporter is responsible for the control of pH and the level of sodium ions in the cytosol of a cell and for cell size by promoting transport of sodium ions from the cytosol to a vacuole through the membrane of a vacuole and exchange of hydrogen ions in a direction opposite to sodium ions (Aronson, P. S., Annu. Rev. Physiol. 1985. 30 47: 545-560; Orlowski, J., Grinstein, S., J. Biol. Chem. 1997. 272: 22373-22376). In particular, it is known that a vacuolar Na+/H+ antiporter present in the membrane of a plant cell vacuole can compartmentalize sodium ions, that are present at high level in a cytosol of a plant cell under high salt concentration, into a vacuole, thus reducing the -2 concentration of sodium ions in the cytosol and increasing the salt tolerance of the plant cell (Zhang, H-X and E Blumwald. 2001. Nat. Biotechnol. 19: 765-768). Further, a yeast complementation study regarding the ability of complementing Na+/H+ antiporter to inhibit sensitivity to sodium ions and hygromycin of a 5 Saccharomyces cerevisiae mutant, lacking Na+/H+ antiporter, has been carried out for AINHXJ (Gaxiola et al. 1999. Proc. NatI. Acad. Sci. USA. 96: 1480-1485), OsNHXI (Fukuda et al. 2004. Journal of Experimental Botany, 55:585-594) and TaNHXJ (Brini et al. 2004. Plant Physiology and Biochemistry 43: 347-354), etc. Further, more evidence has been found that, with compartmentalization of sodium ions that are 10 transported into a yeast vacuole within a cytosol by HvNHXJ, salt sensitivity is reduced and that, with regulation of toxic cations by HvNHXJ in a yeast vacuole, hygromycin sensitivity is also reduced. Recently, by showing increased salt tolerance in a rice plant transformed with Na+/H+ antiporter gene of Atriplex gmelini (AgNHX1) (Ohta M. el al. 2002. FEBS 15 Letters 26785: 1-4), the usefulness of Na+/H+ antiporter gene for improving salt tolerance that had been described for a dicot plant in previous studies, has also been verified for a monocot plant. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. 20 SUMMARY OF THE INVENTION According to a first aspect, the present invention provides a recombinant vector comprising a HvNHXJ gene of Hordeum vulgare. According to a second aspect, the present invention provides a yeast cell 25 transformed with the recombinant vector of the present invention. According to a third aspect, the present invention provides a plant having improved salt tolerance, wherein said plant is transformed with the recombinant vector of the present invention. According to a fourth aspect, the present invention provides a seed wherein the 30 seed is a seed of the plant according to the third aspect of the present invention. According to a fifth aspect, the present invention provides a method for producing a transgenic plant having improved salt tolerance comprising the steps of: transforming a plant cell with the recombinant vector according to the invention; and -3 regenerating the transformed plant cell into a transgenic plant. According to a sixth aspect, the present invention provides a method for improving salt tolerance of a plant comprising a step of transforming a plant cell with the recombinant vector according to the invention to overexpress said HvNHXJ gene. 5 According to a seventh aspect, the present invention provides a plant having improved salt tolerance when produced by any one of the methods according to the sixth aspect of the present invention. According to an eighth aspect, the present invention provides a method of cultivating a plant according to the seventh aspect, wherein said method allows 10 cultivation of said plant in a salt rich reclaimed area or in a region unfavorable for achieving a high harvest yield for said plant. According to a ninth aspect, the present invention provides a composition for increasing salt tolerance of a plant comprising the Hordeum vulgare HvNHXI gene for expression in said plant. 15 Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". 20 BRIEF DESCRIPTION OF THE DRAWINGS Fig. I shows the nucleotide sequence of HvNHXJ cDNA from Hordeum vulgare that is obtained by cDNA library screening, and the amino acid sequence thereof. The arrow indicates the site for the signal peptide of NHX I protein. Fig. 2 shows a constructional diagram of pMG515 vector fbr yeast expression, 25 which comprises a structural element for expressing HvNHXJ cDNA: PMAI, promoter from pRS699; HvNHXI, Hordeum vulgare Na+/H+ antiporter; CYCI, terminator; URA3, yeast selection marker (uracil); and Amp, ampicillin selection marker. Fig. 3 shows the result of yeast culture for three days on YAPD medium with or without hygromycin (50 ug/ml), including: wild type Saccharomyces cerevisiae line 30 K601 having salt tolerance; mutant Saccharomyces cerevisiae line RI00, which is sensitive to salt; and Saccharomyces cerevisiae line Y.PMG515, wherein the vehicle pMG515 comprising recombinant HvNHX] cDNA gene is introduced to Saccharonyces cerevisiae line R100.

-4 Fig. 4(A) shows the growth amount of each Saccharomyces cerevisiae strain cultured in various NaCl concentrations, determined by measuring absorbance at 600 nm for the wild type Saccharomyces cerevisiae line K601 having salt tolerance, for mutant Saccharomyces cerevisiae line RI 00, which is sensitive to salt and for Saccharomyces 5 cerevisiae line Y.PMG515 wherein pMG515 vector comprising recombinant HvNHXI cDNA gene is introduced to Saccharomyces cerevisiae line RI 00. Fig. 4(B) indicates the result of culturing each of the said yeast strains for twenty-four hours on YAPD medium comprising various NaCl concentrations. Fig. 5 is a diagram representing a partial constitution of a binary vector for 10 plant gene expression (pMG-HvNHXI), which comprises a structural element for expression of HvNHX] cDNA: pUbi, maize ubiquitin promoter; HvNHXI, 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, T-DNA right border; and LB, T-DNA left border. 15 Fig. 6 shows (A) selection of a plant cell comprising HvNHXI gene on a medium which contains hygromycin as antibiotics, following co-culture of an endosperm fragment obtained by asceptic germination with Agrobacterium comprising a vector with HvNHXI recombinant gene, (B) new shoots grown from the selected plant cells, (C) a plant regenerated from the root, (D) a nutrient solution for the regenerated 20 plant, (E) adaptation process in a soil pot and in a greenhouse, (F) a transgenic plants at transformed TI generation, and (G) growth of transgenic paddy rice kept under isolated test culture condition. A: Antibiotics selection B: Regeneration 25 C: Rooting D: Pot adaptation E: Greenhouse adaptation F, G: Isolated test culture of transgenic paddy rice. Fig. 7 shows the results of Southern blot analysis for determining the presence 30 or the absence of HvNHXI gene in DNA extracted from a non-transgenic plant (Dongjin variety rice) or from a TI generation rice plant that has been transformed with HvNHXI cDNA. lane 1: Non-transgenic rice -5 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: Non-transgenic rice lane 11: 66-1, lane 12: 70-1, lane 13: 76-1, lane 14: 77-1, lane 15: 81-1, lane 16: 5 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 growth of the plant under salt treatment, wherein seeds of TI generation of a non-transgenic rice (Dongjin variety) and transgenic rice plants (transgenic lines 44-1, 90-1, 90-2, 90-8, 90-10, and 90-11 ) were allowed to germinate 10 and growth of the seedlings was observed for five days under 100mM NaCl condition. Fig. 9 shows shoot length of the plants that are grown under salt treatment, wherein seeds of TI generation of a non-transgenic rice (Dongjin variety) and transgenic rice plants (transgenic lines 44-1, 90-1, 90-2, 90-8, 90-10, and 90-11) were allowed to germinate and then to grow for five days under 100 mM NaCl condition, followed by 15 normal irrigation for fifteen days to observe the growth of the plants. Specifically, the shoot length (i.e., stem length grown up above the ground) of the recovered plants was measured and expressed as percentage ratio. DETAILED DESCRIPTION OF THE INVENTION 20 According to the present invention, which is devised in view of the above problems, a Na+/H+ antiporter gene (HvNHXI) is isolated from Hordeum vulgare (a species of barley) and characterized. In addition, by introducing this gene to a Sacchromyces cerevisiae mutant R100 (Anhxl) lacking the vacuolar Na+/H+ antiporter gene, the salt-related function of the HvNHXI gene was determined by the heterologous 25 expression of the gene. Further, by recombination of the gene in a plant gene expression vehicle, rice plant cells were transformed with the gene using Agrobacterium tumefaciens. Further, with differentiation of the gene-transferred plant cells into a grown rice plant and its progress into next generation, a TI generation rice line, having improved salt tolerance, was obtained. The method described above and the rice line 30 developed by using such method are different from, respectively, a conventional method based on crossing and selecting plants and from a rice variety having a combination of multiple genes obtained by such a conventional method. Thus, the rice variety obtained according to the present invention has increased salt tolerance while favorable properties -6 of the original variety chosen in the invention are maintained. The present invention is advantageous in that, by isolating NHXJ gene from Hordeum vulgare, which has been found to be most effective among all known genes for increasing salt tolerance in a plant, and using it as a gene useful for improving 5 adaptability of a plant to an area that is not conducive to achieving a high harvest yield for a non-transgenic plant of the same species and/or for producing and developing a plant better adapted to such environments, improving land use efficiency of an area that is not conducive to achieving a high harvest yield for a non-transgenic plant of the same species (such as a salt rich reclaimed area, etc). In addition, the invention can be 10 advantageously used for an industrial field such as developing seeds of a new variety of a plant, etc. In one or more preferred embodiments, in order to solve the problems described above, the present invention, provides a recombinant vector comprising Hordeum vulgare Na+/H+ antiporter gene (HvNHXI), which contributes to improvement of salt is tolerance of a plant cell by compartmentalizing sodium ions of the plant cell cytosol into a vacuole. Further, in some embodiments the present invention provides yeast and a plant transformed with the said recombinant vector. Further, in other embodiments the present invention provides a method for 20 increasing salt tolerance of a plant by using the said gene. In further embodiments, the present invention provides a plant having high salt tolerance developed based on the said method and seeds of the plant. In one or more preferred embodiments, the present invention further provides a method of culturing the said plant having improved salt tolerance in a highly saline 25 reclaimed area or in a region which is unfavorable in terms of productivity. Still further, the present invention provides a composition for increasing salt tolerance of a plant comprising the said gene. According to the present invention, it was confirmed that the cDNA nucleotide sequence isolated in the present invention corresponds to cDNA of Hordeun vulgare 30 Na+/H+ antiporter gene (HvNHXI), and this HvNHXI cDNA can restore salt tolerance of a mutant Saccharomyces cerevisiae which lacks the vacuolar Na+/H+ antiporter gene. Under salt stress this cDNA can improve salt tolerance of a plant in a seedling stage. Thus, as a gene highly useful for increasing salt tolerance of a plant, cDNA of Hordeum -7 vulgare Na+/H+ antiporter gene (HvNHXI) can be used for improving adaptability of a plant to an area that is not conducive to achieving a high harvest yield a non-transgenic plant of the same species and for producing and developing a plant better adapted to such environments. In addition, the gene can be advantageously used in an industrial 5 field such as developing new varieties of plant seeds, etc. In order to achieve the above described purpose of the invention, the present invention provides Hordeum vulgare Na+/H+ antiporter gene (HvNHXI) and a vector comprising the same. The present invention relates to a cDNA encoding Hordeum vulgare Na+/H+ 10 antiporter which can compartmentalize sodium ions that are present in high concentration in the cytosol of a plant cell under high salt concentration and can reduce their concentration in the cytosol. Specifically, the ability of HvNHXJ cDNA for restoring NHX function of Saccharomyces cerevisiae has been confirmed by using a yeast mutant Saccharomyces cerevisiae R100 (Anhx I), which lacks vacuolar Na+/H+ 15 antiporter, and the control line K601 (Dr. R. Rao, Johns Hopkins University, USA) so that a function ofNHX is determined based on heterologous expression. Preferably, the above described HvNHXI gene may consist of a nucleotide sequence that is represented by SEQ ID NO: 1. Sequence of the protein that is deduced from the said HvNHXI gene corresponds to an amino acid sequence that is represented 20 by SEQ ID NO: 2. In addition, a mutant of the said sequence is also within the scope of the present invention. The term "mutant" indicates a nucleotide sequence which has a different nucleotide sequence but similar functional characteristics to that of SEQ ID NO: 1. Specifically, HvNHXJ gene may comprises a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, still more preferably at least 25 95% sequence homology with the nucleotide sequence of SEQ ID NO: I. The said "sequence homology %" for a certain polynucleotide is identified by comparing a comparative region with two sequences that are optimally aligned. In this regard, a part of the polynucleotide in the comparative region may comprise an addition or a deletion (i.e., a gap) compared to a reference sequence (without any addition or 30 deletion) relative to the optimized alignment of the two sequences. A recombinant vector according to one embodiment of the present invention can be a yeast expression vector. More preferably, it can be pMG51 5 vector illustrated in Fig. 2. pMG515 vector for expression in yeast is constructed by recombination of cDNA -8 of Hordeum vulgare Na+/H+ antiporter gene (HvNHXI), that is isolated and characterized in the present invention, in pRG410 wherein uracil is used as a selection marker. However, the recombinant vector of the present invention is not limited to the vector of Fig. 2. Any vector which is useful for yeast transformation can be also used. 5 The term "recombinant" indicates a cell which replicates a heterogeneous nucleotide or expresses the nucleotide, a peptide, a heterogeneous peptide, or a protein encoded by a heterogeneous nucleotide. A recombinant cell can express a gene or a gene fragment that are not found in the natural state of the cell, both in a sense or antisense direction. In addition, a recombinant cell can express a gene that is found in its natural 10 state, provided that the said gene is modified and re-introduced into the cell by an artificial means. The term "vector" is used herein to refer DNA fragment(s) and nucleotide molecules that are delivered to a cell. A vector can be used for the replication of DNA and be independently reproduced in a host cell. The terms "delivery system" and 15 "vector" are often interchangeably used. The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and other appropriate nucleotide sequences that are essential for the expression of the operatively-linked coding sequence in a specific host organism. Further, the present invention provides a yeast that is transformed with the said 20 recombinant vector. More specifically, pMG515 vector for yeast expression containing the HvNHXI cDNA is introduced to Saccharomyces cerevisiae R100 lacking Na+/H+ antiporter gene by recombination to generate the Saccharomyces cerevisiae Y.PMG515 strain with restored tolerance to salt. Although Saccharomyces cerevisiae is preferred as a host cell to be used for the 25 present invention, it is not specifically limited thereto. Instead, other host cells such as Pichia pastoris; Hansenula polymorpha;Yarrowia spec.; Schizosaccharomyces pombe; Kluyveromyces spec. including K. lactis, K. fragilis, K. bulgaricus, K. wickeramii, K. waltii, K. drosophilarum, K. thermotolerans, K. marxianus can be also used. With respect to a method for transforming a host cell with the recombinant 30 vector of the present invention, a method that is generally used in the pertinent art, such as an electroporation method, etc., can be used. The recombinant vector of the present invention can be a plant expression vector.

-9 A preferred example of a plant expression vector is a Ti-plasmid vector which can transfer a part of itself, i.e., so-called T-region, to a plant cell when the vector is present in an appropriate host such as Agrobacterium tumefaciens. Other types of Ti plasmid vector (see, EP 0 116 718 B1) are currently used for transferring a hybrid gene 5 to protoplasts that can produce a new plant by appropriately inserting a plant cell or hybrid DNA to a plant genome. Especially preferred form of Ti-plasmid vector is a so called binary vector which has been disclosed in EP 0 120 516 BI and USP No. 4,940,838. Other appropriate vectors that can be used for introducing the HvNHXJ gene of the present invention to a host plant can be derived from a double-stranded plant virus 10 (e.g., CaMV), a single-stranded plant virus, and a viral vector which can be originated from Gemini virus, etc., for example a non-complete plant viral vector. Use of the said vector can be especially advantageous when a plant host cannot be appropriately transformed. An expression vector preferably comprises at least one selection marker. The 15 said selection marker is a nucleotide sequence having a property which allows selection based on a common chemical method. Any kind of gene that can be used for the differentiation of transformed cells from non-transformed cells can be a selection marker. Examples include, a gene resistant to herbicide such as glyphosate and phosphinotricin, and a gene resistant to antibiotics such as kanamycin, G418, bleomycin, 20 hygromycin, and chloramphenicol, but are not limited thereto. For the plant expression vector according to one example of the present invention, hygromycin resistant gene is used as a selection marker. For the plant expression vector according to one embodiment of the present invention, a promoter can be any one of the CaMV 35S, actin, ubiquitin, pEMU, MAS 25 or histone promoters, but is not limited thereto. The term "promoter" means a DNA molecule to which RNA polymerase binds in order to initiate transcription and corresponds to a DNA region upstream of a structural gene. The term "plant promoter" indicates a promoter which can initiate transcription in a plant cell. The term "constitutive promoter" indicates a promoter which is active in most of environmental 30 conditions and developmental states or cell differentiation states. Since a transformant can be selected with various mechanisms at various stages, a constitutive promoter can be preferable for the present invention. However, the choice of a constitutive promoter is not limited in the present invention.

- 10 Any conventional terminator can be used for the present invention. Examples include, the nopaline synthase (NOS), rice a-amylase RAmyl A terminator, phaseoline terminator, and a terminator for an optopine gene of Agrobacterium tumefaciens, etc., but are not limited thereto. Regarding the necessity of a terminator, it is generally known 5 that such a region can increase reliability and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly preferable in view of the contexts of the present invention. Preferably, a recombinant plant expression vector according to one embodiment of the present invention can be pMG-HvNHXI vector as illustrated in Fig. 5. This pMG 10 HvNHXI vector is a binary vector for plant gene expression, which is constructed by recombination of HvNHXI cDNA from Hordeun vulgare in pGA1611. However, the recombinant vector of the present invention is not limited to the vector of Fig. 5. Any vector which is useful for plant transformation can be also used. Further, the present invention provides a plant having high salt tolerance that is 15 transformed with the recombinant vector of the present invention. More specifically, by using the plant transformation vector pMG-HvNHXI to express HvNHXI in the plant thereby providing a transgenic plant having improved salt tolerance with homeostasis to sodium ions. The plant according to the present invention can be food crops including rice, 20 wheat, barley, corn, soy bean, potato, red bean, oat and millet; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, hot pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, zucchini, scallion, onion and carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, wild sesame, peanut and rapeseed; fruits including apple, pear, date, peach, kiwi, grape, tangerine, 25 orange, persimmon, plum, apricot and banana; flowers including rose, gladiolus, gerbera, carnation, chrysanthemum, lily, and tulip; and feed crops including rye grass, red clover, orchard grass, alfalfa, tall fescue, and perennial rye grass. Preferably, the plant can be a monocot plant such as rice, barley, corn, wheat, rye, oats, grass, hay, millet, sugar cane, rye grass, orchard grass and the like. More preferably, the plant is 30 rice. Further, the present invention provides seeds of the above described plant. Preferably, the said seeds are rice seeds. Further, the present invention provides a method for producing a plant with salt - 11 tolerance comprising the steps of: - transforming a plant cell with the recombinant vector of the present invention, and - regenerating the above described transformed plant cell into a transgenic plant. "Plant transformation" means any method by which DNA is delivered to a plant. 5 Such transformation method does not necessarily have a period for regeneration and/or tissue culture. Transformation of plant species is well known in the art for both dicot and monocot plants. In principle, any transformation method can be used for introducing a hybrid DNA of the present invention to an appropriate progenitor cell. It can be appropriately selected from a calcium/polyethylene glycol method for protoplasts 10 (Krens, F.A. el al., 1982, Nature 296, 72-74; Negrutiu I. el al., June 1987, Plant Mol. Biol. 8, 363-373), an electroporation method for protoplasts (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), a microscopic injection method for plant components (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), a particle bombardment method for various plant components (DNA or RNA-coated) (Klein T.M. et al., 1987, 15 Nature 327, 70), or a (non-complete) viral infection method in Agrobacterium tumefaciens mediated gene transfer by plant invasion or transformation of fully ripened pollen or microspore, etc. A method preferred in the present invention includes Agrobacterium mediated DNA transfer. In particular, so-called binary vector technique as disclosed in EP A 120 516 and USP No. 4,940,838 can be preferably adopted for the 20 present invention. The "plant cell" that is used for the plant transformation according to the present invention can be any plant cell. The plant cell can be a cultured cell, a cultured tissue, a cultured organ, or a whole plant, preferably a cultured cell, a cultured tissue or a cultured organ, and more preferably any form of a cultured cell. Preferably, the plant is rice. 25 The "plant tissue" includes either differentiated or undifferentiated plant tissue, including root, stem, leaf, pollen, seed, cancerous tissue and cells having various shape that are used for culture, i.e., single cell, protoplast, bud and callus tissue, but is not limited thereto. Plant tissue can be in planta or in a state of organ culture, tissue culture or cell culture. 30 The method of the present invention comprises a step of transforming a plant cell with the recombinant vector of the present invention, and such transformation may be mediated by Agrobacterium tumefaciens. In addition, the method of the present invention comprises a step of regenerating a transformed plant cell to a transgenic plant.

- 12 A method of regenerating a transformed plant cell to a transgenic plant can be any method that is well known in the pertinent art. According to the present invention, the recombinant gene pMG-HvNHXI, that is constructed by recombination of cDNA of.Hordeum vulgare HvNHXI in pGAl611 as 5 a vehicle for plant gene expression, is introduced to Agrobacterium lumefaciens, and the resulting Agrobacterium lumefaciens comprising the recombinant gene is co-cultured with rice callus having a diameter of 1-2 mm. As a result, a transgenic rice line having improved sodium ion homeostasis against toxicity of high-level sodium ions that are transported into the cytosol of a plant, is developed. Since such a transgenic rice plant is 10 a crop better adapted to certain environments, which can be used in highly saline reclaimed areas and in an area that is not conducive to achieving a high harvest yield for a non-transgenic plant of the same species, it also has practical value in terms of maximizing land use efficiency of highly saline reclaimed area and in an area that is not conducive to achieving a high harvest yield for non-transgenic plant of the same species. 15 Further, based on teachings of the present invention, a method for the development of a plant variety having improved salt tolerance can be also provided for a plant other than rice. Thus, the present invention can be also useful for an industrial field relating to development of seeds of a new variety of a plant. Further, the present invention provides a method for improving salt tolerance of 20 a plant comprising a step of transforming a plant cell with the recombinant vector of the present invention to overexpress or regualate the expression of HvNHXI gene. A method of transforming a plant, a recombinant vector used for transformation and plants that can be used as a host cell are the same as those described in the above. Further, the present invention provides a plant having improved salt tolerance 25 that is produced with the method described in the above. The plant having improved salt tolerance can be a monocot plant such as rice, barley, corn, wheat, rye, oats, grass, hay, millet, sugar cane, rye grass, orchard grass and the like. More preferably, the plant is rice. Further, the present invention provides a method of cultivating the said plant 30 having improved salt tolerance in a salt rich reclaimed area or in a region which is not conductive to achieving a high harvest yield for the plant. A typical non-transgenic plant of the same species cannot grow normal in a salt rich reclaimed area or in a region which is not conducive to achieving a high harvest yield. However, since the plant having - 13 improved salt tolerance provided by the present invention can grow normally even in such areas, efficient use of a salt rich reclaimed area or a region which is not conducive to achieving a high harvest yield for a non-transgenic plant of the same species can be maximized. 5 Still further, the present invention provides a composition for increasing salt tolerance of a plant comprising HvNHX] gene of Hordeum vulgare NHX (Na+/H+ antiporter). Regarding the composition of the present invention, the said HvNHXI gene may preferably consist of a nucleotide sequence that is represented by SEQ ID NO: 1. The composition for increasing salt tolerance of a plant comprises HvNHXJ gene of 10 Hordeum vulgare NHX (Na+/H+ antiporter) as an effective component. With transformation of a plant with the said HvNHXI gene, salt tolerance of a plant can be improved. The present invention will now be described in greater detail with reference to the following examples. However, it is only to specifically exemplify the present 15 invention and in no case the scope of the present invention is limited by these examples. Examples <Example 1> Obtainment of the nucleotide sequence of HvNHX1 cDNA from Hordeum vulgare based on cDNA library screening 20 By using mRNA that had been extracted from Hordeum vulgare (Dongbori 1) with uni-ZAP cDNA library biosynthesis kit (Stratagene, California, USA), cDNA library for Hordeum vulgare was constructed. Then, using a probe for partial nucleotide sequence of HvNHXJ gene that has been already established, cDNA library of Hordeum vulgare (4x1 06 pfu) was searched. Lambda phages that are believed to comprise a 25 nucleotide sequence of Hordeum vulgare Na+/H+ antiporter gene were isolated and converted into a plasmid vector (pBluscript SK(+)) clone by utilizing specific characteristics of lambda Zap II vector. Thus obtained clones were sequenced using an automated sequencer (Model No. ABI 3730xl, manufactured by Macrogen) with primers as follows; primer T7 (5'-AATACGACTCACTATAG-3'; SEQ ID NO: 3) that is present 30 within the vector and primer T3 (5'-AATTAACCCTCACTAAAGGG-3'; SEQ ID NO: 4) that is used for nucleotide sequencing. As a result of nucleotide sequencing, clones that are identified as containing cDNA of Na+/H+ antiporter gene were named HvNHXI of Hordeum vulgare Na+/H+ - 14 antiporter gene. Thus identified nucleotide sequence of the gene and the amino acid sequence corresponding to the open reading frame are indicated in Table 1. Homology analysis between nucleotide sequences was carried out by using the DNA sequence obtained from GenBank based on BLAST program [Altschul el al., 5 Nucleic Acid Res, 25:3389-3402 (1997)]. Analysis for the nucleotide sequence and the amino acid sequence was carried out based on DNASIS program (Hitachi, Yokohama, Japan). In order to predict a hydrophobic polypeptide, mutation site after translation and a secondary structure of the protein, etc., programs as follows were used; SIGFIND [Reczko et al., Version 2.10, Lecture Note in Computer Science, 2452:60-67 (2002)], 10 SignalP 3.0 [Bendtsen el al., Proteomics, 4:1633-1649 (2004)], NetPhos 2.0 [Chen et al., J. Mol. Biol. 40:247-260 (2007)], NetNGlyc 1.0 [Blom el al., Proteomics, 4:1633-1649 (2004)], WoLFPSORT (http://wolipsort.seq.cbrc.jp), PREDATOR [Frishman and Argos, Protein Engineering, 9:133-142 (1996)], SOPMA [Geourjon and Deleage, Comput. Apple. Biosci., 11:681-684 (1995)] and the like. Homology analysis among 15 various NHX sequences were carried out based on AliBee program [Brodsky et al., Biosystems, 30:65-79 (1993)]. <Example 2> Production of Saccharomyces cerevisiae Y.PMG515 comprising recombinant vector pMG515 for yeast expression comprising HvNHXJ 20 cDNA from Hordeum vulgare and determination of NHX function based on heterologous expression (Step 1) Construction of a recombinant for Saccharomyces cerevisiae expression comprising HvNHXJ cDNA from Hordeum vulgare and introduction of the recombinant to Saccharomyces cerevisiae 25 In order to express HvNHXI cDNA in Saccharomyces cerevisiae, the open reading frame (ORF) sequence of HvNHXI cDNA was inserted to pRG410 [generously provided by Mr. Roberto Gaxiola at University of Connecticut, USA], which is a vehicle for gene expression in Saccharomyces cerevisiae. First, in order to align restriction enzyme sites, the ORF region was amplified by PCR with Extaq (Takara, Shiga, Japan) 30 using HvNHXI as a template. The nucleotide sequence of the primers that were used for the amplification was as follows: 5'-TTCGGGCGGCCGCAAGGAAGAA-3' (SEQ ID NO: 5) and 5'-CATCTTCAGTCGACACTCTGAA-3' (SEQ ID NO: 6). Thus amplified PCR product was subjected to sequencing according to a general method, following the - 15 cloning into pGEM T-Easy vector (Promega, Madison, USA). HvNHXJ cDNA for recombination as identified in the above was then inserted to pRG410 vector at the restriction enzyme sites of Not I-Sal 1. As a result, the recombinant vehicle pMG515 for HvNHXJ gene was constructed (Fig. 2). 5 By using an electroporation method, pMG515 comprising the HvNHXI gene was introduced to mutant Saccharomyces cerevisiae line RI 00(Anhx I), which lacks vacuolar Na+/H+ antiporter gene, to give Saccharomyces cerevisiae line Y.PMG515. It was then determined whether or not HvNHX 1 produced by Saccharomyces cerevisiae line Y.PMG515 can restore the mutated property of Saccharomyces cerevisiae line 10 R100. In this case, as selection culture medium for Saccharomyces cerevisiae, SD medium (comprising 0.17 % Yeast nitrogen base without amino acid and ammonium sulfate (AS), 5% (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, and 2% Glucose) from which uracil was removed was used. 15 (Step 2) Determination of the activity of NHX that is produced by Saccharomyces cerevisiae Y.PMG515 comprising the HvNHX1 cDNA by using culture medium to which hygromycin is added Restoration of the mutated property of Saccharomyces cerevisiae by HvNHXI 20 cDNA that is produced by Saccharomyces cerevisiae Y.PMG515 comprising pMG515 was determined. Specifically, wild type Saccharomyces cerevisiae K601 as a control, the mutant Saccharomyces cerevisiae RI 00, and Saccharomyces cerevisiae Y.PMG515 transformed with HvNHX/ cDNA were inoculated to 5 ml YPAD medium (comprising I% Bacto 25 yeast extract, 2% Bacto peptone, 0.0075% Adenine hemisulfate, 2% Glucose, respectively). The cells were cultured for 24 hours at 28'C and 250 rpm until measured

OD

6 00 value of about 1.0-1.8 was obtained. Hygromycin is a high molecular weight alkaline cation having toxicity, which can accumulate in cell cytosol in response to electrochemical proton gradient. Saccharomyces cerevisiae R100 having nhxI mutation 30 has supersensitivity to hygromycin (McCusker et al., (1987) Mol. Cell. Biol. 11, 4082 4088). YPAD solid medium with or without hygromycin (0.05 mg/ml) was inoculated with yeast cells that had grown to OD 600 value of 0.3 (2.5 t) followed by culturing at 28'C for 48 hours in the dark. After that, a growth property of the yeast cells on the - 16 selection medium comprising 0.05 mg/ml hygromycin was determined to see whether or not HvNHXI cDNA can complement the supersensitivity of yeast cells to hygromycin. As shown in Fig. 3, the results of Example 2 indicate that, all yeast cells including the mutant Saccharomyces cerevisiae RI 00, having nhxl mutation and being 5 supersensitive to hygromycin, were able to grow on YAPD medium not comprising any hygromycin. However, on YAPD medium comprising 0.05mg/ml hygromycin, only the wild type Saccharomyces cerevisiae K601 and Saccharomyces cerevisiae Y.PMG515 transformed with HvNHX] cDNA were able to grow. These results suggest that the expression of HvNHXJ cDNA may inhibit the supersensitivity of mutant Saccharomyces 10 cerevisiae having defective Na+/H+ antiporter gene to hygromycin. (Step 3) Determination of salt tolerance of Saccharomyces cerevisiae Y.PMG515 having HvNHX1 gene based on growth on medium comprising NaCl Transformed Saccharomyces cerevisiae Y.PMG515, NHX mutant 15 Saccharomyces cerevisiae RI 00, and the wild type Saccharomyces cerevisiae K601 were cultured for 24 hours at 28"C until the measured OD 6 00 values reached about 1.0-1.8 and were subsequently used to inoculate 5ml YPAD medium, respectively. Then, YPAD medium comprising various concentrations of sodium chloride (i.e., 0, 300, 600, 1200 mM NaCl), was inoculated with each type of the above yeast 20 cells that had grown to OD 6 00 value of 0.12 (100 t.) followed by culturing at 28'C for 48 hours. After that, absorbance of OD 60 0 for each type of the cells was measured to see the recovery of salt tolerance of Saccharomyces cerevisiae Y.PMG515 by the expression of HvNHX cDNA. To further confirm the results described above, each type of the above yeast 25 cells that had grown to OD 6 00 value of 0.3 were spotted (2.5 0i) on YPAD solid medium comprising various concentrations of sodium chloride (i.e., 0, 300, 600 mM NaCl), followed by culturing at 28 'C for 48 hours to see the recovery of salt tolerance of Saccharomyces cerevisiae Y.PMG515 by the expression of HvNHX] cDNA. As a result, all yeast cells were able to grow on YAPD medium comprising 0 or 30 300 mM NaCI. However, on YAPD medium comprising 600 or 1200 mM NaCl only the wild type Saccharomyces cerevisiae K60 1 and the Saccharomyces cerevisiae transformed Y.PMG515 were able to grow (see Fig. 4A). Specifically, while all yeast cells were able to grow on YAPD medium - 17 comprising no NaCl, on YAPD solid medium comprising 300 or 600 mM NaCl, growth of the salt-sensitive mutant Saccharomyces cerevisiae R100 was inhibited while both the wild type Saccharomyces cerevisiae K601 and the transformed Saccharomyces cerevisiae Y.PMG515 were able to grow normally, showing substantial tolerance (see 5 Fig. 4B). These results, indicating increased inhibition of the transformed Saccharomyces cerevisiae Y.PMG515 on sensitivity to hygromycin and high salt, suggest that, HvNHXI cDNA isolated from Hordeum vulgare can complement the hygromycin related defect of a mutant Saccharomyces cerevisiae which lacks Na+/H+ antiporter, and it corresponds 10 to a gene which can provide salt tolerance. <Example 3> Insertion of HvNHXI cDNA from Hordeum vulgare to rice cells and transformation (Step 1) Construction of a recombinant for plant transformation 15 comprising HvNHXJ cDNA in a plant gene expression vehicle pGA1611 The plant gene expression vehicle pGA1611 was obtained from Prof. Jinhung Ahn at POSTECH (Pohang, South Korea). After analyzing the nucleotide sequence of HvNHXI cDNA and the restriction enzyme sites included therein, a fragment which comprises the entire open reading frame to obtain normal gene expression was prepared, 20 followed by its recombination in pGA1611 to construct a gene recombinant for transformation (pMG-HvNHX I) (see Fig. 5). (Step 2) Introduction of a recombinant gene delivery vehicle to rice cells and selection of the cells comprising the vehicle 25 By using a freezing-thawing method (An G, Ebert PR, Mitra A, Ha SB, 1988, Plant Molecular Biology Manual A3: 1-19. (Gelvin., S.B., Schilperoort, R., and Verma, D. P., Eds.), Kluwer Academic Pub., The Netherlands), pMG-HvNHX I that had been prepared in the above Step I was introduced to Agrobacterium tumefaciens LBA 4404 followed by selection of the cells comprising the recombinant gene delivery vehicle on a 30 medium including hygromycin. Thus selected cells were cultured, and pMG-HvNHXI was separated therefrom, treated with restriction enzymes, etc. to confirm that HvNHXJ gene was indeed introduced to the bacterial cells. The Agrobacterium iumefaciens LBA 4404 to which pMG-HvNIX I cDNA -18 recombinant had been successfully introduced was added to liquid medium YEP (1% yeast extract, 1% peptone, 0.5% NaCI) containing 50 mg/L of hygrornycin. Then, the cells were incubated under shaking at 250 rpm, 28 *C for more than 48 hours in the dark. Once OD 6 oo of the Agrobacterium culture reached 0.6, the culture solution was diluted 5 with AAM liquid medium (x10 dilution) and a plant callus having a diameter of 1-2 mm was inoculated with the diluted solution for 10 minutes. The callus inoculated with Agrobacterium was subjected to the first selection by using basic N6 medium to which 2,4-D (2 mg/L), cefotaxime (250 mg/L), and hygromycin (40 mg/L) were added, and then subjected to the second selection by using basic N6 medium to which 2,4-D (2 10 mg/L), cefotaxime (250 mg/L), hygromycin (40 mg/L) and BA (0.5 mg/L) were added (see Fig. 6). (Step 3) Regeneration of the rice cells comprising the vehicle into a rice plant 15 New shoots that had been selected from the above Step 2 and had been differentiated were transplanted to a basic MS medium comprising NAA (0.1 mg/L), kinetin (2.0 mg/L), and cefotaxime (125 mg/L) to induce root formation. Thus obtained young plants were transplanted in soil and grown to normal rice plants to bloom (Fig. 6). Seeds were harvested from the rice plants to obtain seeds at To generation of 121 lines. 20 In order to select the seeds at To generation using hygromycin, seeds of a non-transgenic rice plant and of the transgenic rice were subjected to a sprouting test in the presence of hygromycin (50 mg/ml). As a result, 50 lines of hygromycin resistant To generation and 9 lines of hygromycin sensitive To generation were selected. Thus selected young plants at To generation were transplanted in soil and grown to a normal rice plant. Then, by 25 using PCR and Southern blot analysis, insertion of a transfer gene was determined (see Fig. 7). Lines having confirmed transfer gene insertion and lines selected against hygromycin were sown in the experimental paddy field in an isolated area for testing paddy rice. Seeds at T, generation of 231 lines were harvested. 30 (Step 4) Determination of salt tolerance and selection of the lines having unique characteristics based on observation of growth of seedlings and recovery ratio of a transgenic rice plant with confirmed insertion of the recombinant gene under salt stress condition - 19 In order to confirm whether or not the introduction of a recombinant gene provides a regenerated rice plant with salt tolerance, seeds of the transgenic lines at T, generation and the non-transgenic plant were subjected to surface sterilization using 70% ethanol for 5 minutes, followed by another surface sterilization using 0.1% sodium 5 hypochlorite for 20 minutes. The sterilized seeds were washed more than three times with tap water and distilled water. Twelve seeds of each of the non-transgenic rice (Dongjin variety) and transgenic rice plants (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) were germinated at 25 'C in the dark for three days in triplicate. Then, for five days (two days in the dark followed by three days in the light), 10 the germinated seedlings were treated with 100 mM NaCl and their growth was observed. For fifteen days after the salt treatment, the plant was grown with normal irrigation and their recovery ratio was observed. Specifically, after measuring the length of the plant shoot, that is grown the length above the ground, it was converted to percentage ratio compared to the non-transgenic control rice group that had not been 15 treated with any NaCl. After the salt stress treatment for five days, the transgenic rice seedlings showed better growth than the non-transgenic rice plant (Fig. 8). In addition, for the salt stress treatment for five days followed by normal irrigation for 15 days, the transgenic rice seedlings show better growth than the non-transgenic rice plant and have higher 20 recovery ratio (Fig. 9). In particular, after the normal irrigation for 1 5 days, the shoot length for the lines 44-1, 90-1, 90-2, 90-8 and 90-11 that had been subjected to salt stress show significantly faster growth compared to the seedling of other lines, and have increased growth as much as 47.3, 85.5, 92.4, 91.5, and 83.1%, respectively, compared to the seedlings of the non-transgenic plant. Recovery ratio of the entire transgenic 25 plants increased by an average of 48.5% compared to the non-transgenic control rice group that had not been treated with any NaCl (Table 1). 30 - 20 Table 1. Shoot length and recovery ratio of young seedlings cultivated for 15 days with normal irrigation after the stress treatment with 100 mM NaCl for 5 days Line Shoot length (cm) Recovery ratio (%) Line H20 120 + NaCl 120 H20 + NaCl Non-transgenic plant 4.

52

±

0

.

84 3.95+1.45 0.00 0.00 (Dongjin variety rice) 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 Transgenic 90-8 5.62±1.24 7.57±0.15 4.26 1.49 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 .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 Average of the erage ofites 5.88±1.21 5.76 72 29.97 45.77 transgenic lines The above results indicate that tolerance to salt stress is actively obtained 5 through the expression of HvNHXI cDNA, and also imply that for any plant species that is transformed with HvNHXJ cDNA salt tolerance can be improved in a similar manner.

Claims (20)

1. A recombinant vector comprising a HvNHXI gene of Hordeum vulgare.

2. The recombinant vector of Claim 1, wherein said HvNHXI gene consists of a 5 nucleotide sequence represented by SEQ ID NO: 1.

3. The recombinant vector of Claim 1, wherein said vector is a yeast expression vector.

4. The recombinant vector of Claim 3, wherein said vector is pMG515.

5. A yeast cell transformed with the recombinant vector of Claim 3 or 4. 10

6. The recombinant vector of Claim 1, wherein said vector is a plant expression vector.

7. The recombinant vector of Claim 6, wherein said vector is pMG-HvNHX 1.

8. A plant having improved salt tolerance, wherein said plant is transformed with the recombinant vector of Claim 6 or 7. 15

9. The plant of Claim 8, wherein said plant is a monocot plant.

10. The plant of Claim 9, wherein said monocot plant is a rice plant.

11. A seed of the plant of Claim 8.

12. A method for producing a transgenic plant having improved salt tolerance comprising the steps of: 20 - transforming a plant cell with the recombinant vector of Claim 6 or 7; and - regenerating the transformed plant cell into a transgenic plant.

13. A method for improving salt tolerance of a plant comprising a step of transforming a plant cell with the recombinant vector of Claim 6 or 7 to overexpress said HvNHXI gene. 25

14. The method of Claim 13, wherein said plant is a monocot plant.

15. The method of Claim 14, wherein said monocot plant is a rice plant.

16. A plant having improved salt tolerance when produced by a method according to any one of Claims 13 to 15.

17. A method of cultivating a plant of Claim 16 wherein said method allows 30 cultivation of said plant in a salt rich reclaimed area or in a region not conducive to achieving a high harvest yield for a non-transgenic plant of the same species.

18. A composition for increasing salt tolerance of a plant comprising the Hordeum vulgare HvNHXI gene for expression in said plant. - 22

19. The composition of Claim 18, wherein said HvNHXJ gene consists of a nucleotide sequence represented by SEQ ID NO: 1.

20. A recombinant vector; a yeast cell; a plant having improved salt tolerance; a seed; a method of producing a transgenic plant; a method for improving salt tolerance of 5 a plant; a method of cultivating a plant; or a composition for increasing salt tolerance of a plant, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.