KR100695072B1 - - 1 Stress-Inducible 1 Gene and Protein Enhancing Tolerance to Abitoic Stress - Google Patents

Abstract

The present invention relates to abiotic stress-induced OsAsr1 genes and proteins that promote resistance to low temperature, salt or dry stress in plants when overexpressed in plants, wherein the OsAsr1 genes and proteins of the present invention are low temperature, salts in plants. Or very effective in enhancing resistance to dry stress.

Plant, paddy, low temperature, dry, salt. Stress, OsAsr1, transformation

Description

Stress-Inducible Oss1 Gene and Protein Enhancing Tolerance to Abitoic Stress

1 is a view showing the amino acid comparison of the OsAsr1 protein and another known Asr protein carried out by the CLUSTALW program. The putative Zn ²⁺ DNA binding site and intranuclear positioning signal (NLS) are underlined. White bodies in black boxes show sequences that match completely in all 16 sequences.

2 is a dendrogram showing genetic analysis of various Asr proteins in rice analogs. Information on rice analogs was obtained from the TBLASTN search for indica rice WGS contig. Dendrograms were constructed using the ClustalX and Mega2 programs. Each accession number is listed next to the plant name. The length of the horizontal line represents the evolutionary distance.

3 shows Southern blot analysis of OsAsr1 . Three Japonica cultivars, Namyang 21 (1), Odae (2) and Dongjin (3) were digested with Eco RI (E), Hind III (H) and Bam HI (B) and hybridized with radiolabeled OsAsr1 probes. The location and kb size of OsAsr1 DNA digested with Hind III are shown.

4A-4E show the results of OsAsr1 expression analysis under various abiotic stress and ABA treatments. FIGS. 4A-4C show Northern blot analysis of OsAsr1 . 30 μg (A) or 10 μg (B and C) of total RNA were isolated, blotted and hybridized with radiolabeled OsAsr1 probes. 4A shows OsAsr1 expression in various organs or tissues. Lanes: 1, callus; 2, shoots of seedlings; 3, the root of the seedlings; 4, mature leaves; 5, leaf leaf of the flag leaf; 6, internode between nodes I and II in the freehead stage; 7, 1-2 cm cone inflorescences; 8, 3-8 cm conical inflorescences; 9, mature cone inflorescence before flowering; 10, seed during development on day 3 (DAP); 11, 6 seeds during development of DAP. Figure 4b shows a low-temperature inducible expression of OsAsr1. Mature plants were treated at 12 ° C. for 4 days. L, leaves; CL, pasteurized leaves; F, florets; CF, low temperature treated florets. 4C shows the expression of OsAsr1 with different temperatures and by ABA. Seedlings grown at 30 ° C. were treated with 10 μM ABA for 4 ° C., 12 ° C. or 3 h, 6 h and 20 h. Figure 4d is a semi OsAsr1 - it was used as a control group and ABA- reaction-drying SalT quantitative RT-PCR analysis, a low-temperature-salt. Transcripts of the rice actin gene represent intrinsic controls in PCR analysis. Figure 4E shows the analysis of OsAsr1 expression levels using real-time PCR. Error bars represent standard deviations. 8-day seedlings were treated with 4 ° C. (c), 250 mM NaCl (s), dry (d) or 100 μM ABA (A) and harvested periodically. Transcripts of the rice actin gene represent intrinsic controls in PCR analysis.

5A and 5B are photographs showing in situ localization of OsAsr1 mRNA. Sections of rice flowers at 4 days before heading and leaves exposed to 12 ° C. for 4 days were treated with digoxigenin-labeled antisense (A, C, E, and G) or sense (B, D, F and H) OsAsr1 probes. Hybridized. High magnifications of the cut planes are shown (C, D, G and H). an anther; lm, external appearance; pa, inner spirit; lep, lower epidermis of inner ear; LVB, large flow rate; mc, motor cell; xy, neck; ph, quadruplet; me, lobules. Rod = 0.3 mm

6A-6C show the analysis results of transgenic plants expressing OsAsr1 in the sense and antisense directions. Figure 6a is a structure of OsAsr1 sense (pSK167) and antisense (pSK168) expression vector for the transfection of rice conversion. P _UBI , corn ubiquitin promoter; P _35S , CaMV 35S promoter; T _NOS , nopaline synthase terminator sequence; T7, terminator sequence of transcript 7; hph , hygromycin phosphotransferase gene to select for transforming callus; RB and LB, right and left border sequences, respectively, of Ti plasmids of Agrobacterium tumefaciens . 6B shows Southern blot analysis of the transgene. Southern blots were performed using 10 μg of the genome DNA obtained from the transgenic plant with Hin dIII. Ubiquitin promoter fragments were used as probes in the transformation vectors. The position and kb size of OsAsr1 DNA digested with Hind III are indicated. Figure 6c is a photograph showing OsAsr1 expression of the sense transgenic rice (Ubiquitin :: OsAsr1). Total RNA of leaves obtained with wild type separator (WS) and sense transgenic plants under normal growth conditions was isolated, blotted and hybridized with radiolabeled OsAsr1 probe. Numbers represent each transformant. EtBr-stained rRNA bands show that the same amount was loaded.

7 is a graph showing the stress resistance of Ubiquitin :: OsAsr1 plants as determined by chlorophyll fluorescence measurements. Changes in chlorophyll fluorescence of elongated leaves under cold stress were measured. The functional impairment of photosynthesis was assessed by measuring the mean and standard deviation of the Fv / Fm values.

8 is a photograph showing the OsAsr1 expression in transgenic rice CBF1. Here expression (18-2 and 18-3) and non-transfected be saehyeong the total RNA obtained from the mature leaf of the switching state (NT) radiolabeled OsAsr1 - two kinds of strong CBF1 in the control group (30 ℃) or low-temperature treatment (4 ℃) Hybridization with probes. EtBr-stained rRNA bands show that the same amount was loaded.

Figure 9 shows the salt resistance of transgenic rice expressing OsAsr1 in the sense (9a) or (9b) antisense direction: 12 day old seedlings at 200 mM NaCl for 1 day (sense plant) or 12 hours (antisense plant) And recovered for 2 days under normal conditions.

FIG. 10 shows the fake rate of rice varieties and transgenic OsAsr1 rice plants: 12 day old seedlings were treated in 200 mM NaCl for 1 day (sense plant) or 12 hours (antisense plant) and recovered for 2 days under normal conditions. .

FIG. 11 shows the dry resistance of transgenic rice expressing OsAsr1 in the sense (11a) or (11b) antisense direction: 14 days old seedlings without water for 4 days, again restored for 7 days under normal conditions .

FIG. 12 shows the forgery rates of rice varieties and transgenic OsAsr1 rice plants: 14 days old seedlings were not fed with water for 4 days, and then recovered for 7 days under normal conditions.

The present invention relates to abiotic stress-induced OsAsr1 genes and proteins that promote resistance to cold, dry or salt stress.

Plants as fixed organisms must cope with environmental stresses such as non-biological stresses such as low temperature, drying and salting. After this exposure, a series of changes occur in the pattern of gene expression (Guy, 1999). This reaction affects growth rate, productivity and species distribution. Rice, as cereals with tropical or subtropical origin, is often subjected to cold damage, and the damaged plants show various symptoms such as medulla, necrosis or delayed listing (De Datta, 1981). In temperate zones, rice is often faced cold during the flowering period as well as cold-summer damage.

To understand the abiotic stress-response mechanism, the researchers isolated a number of genes encoding abiotic-induced proteins in several plants (Guy, 1999; Thomashow, 1999). The calcineurin B-like 1 (CBL 1) gene is significantly induced by dryness, salt and cold stress. In addition, CBL1-overexpressing plants showed enhanced salt and dry resistance (Cheong, YH, Kim, KN, Pandey, GK, Gupta, R., Grant, JJ, and Luan, S. 2003. CBL1, a calcium sensor that differentially regulates salt, drought, and cold responses in Arabidopsis .Plant Cell 15: 1833-1845). Genes derived under low temperatures are expected to function to protect cells by producing a variety of genetic products. For example, Arabidopsis (Arabidopsis) MAP kinases (ATMPK4 and ATMPK6) and C-repeat / DRE binding factor (CBF / DREB) transcription factors are involved in cold stress signaling (Mizoguchi et al., 1993; Stockinger et al., 1997), lipid desaturase Is involved in membrane modification during cooling (Gibson et al., 1994). Cold-induced onset-related proteins such as chitinase-like and tautin-like proteins act as antifreeze proteins (Hiilovaara-Teijo et al., 1999). It is also predicted to enhance chaperone, late embryogenic abundance (LEA) protein, calmodulin-related protein and freeze resistance (Thomashow, 1999). Finally, enzymes necessary for biosynthesis of various osmoprotectants such as sugars, proline and betaine are also very important for regulating plant osmotic pressure after cold exposure (Kishor et al., 1995; Igarashi et al., 1997; Scott et al. , 1999).

A variety of species such as ABA Asr genes showing the reactivity (abscisic acid), osmotic stress (osmotic s tress) and mature (r ipening) are tomatoes, potatoes, apricots, Te somewhat trees, lily, corn, Firm booties, and grapes and rice (Iusem et al., 1993; Canel et al., 1995; Silhavy et al., 1995; Wang et al., 1998; Chang et al., 1996; Mbeguie-A-Mbeguie et al., 1997; Vaidyanathan et al., 1999; Hong et al., 2002; Jeanneau et al., 2002; Cakir et al., 2003). High levels of tomato Asr1 mRNA were detected in mature fruits and leaves under water stress (Hagit et al., 1995). The amount of Lilium ASR protein is increased by drying during pollen maturation (Wang et al., 1998). However, the physiological role of ASR protein is still unknown. The localization of tomato ASR1 protein in the nucleus suggests that tomato ASR1 protein acts as a nonhistone chromosome protein (Rossi and Iusem, 1994). In addition, from the fact that several ASR proteins are structurally and functionally similar to late developmental rich (LEA) proteins or dihydrin proteins, one can infer that ASR is involved in seed development (Maskin et al., 2001). Silhavy et al., 1995). Many known ASR proteins have two conserved sites corresponding to the putative Zn-binding position at the N-terminal site and the putative in-nuclear positioning sequence (NLS) of about 70 amino acids at the C-terminus (Cakir et al., 2003; Silhavy et al., 1995). From these facts and the fact that grape ASR (VvMSA) binds to the promoter sequence of the monosaccharide transport gene, one can infer that VvMSA is a component of the transcriptional-regulatory complex involved in sugar and ABA signaling (Cakir et al. , 2003).

Vaidyanathan et al. (1999) were identified Po Carly (Pokkali) rice from the ASR cDNA, OsAsr1, this gene ABA- and osmotic pressure (NaCl) stress-induced were identified for ordination. However, the function of OsAsr1 with a hydrophilic alpha helix structure is largely unknown.

Throughout this specification, numerous papers and patent documents are referenced and their citations are indicated in parentheses. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors abiotic efforts example to identify genes which can enhance resistance results against sex stress, rice (OsAsr1 induced by stress from (Oryza sativa) Oryza sativa responsive to ABA, osmotic s tress, and r ipening gene 1 ) The present invention was completed by discovering a gene and confirming that the plant transformed by the gene exhibits enhanced resistance to low temperature, salt, or drying.

Accordingly, it is an object of the present invention to provide abiotic stress-induced OsAsr1 protein that enhances resistance to cold, salt or dry stress.

Another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding a non-biological stress-induced OsAsr1 protein.

It is still another object of the present invention to provide a vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a non-biotic stress-induced OsAsr1 protein.

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

Another object of the present invention to provide a method for producing a transgenic plant having enhanced low temperature, salt or dry stress resistance.

Another object of the present invention is to provide a transgenic plant having low temperature, salt or dry stress resistance.

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

According to one aspect of the invention, the invention comprises the amino acid sequence of SEQ ID NO: 2 sequence and when overexpressed in a plant abiotic stress-induced OsAsr1 which enhances resistance to cold, salt or dry stress in the plant Provided is a protein or a protein that exhibits at least 80% amino acid sequence homology with the protein.

The present inventors have cold, salt or effort results to identify genes which can enhance the resistance to drought stress, rice Oryza sativa responsive to ABA, OsAsr1 (induced by stress from (Oryza sativa) osmotic s tress, and r ipening gene 1 ) The gene was excavated and confirmed that the plant transformed by the gene exhibits enhanced resistance to low temperature, salt or drying.

As used herein, the term “abiotic stress” refers to stress induced by abiotic factors such as low temperature, salt and drying.

As used herein, the term "cold stress" means a stress caused by a low temperature, preferably 15 ° C. or less, more preferably 12 ° C. or less, and most preferably 10 ° C. or less.

The non-biological stress-induced OsAsr1 protein of the present invention exhibits a substantial identity to the above-described amino acid sequence, and at the same time the amino acid sequence within a range capable of enhancing resistance to low temperature, salt or dry stress. It is to be interpreted as including. Such substantial identity is to align the amino acid sequence of the present invention with any other sequence to the maximum correspondence, and to analyze the aligned sequence using programs commonly used in the art (eg, ClustalX and PROSIS). In one case it is meant an amino acid sequence which exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology.

(. Oryza sativa L): - On the other hand, according to the present invention stress inducible OsAsr1 protein can be specified as follows: separation from (a) rice; (b) there is one Zn-binding motif at the N-terminal site; And (c) there is one nuclear localizaton domain at the C-terminal site.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the abiotic stress-induced OsAsr1 protein.

As used herein, the term “nucleic acid molecule” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic building blocks of nucleic acid molecules are naturally modified nucleotides, as well as modified sugar or base sites. Analogs are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

Most preferably, the nucleic acid molecule of the present invention comprises the nucleotide sequence of SEQ ID NOs: 67-480 in the nucleotide sequence of SEQ ID NO: 1. Nucleic acid molecules of the present invention encoding non-biogenic stress-inducing OsAsr1 proteins are also construed to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences described above. The substantial identity above aligns the nucleotide sequence of the present invention with any other sequence to the maximum correspondence, and analyzes the aligned sequence using programs commonly used in the art (eg, ClustalX and DNASIS). In one case it is meant a nucleotide sequence that exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology.

According to another aspect of the invention, the invention provides a vector comprising a nucleotide sequence encoding the abiotic stress-induced OsAsr1 protein described above. According to a preferred embodiment of the present invention, the vector of the present invention comprises (a) a nucleotide sequence encoding a non-biological stress-induced OsAsr1 protein and (b) a promoter operably linked to the nucleotide. Provide a vector

As used herein, the term “operably linked” refers to a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, whereby such regulation The sequence will control the transcription and / or translation of said other nucleic acid sequence.

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

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

For example, the vector is an expression vector of the present invention, in the case of a prokaryotic cell as a host, the strong promoter that can proceed with the transfer (e.g., p _L ^λ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc. ), It is common to include ribosomal binding sites and transcription / detox termination sequences for initiation of translation. When E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol. , 158: 1018-1024 (1984)) and the leftward promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14: 399-445 (1980)) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) which are often used in the art, phage (e.g. λgt4.λB , λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

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

Vectors of the invention may also be fused with other sequences to facilitate purification of the non-biological stress-inducing OsAsr1 protein expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA). Because of the additional sequence for this purification, the protein expressed in the host is purified quickly and easily through affinity chromatography.

The vector of the present invention may include an antibiotic resistance gene commonly used in the art as an optional marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neo There are genes resistant to mycin and tetracycline.

The genes of the present invention have been isolated from plants and have the most desirable utility for plants as they can act to enhance the plant's resistance to cold, salt or dry stress. Thus, the vectors of the present invention may comprise (i) nucleotide sequences encoding abiotic stress-induced OsAsr1 proteins; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause polyadenylation of the 3'-end of the RNA molecule.

According to a preferred embodiment of the present invention, a promoter suitable for the present invention may be used any conventionally used in the art for the gene introduction of plants, for example, the ubiquitin promoter of corn, cauliflower mosaic virus (CaMV) 35S promoter, nopalin synthase (nos) promoter, pigwart mosaic virus 35S promoter, sugacran basilisform virus promoter, commelina yellow mottle virus promoter, ribulose-1,5-bis-phosphate carbide Photoinducible promoter of the carboxylase small subunit (ssRUBISCO), rice cytosolic triosphosphate isomerase (TPI) promoter, adenine phosphoribosyltransferase (APRT) promoter of Arabidopsis and octopine synthase promoter do.

According to a preferred embodiment of the present invention, the 3'-non-detoxification site which causes polyadenylation suitable for the present invention is derived from the nopaline synthase gene of Agrobacterium turmeration (nos 3 'end). (Bevan et al., Nucleic Acids Research , 11 (2): 369-385 (1983)), derived from the Octopine synthase gene of Agrobacterium tumersions, protease inhibitor I or II gene of tomato or potato 3 'terminal portion of and CaMV 35S terminator.

Optionally, the vector additionally carries a gene encoding a reporter molecule (eg, luciferase and β-glucuronidase). In addition, the vector of the present invention may be selected as an indicator for antibiotics (e.g. neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, and the like) and resistance genes (e.g. neomycin phosphotransferase ( nptII ), hygro Mycin phosphotransferase ( hpt ), and the like).

According to another aspect of the present invention, the present invention provides a transformant transformed with the vector of the present invention described above.

Host cells capable of stable and continuous cloning and expression of the vectors of the present invention are known in the art and can be used with any host cell, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. strains of the genus Bacillus, such as coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas species. Enterobacteria and strains.

In addition, when transforming the vector of the present invention into eukaryotic cells, the host cells may be yeast ( Saccharomyce cerevisiae ), insect cells, human cells (eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells and the like can be used. On the other hand, since the Oslti 32 gene of the present invention is highly useful in plants, the transformants include not only cells but also callus derived from plant cells or tissues.

The method of carrying a vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973), when the host cell is a prokaryotic cell). ), One method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol. , 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res. , 16: 6127-6145 (1988)) and the like. In addition, when the host cell is a eukaryotic cell, fine injection method (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology , 52: 456 (1973)), Electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene , 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol. , 5: 1188-1190 (1985)), and gene balm (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568-9572 (1990)) Can be injected into the host cell.

The vector injected into the host cell is expressed in the host cell, in which case a large amount of abiotic stress-induced OsAsr1 protein is obtained.

According to another aspect of the present invention, the present invention comprises the steps of (a) transforming a plant cell or plant tissue with the vector of the present invention; (b) selecting the transformed plant cells or plant tissues; And (c) regenerating the plant from the transformed plant cells or plant tissues to obtain a transformed plant, thereby providing a method for producing a transformed plant having enhanced low temperature, salt or dry stress resistance.

According to another aspect of the invention, the invention provides a transgenic plant having low temperature, salt or dry stress resistance.

The plant used in the present invention is a plant cell or plant tissue, and in the case of using plant tissue, it is preferable to use callus.

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

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent (eg metabolic inhibitors, antibiotics and herbicides). Plant cells that are transformed and stably contain marker genes that confer selective resistance are grown and divided in the cultures described above. Exemplary labels include, but are not limited to, hygromycin phosphotransferase genes, glycophosphate resistance genes, and neomycin phosphotransferase (nptII) systems.

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

The method of the present invention is applied to a variety of plants, but is preferably applied to plants of cereals such as rice, barley, wheat and corn, more preferably to rice, and most preferably to japonica cultivars.

In the present invention, the preferred transformation method is carried out using the Agrobacterium system, more preferably using the Agrobacterium tumefaciens -binary vector system.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') an Agrobacterium tumer embedded in a genome DNA of a plant cell and having a vector having the sequence Infecting the plant's plant with Agrobacterium tumefaciens : (i) a nucleotide sequence encoding the abiotic stress-induced OsAsr1 protein; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; (Iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule; (b ') regenerating the infected plant in regeneration medium to obtain a transgenic plant.

Transformation of plant cells is carried out with Agrobacterium trimers comprising Ti plasmids (Depicker, A. et al., Plant cell transformation by Agrobacterium plasmids.In Genetic Engineering of Plants, Plenum Press, New York (1983) ).

More preferably, binary vector systems such as pBin19, pRD400, pRD320, pGA1611 and pGA1991 are used (An, G. et al., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986) An et al., 1988 and Lee et al., 1999. Binary vectors suitable for the present invention include (i) a promoter that operates in a plant; (ii) a structural gene operably linked to the promoter; and (iii) Optionally, the vector additionally carries a gene encoding a reporter molecule (eg, luciferase and glucuronidase) An example of a promoter used in a binary vector is CaMV 35S. Promoters, 1 promoter, 2 promoters and nopaline synthase (nos) promoters.

Infection of implants with Agrobacterium tuberculosis includes methods known in the art. Most preferably, the infection process comprises coculture with an implant impregnated with a culture of Agrobacterium tuberation. As a result, Agrobacterium trimmers are infected into plants.

Plants transformed by Agrobacterium tumerization re-differentiate in regeneration medium, which finally forms transgenic plants.

Plants transformed according to the present invention is confirmed whether or not transformed by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transformed plants to identify foreign genes inserted into the genome of the transformed plants. Alternatively, Northern or Southern blotting can be performed to confirm transformation (Maniatis et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).

OsAsr1 genes and proteins of the present invention are very effective in enhancing resistance to low temperature, salt or dry stress in plants.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experimental Materials and Methods

Plant Samples and Bacterial Strains

Dongjin, a cultivar of Oryza sativa , was used to construct a seed-coat cDNA library and to prepare transgenic plants. Other cultivars or wild species used in the cold resistance test are listed in Table 1. Seeds were surface-sterilized and germinated in water or MS agar medium (Murashige and Skoog, 1962). Alternatively, they were germinated in Yoshida solution (Yoshida et al., 1976), and these were grown in hydroponic cultures including 29 ° C./21° C. night temperature, 16-hour-light / 8-hour-cancer cycle, and 83% relative humidity conditions. Incubated under controlled conditions. The seedlings were transferred to the soil and placed in a growth chamber under 30 ° C. continuous light (intensity 60 to 70 μmol m ⁻² sec ⁻¹ ). For low temperature treatment, 14-day-old seedlings were harvested exposed to 4 ° C. or 12 ° C. under dark conditions. For ABA treatment studies, 5 day old seedlings were harvested transferred to 10 μM ABA-containing MS liquid medium. For salt or ABA treatment, the seedlings were transferred to a Yoshida solution containing 250 mM NaCl or 100 μM ABA and then harvested periodically. For the drying treatment, the seedlings were dried in air and then harvested periodically. To study the effects of cold stress on the reproductive stage, rice plants growing on the 4th day before heading were transferred to a greenhouse (30 ° C., 14 hr light / 10 hr cancer) and acclimated for 2 days. The plants were then treated for 4 days at 12 ° C. with a 14 hr photoperiod (ca. 300 μmol m ⁻² sec ⁻¹ ). All tissue samples were immediately frozen with liquid nitrogen and stored at -70 ° C. E. coli strain, XL-1 Blue MRF '{Δ (mcrA) 183Δ (mcrCB-hsdSMR-mrr) 173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F' proAB, lacIqZΔM15 Tn10 (Tetr)]} (Stratagene, USA) It was used as a host for cloning.

cDNA library construction and EST analysis

Coats of 4 or 8 day old seeds were digested by hand in the microscope section. The cDNA library was then constructed using poly-A RNA from seed coat. CDNA clones were randomly selected from the library and their 5′-ends were sequenced. DNA preparation, sequencing and computer analysis are described in Hong et al. (1998), according to the method disclosed. First, template DNAs were prepared by alkaline digestion, and insertion sequences were sequenced using the ABI PRISM ^™ BigDye ^™ Terminator Cycle Sequencing Kit (Amersham). Sequencing was performed using computer software such as DNAsis, Prosis (Hitachi), ClustalX, ClustalW and GeneDoc (Thompson et al., 1994; Nicholas and Nicholas, 1997). Genbank, EMBL and Swiss-Prot databases were retrieved upon amino acid sequence homology investigation using the BLASTX algorithm (Altschul et al., 1997).

DNA and RNA Gel-Blot Analysis

DNA gel-blot analysis was performed on three Japonica cultivars, Namyang 21, Dongjin and Odae. Namyang 21 is cold-sensitive (low temperature tolerance, CTI = 7), the fifth is cold-resistant (CTI = 3), and the east is medium (CTI = 5) (Lim, 1998). For CTI values between 0 and 9, 0 is the most resistant and 9 is the most sensitive. The genome DNA was isolated from the seedlings according to the cetyltrimethylammonium bramide method (Roger and Bendich, 1988). 10 μg of DNA digested for 6 hours at 37 ° C. with restriction enzymes was isolated on a 0.8% agarose gel and transferred to a Hybond-N membrane (Amersham) using a vacuum transfer system (Hefer). For RNA gel-blot analysis, 10 μg of total RNA was isolated on 1.3% formaldehyde agarose gel and blotted with nylon membrane. DNA and RNA blot analyzes were performed using radiolabeled OsAsr1 probes. To prepare the probes, according to a random priming method, OsAsr1 cDNA fragments were radiolabeled with [α- ³² P] dCTP (3000 ci mmol- ¹ ). Uninserted nucleotides were removed via G-50 Sephadex column chromatography. After hybridization, the membranes were placed for 15 minutes at room temperature in 2 × SSC, 0.1% SDS; 15 min at RT in 1 x SSC, 0.1% SDS; And washed at conditions of 15 minutes at room temperature in 0.1 x SSC, 0.1% SDS. Hybridization signals were detected with a phase analyzer (BAS-1500, Fuji) and exposed to Hyperfilm ^™ MP film (Amersham).

RNA In Situ  Hybridization

Rice flowers and leaves were immobilized with 2% (w / v) paraformaldehyde and 2.5% (v / v) glutaraldehyde overnight at 4 ° C. in 50 mM PIPES buffer, pH 7.2. Tissues fixed at a graded concentration of ethanol were dehydrated and embedded in paraplast medium (Oxford labware, USA). Embedded tissue was cut into 7 μm sections with a rotating microtome (Leica, Germany) and the sections were attached to silanized glass slides (Matsunami, Japan). Paraffin was removed using a graded concentration of ethanol and then the sample was dried for 1 hour. T3 or T7 RNA polymerase using the (Roche, USA), digoxigenin from the linearized pBluscript with OsAsr1 cDNA - to prepare a labeled sense or antisense RNA probe. The sections were hybridized by treatment at 48 ° C. for 16 hr with the prepared probes in the hybridization solution and washed at 50 ° C. for 15 minutes in a solution containing 2 × SSC, 1 × SSC and 0.1 × SSC. Hybridized probes were detected by color development using anti-DIG conjugated alkaline phosphatase (Boheringer Mannheim, Germany). The photograph was obtained using a bright-field microscope (Nikon Eclipse 600, Japan).

Preparation and Analysis of Transgenic Plants

The full-length OsAsr1 cDNA was inserted in the sense and antisense direction downstream of the corn ubiquitin promoter (Christensen, Sharrock & Quail 1992) of binary vector pGA1611 (Professor Jin-heung Ahn) (An et al . 1988; Kim et al., 2003). . Rice transformation is described in Jeon et al., 1999; According to the method disclosed in Lee et al., 1999, it was carried out via the Agrobacterium-mediated coculture method. Rice seeds removed from external and internal bodies were injured in 2N6 medium containing 2 mg L ^-1 2,4-D to induce callus for one month, and 30 mg L ^-1 hygromycin B and 3 mg L ^-1 tetracycline Agrobacterium grown for 3 days in this added AB medium and 3 days coculture in 2N6-ASB medium in the dark at 20 ° C. for 3 days, followed by 40 mg L- ¹ hygromycin B and 250 mg L- ¹ cell suspension After 3 weeks in shim added 2N6-CH40 medium, select well-grown callus for 2-3 weeks for 0.1 mgL ^-1 NAA, 2 mg L ^-1 kinetin, 2% sorbitol, 1.6% petagar, 50 mg L ^-1 hydro In the MS medium mixed with the mycin B and 250 mg L ^-1 cell Taxim, it was re-differentiated in the continuous light state, the re-differentiated individuals were transferred to the soil and grown in the greenhouse. All transgenic rice plants were 40-mg L ^-1 hygromycin Prepared in B-containing medium and transferred to the greenhouse after regeneration. PCR analysis to identify transgenic plants was carried out using forward primers (5-CAC CCT GTT GTT TGG TG-3) and reverse primers (5-GCG GGA CTC TAA TCA TAA AAA CC-3). PCR conditions were 30 cycles by the cycle of 94 degreeC 1 minute, 54 degreeC 1 minute, and 72 degreeC 1 minute.

Measurement of Chlorophyll Fluorescence

About 5-cm-length fragments of the elongated leaves obtained from mature plants were suspended in MS liquid medium at 4 ° C. under white fluorescence (260 mol m ⁻² sec ⁻¹ ) for 0, 6, 12 or 24 hours. After 30 minutes of cancer-adaptation, the chlorophyll signal was measured using the Plant Efficiency Analyzer (Hansatech, UK). All experiments were repeated four times.

Cold stress determination

The extent of wilting (Saijo et al., 2000) or the amount of regrowth (Lee et al., 1993) was evaluated to calculate the survival rate of plants exposed to cold stress. 10 day-old seedlings grown in a growth chamber (16-h light / 8-h arm; light intensity 60 μmol m ⁻² sec ⁻¹ ; 30 ° C.) (3 lobe stages) were subjected to continuous light (60 μmol m ⁻² sec ^{− 1} ) was exposed to 4 ° C. for 3, 4, 5, 6, 7, 10 or 12 days. The plants were then returned to standard growth-chamber conditions for 10 days to recover. The counterfeiting rate was based on the apparent degree of receding and leaf regression data. To analyze the regrowth, the seedlings exposed to 4 ° C. for 4 days were returned to the growth chamber and recovered for 13 days. Regrowth was defined as the production of new four lobes on seedlings.

Determination of Dry and Salt Stress

The plants were incubated for about two weeks in a growth chamber (30 ° C., light / dark cycle of 16/8 hours) and the survival rate after stress treatment was measured. For the dry-survival test, no water was supplied for 4 days. Incubate again for 7 days under normal growth conditions and determine the number of wilted and healthy plants (Lee, SC, Huh, KW, An. K., An. G., and Kim, SR 2004b Ectopic expression of a cold-inducible transcription factor.CBF1 / DREB1b, in transgenic rice ( Oryza sativa L.) Mol. Cells 18: 107-114). For salt stress treatment, 10-day-old seedlings were incubated for 2 days in 0.1% (w / v) Hyponex (Hyponex) nutrient medium and transferred to fresh nutrient medium containing 200 mM NaCl, either 1 day (with respect to sense plants) or 12 Incubated for 16 hours at 30 ° C. in a photoperiod (50-60 μmol m ⁻² sec ⁻¹ light) for time (for antisense plants). After salt stress treatment, the roots of the seedlings were washed with water and incubated in fresh medium without NaCl under normal conditions. When the three to four leaves of the fully forged plant showed obvious regression, forgery was determined after stress.

Experiment result

From the Rice Seed-Coat Library OsAsr1  Identification of cDNA

The cDNA clones isolated from the seed coats developed by the present invention are described in Vaidyanathan et al. (1999) has a high homology with OsAsr1 . Clones of the present invention are 838 bp long and include 66-bp, 5'-nontranslated site (UTR), 417-bp open reading frame, 325-bp 3'-UTR, and poly (A) -tail. Compared with known clones, the 5'-end is about 4 bp short and is completely consistent with OsAsr1 except that T at 3'-UTR is changed to C (304 bp from downstream of stop codon). Since the known cDNA was isolated from Pokkali, Indica rice and from the Japonica type Dongjin isolated, the single-nucleotide variation in 3'-UTR is due to differences in cultivars. I think it was.

OsAsr1 genome sequences were recovered from the Indica rice WGS genome database (Yu et al., 2002). In -silico analysis of the clones predicted that the central sequence of the ABA response element (ABRE), ACGT, was at the -261 position, ie 610 and 616 bp away from the original ATG codon. Important cold-reactive cis element, C-repeat / dry reactive element (CRT / DRE) sequence, GCCGAC, was found at position 714 and putative Myb (143, -149, and 698), Myc (71, 512, and 694) And bZIP (202 and 711) binding sites were also found. The putative Zn-binding His-residue and putative NLS domains are located at the N-terminus and C-terminus of OsAsr1 protein, respectively (FIG. 1). from cDNA and genomic sequence comparison, the intron of 119-bp- length we can be seen that the OsAsr1 gene. TBLASTN search (Altschul et al., 1997) using the questionnaire OsAsr1 protein sequence (accession no. AAB96681) identified five analogs in scaffolds (002913, 037286, 026604, 023736, and 081294). . Phylogenetic relationships presented in dendrograms show that OsAsr1 has the highest homology with ZmAsr1 in corn (FIG. 2). ASR proteins are classified into four main groups (Hong et al., 2002), and OsAsr1 belongs to group I related to ZmAsr1. More than 100 OsAsr1 cDNA sequences are registered in the GenBank database, indicating that the genes of the invention belong to the abundance-expressing gene family. In addition, OsAsr1 analogs were found only in plant species.

OsAsr1 DNA gel-blot analysis

DNA gel-blot analysis of three Japonica cultivars showed only a single copy of the OsAsr1 gene (FIG. 3). These results are in agreement with the results of the Pokary variety test (Vaidyanathan et al., 1999). Restriction-enzyme-fragment lengths for these three cultivars with different low temperature resistance showed no clear polymorphism.

Due to cold stress OsAsr1 Induction of

We used RNA gel-blot analysis to examine genes expressed in other tissues and under various abiotic conditions. OsAsr1 transcripts were detected in all tissues and organs except callus (FIG. 4A), and OsAsr1 transcripts were induced by low temperature, drying, salt and ABA treatment, and at 3 hours after drying and salt stress treatment, Reached (FIGS. 4D and 4E). The size of the transcript is about 0.9 kb, indicating that the OsAsr1 cDNA clone of the present invention is full length. The transcripts were present at high levels in shoots, roots and flap leaves of seedlings, most abundantly in the internodes between nodes I and II, indicating that the OsAsr1 gene exhibits organ-preferred expression.

OsAsr1 was expressed at ground level in the leaves of mature plants, but more abundantly in mature flowers. Interestingly, low temperatures raised the overall transcript levels in both organs (FIG. 4B). Cold treatment increased the amount of transcript in the seedling stage, and the increase in the amount of transcript was more apparent at 12 ° C. than at 4 ° C. (FIG. 4C). Cold-reactive OsAsr1 accumulation is limited to shoots, and this shows the organ-specific stress response of gene expression.

Since OsAsr1 is ABA-induced (Vaidyanathan et al., 1999), we compared induced kinetics of cold stress versus induced kinetics of ABA. Transcript levels peaked after 3 hours of cold stress, whereas ABA treatment increased at a slower rate and peaked after 6 hours (FIG. 4C). OsAsr1 expression in the physiological range of growth temperatures (12, 20, and 30 ° C.) was compared between cold-resistant cultivars and cold-sensitive cultivar Namyang 21. However, we could not observe any clear difference in gene expression between these cultivars.

To identify the cold-induced expression pattern of OsAsr1 gene at the tissue level, RNA in situ hybridization was performed. In leaves, induction was confined to lobules tissue and was not found in epidermal or tubular tissues (E and G in FIG. 5). Since flowers are the main target organs of low temperature stress, hybridization was performed on the flowers. Immature flowers were cold treated at 12 ° C. for 4 days before heading. Then, from the palea and oeyoung of flowers mesophyll stressed cells could be detected OsAsr1 transfer member (A and C in Fig. 5). OsAsr1 expression patterns in control flowers that were not cold stressed were similar to those that were cold stressed, but the expression was much lower than that of flowers that were cold stressed.

In transgenic plants OsAsr1 expression of cDNA

In order to study the function of OsAsr1, OsAsr1 a cDNA, a sense or antisense orientation, such that by positioning control by the maize ubiquitin promoter (P _UBI), finally obtaining the vector pSK167 and pSK168, respectively (Fig. 6a). Agrobacterium-mediated co-culture method was used to generate 20 transgenic plants, and DNA gel-blot analysis confirmed whether the transgene was inserted in the rice genome. The copy number was one to two in one, but more than three copies were also detected (FIG. 6B). Transgenic plants showed no significant morphological changes in T1 and T2 generations.

To examine whether the altered expression of OsAsr1 results in cold resistance in transgenic rice, RNA gel-blot analysis was performed (FIG. 6C). Overall, OsAsr1 transcript amounts were higher than wild type in transgenic plants. The size of hybridized transcripts in transgenic plants was slightly larger than wild type. Two strong overexpressors, S2 and S15, were selected for further experiments.

Chlorophyll Fluorescence Measurement of Transgenic Plants Under Cold Stress

Chlorophyll fluorescence as a low temperature resistant marker after low temperature treatment (4 ° C) was measured. The ratio of Fv to Fm indicating activity of light system II is used to assess functional impairment in plants (Genty et al., 1989). In our wild type separator, Fv / Fm gradually decreased due to low temperature. This decrease shows the degree of light inhibition caused by cold stress (Krause, 1994). Before inducing stress, the Fv / Fm value was 0.84 ± 0.01. After 6 hours of cold treatment, Fv / Fm decreased slightly and no clear change in value was observed in both transformants and wild type. However, after 24 hours of treatment, the Fv / Fm values for wild type decreased significantly, reaching 0.25 ± 0.021 (FIG. 7). In contrast, the Fv / Fm values for overexpressing S2 and S15 were 0.53 ± 0.049 and 0.60 ± 0.067, respectively. This ratio is about twice the value of the wild type (FIG. 7), indicating that the transgenic plants have high low temperature resistance.

Survival rate

Survival of the transformed plants at the seedling stage was measured by forgery and regrowth tests. First, the amount of forgery after stress (ie, the ratio of the number of counterfeit seedlings to the total number of cold treated seedlings) was evaluated to determine the critical length of cold treatment. After 5 days of exposure to cold, cold-resistant cultivars, larvae and Stejaree 45 exhibited counterfeit rates of 10/18 (55.6%) and 8/20 (40%), respectively (Table 1). . This forgery rate increased to about 75% after 6 days of stress. On the contrary, in the parent strain and oscillation of the OsAsr1 transformation of the present invention, 12 out of 17 plants (70.6%) exhibited fake symptoms after 5 days of stress. The forgery rate increased to about 94% after 6 days of stress. The cold-sensitive Indica x Japonica cultivar, Milyang 23, exhibited a 100% forgery after only 4 days of stress. Therefore, it can be seen that the critical time length for cold stress resistance by vibrating is about 5 to 6 days. Based on this presumption, the T2 transgenic plants were cold treated for 6 days. Twenty-three (68%) of the 34 seedlings obtained from the S2 plant showed forgery after cold treatment, and ten of the 15 seedlings of the S15 plant (67%) also exhibited this symptom. In contrast, 63 out of 66 seedlings of wild-type plants (96%) showed forgery. Antisense transgenic plants were also investigated, and counterfeit development was 9/9 (100%), 9/11 (82%), and 10/11 (91%) in A3, A12 and A14, respectively.

Regrowth tests conducted in the T3 generation included analyzing homozygous strains for the transgene after the selection of hygromycin. Fourteen of the 35 seedlings (40%) of the S15 plant had active development of four lobes. In contrast, S18 plants derived from weak OsAsr1 -expressing strains showed regrowth of only 7 of 20 plants (20%). Nevertheless, wild-type control plants showed a much lower regrowth rate, i.e. 9 out of 105 plants (9%). Similar results were observed in A3 plants, with only seven out of 70 plants (10%) responding. Taken together, the experimental results confirmed that the transgenic plants overexpressing OsAsr1 transcript have increased resistance to cold stress. However, when OsAsr1 expression was suppressed as in antisense plants, the degree of resistance did not change significantly.

Cold Stress Tolerance of Rice Cultivars and Transgenic OsAsr 1 Rice Cultivars or lines Cryogenic Treatment Period (Days) 0 3 4 5 6 7 10 12 Odae 0/18 ^* 2/20 5/20 10/18 13/17 17/18 19/19 19/19 Stejaree 45 0/18 0/20 6/20 8/20 15/20 18/19 20/20 19/19 Hapchunaengmi 0/18 0/20 4/20 8/20 10/20 18/19 20/20 19/19 Chuchung 0/18 0/19 5/20 9/20 18/19 19/19 20/20 20/20 Dongjin 0/20 6/20 6/20 12/17 17/18 17/18 20/20 19/19 Namyang21 0/19 10/16 12/20 16/18 18/18 19/19 19/19 20/20 Milyang23 0/19 13/18 17/17 17/17 17/17 19/19 19/19 20/20 Transformation S2 - - - - 23/34 - - - Transformation S15 - - - - 10/15 - - - Wild type - - - - 63/66 - - - * Number of forged seedlings / Number of cold-treated seedlings

Arabidobsid CBF1 In transgenic plants expressing OsAsr1  expression of cDNA

CRT / DRE sequence center is located in the promoter region of the estimated OsAsr1, the gene is controlled by CBF1. Thus, the present inventors have prepared a transgenic rice plant expressing Arabidopsis CBF1 cDNA. RNA gel-blot through the experiment, it was found that increasing the expression of genes in transgenic plants OsAsr1 (18-2 and 18-3) that strongly expressed the CBF1 transcription factor (Figure 8). The expression of OsAsr1 in transgenic plants was further increased by cold stress.

Salt Tolerance of Transgenic OsAsr1 Plants

The survival rate of the transformed plants at the seedling stage was measured by a wilting test. For accurate experiments, two natural rice varieties, Japonica varieties 'Dongjin' and Indica varieties 'Tachung Native' (TN1) were used to show salt tolerance. The 10 day old seedlings were transferred to a nutrient medium, 0.1% (w / v) Hyponex solution containing 200 mM NaCl. Sense transformed plants were treated for 1 day and antisense transformants at 30 ° C. for 12 hours, then transferred to fresh nutrient medium without NaCl and incubated for 2 days under normal conditions. When the three to four leaves of the fully forged plant showed obvious regression, forgery was determined after stress.

Salt-resistant varieties, TN1, had a wilting ratio of 36.1 ± 8.4% after recovery (Figure 9, Figure 10, Table 2). In contrast, the parental line of the OsAsr1 transformant of the invention, 'dongjin', showed fake symptoms in 66 out of 86 plants (77 ± 22.5%) two days after recovery. Of the 58 seedlings derived from spontaneous isolates, 39 seedlings (67 ± 4.1%) were forged two days after recovery. Two powerful overexpressors, S2 and S15, were used for the stress tolerance test. Each T3 line plant was a homozygous plant. Homozygous T3 transgenic plants were treated with salt stress, 1 day for sense plants and 12 hours for antisense plants. This salt-treatment time is referenced in the literature (Kim SH, PhD thesis, Sogang University, 2004). Of the 94 seedlings obtained from S2-1 plants, 26 seedlings (27 ± 11%) showed forgery. In addition, 27 of the 88 seedlings obtained from S2-2 plants (31 ± 8%) showed forgery. However, 33 seedlings (45 ± 22%) of the 73 seedlings from the S15-1 plant had such symptoms, and 34 seedlings (39 ± 5%) of the 88 seedlings from the S15-2 plant had forgery. Indicated. In contrast, the antisense transgenic plants were examined 12 hours after stress treatment, and the degree of forgery in A3-1 and A2-1 was 38/53 (72 ± 3%) and 30/67 (45 ± 23%), respectively. After 5 days of recovery, the counterfeit rate was 64/73 (73 ± 4%) TN1 plant, 86/86 (100 ± 0%) sinus plant, 58/58 (100 ± 0%) NT-S2 plant, 87/94 (93 ± 3%) S2-1 plant, 75/88 (85 ± 2%) S2-2 plant, 71/73 (97 ± 4%) S15-1 plant and 87/88 (99 ± 2%) S15- Increased to 2 plants. All experiments were repeated three times. Based on these results, it can be seen that overexpression of OsAsr1 confers salt resistance to rice, while the OsAsr1 antisense line shows increased salt sensitivity compared to natural form.

Salt Stress Tolerance of Rice Varieties and Transgenic OsAsr1 Rice line Forged number count forgery  ^a  (%) TN1 32 88 36 ± 8 easting 66 86 77 ± 3 NT-S2 39 58 67 ± 4 S2 -1 26 94 27 ± 11 S2 -2 27 88 31 ± 8 S15-1 33 73 45 ± 22 S15-2 34 88 39 ± 5 A3 -1 38 53 75 ± 3 A3 -2 30 67 45 ± 23 NT-S2 17 50 34 ± 4

^{a Number of} counterfeit plants / total number of plants (percentage of counterfeit plants). The number of counterfeit plants indicates that three to four leaves of the plant were forged upon 2 days recovery after salt stress treatment.

Transformation OsAsr1 Drying resistance of plants

Dry resistance of the transgenic OsAsr1 plants was evaluated. For accurate experiments, dry tolerance was shown using two native rice varieties, Sangnambatbyeo and Dongjin (Table 3, Figures 11 and 12). The 14-day-old seedlings were incubated in the growth chamber (30 ° C., light / dark cycle 16/8 hours) and no water was supplied for the next 4 days. T3 transgenic plants of the invention were dried for 4 days. When three to four leaves of completely forged plants exhibited apparent regression, forgery was determined after stress (Lee, SC, Huh, KW, An.K., An.G., and Kim, SR 2004b Ectopic expression of a cold-inducible transcription factor.CBF1 / DREB1b, in transgenic rice ( Oryza sativa L.) Mol. Cells 18: 107-114).

Of the 60 S2-1 seedlings, 11 seedlings (18 ± 12%) were forged. Of the 61 S2-2 seedlings, 15 seedlings (25 ± 10%) showed forgery. However, after 4 days of stress, 14 out of 51 plants (27 ± 13%) from 51 plants from S15-1 plants had such symptoms, and 17 out of 58 plants from 29 plants (29 ± 4%) from S15-2 plants. ) Forged. In addition, antisense transgenic plants were examined for dry resistance. Antisense plants A3-1 and A3-2 exhibited sensitivity of 41/45 (91 ± 12%) and 38/51 (75 ± 3%), respectively. All experiments were repeated three times. These data suggest that overexpression of OsAsr1 confers drying resistance to rice, while the OsAsr1 antisense line shows higher drying sensitivity compared to natural form.

Salt Stress Tolerance of Rice Varieties and Transgenic OsAsr1 Rice line Forged number  ^a count Counterfeit (%) SNB 25 57 44 ± 6 easting 64 100 64 ± 6 NT-S2 35 59 59 ± 3 S2 -1 11 60 18 ± 12 S2 -2 15 61 25 ± 10 S15-1 14 50 28 ± 13 S15-2 17 58 29 ± 4 A3 -1 38 53 90 ± 12 A3 -2 30 67 79 ± 3

^{The number of} WT and T3 plants that were completely forged when 3 to 4 leaves of WT and T3 plants recovered 7 days after dry stress treatment.

As mentioned above, the present invention provides abiotic stress-induced OsAsr1 protein that enhances resistance to cold, salt or dry stress. The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a non-biotic stress-induced OsAsr1 protein. The present invention provides a vector, a transformant. On the other hand, the present invention provides a transgenic plant with improved low temperature, salt or dry stress resistance and a method for producing the same. OsAsr1 genes and proteins of the present invention are very effective in enhancing resistance to low temperature, salt or dry stress in plants.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (8)

delete delete delete A vector comprising an OsAsr1 nucleic acid molecule represented by the nucleotide sequence of SEQ ID NOs: 67-480 in the nucleotide sequence of SEQ ID NO: 1. A transformant transformed by the vector of claim 4. A method for preparing a transgenic plant having enhanced salt or dry stress resistance comprising the following steps: (a) transforming a plant cell or plant tissue with the vector of claim 4; (b) selecting the transformed plant cells or plant tissues; And (c) regenerating the plant from the transformed plant cell or plant tissue to obtain a transformed plant. 7. The method of claim 6, wherein the plant is rice, barley, wheat or corn. A transgenic plant having enhanced resistance to salt or dry stress prepared by the method of claim 6 or 7.

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