KR101630839B1 - DNAJ like protein from medicago sativa and uses thereof - Google Patents

Abstract

The present invention relates to a method for enhancing abiotic stress tolerance, comprising the step of overexpressing an MsDNAJ protein derived from an aliphatic stress tolerance-related alfalfa ( Medicago sativa L.), a gene encoding the same, and a MsDNAJ gene and overexpressing the same in a plant . The MsDNAJ gene of the present invention increases expression in various abiotic stress environments such as high temperature, low temperature, salt, drying, or heavy metals. Therefore, the MsDNAJ gene can be used for molecular sowing of herbaceous plants and can be useful for the development of transformants having strong abiotic stress tolerance. In addition, the present invention can greatly contribute to increase in productivity of crops or mass production of useful components.

Description

DNAJ-like proteins from alfalfa and their uses {DNAJ like protein from medicago sativa and uses thereof}

The present invention relates to a gene which confers resistance to various abiotic stresses, and more particularly to a method for enhancing tolerance to abiotic stress using a specific gene or protein.

Stress has biotic stresses caused by other organisms and abiotic stresses caused by changes in the physical or chemical environment. It is very important to understand the ability of plants to adapt to environmental stresses at the molecular level and cultivate varieties capable of maintaining and improving plant productivity under environmental stress conditions through molecular breeding. It is emerging as an alternative to overcome the limitation of development of disaster tolerant crops.

Recently, studies on abiotic stresses have been actively conducted in plants and identification and functional analysis of various kinds of abiotic stress inducible proteins such as heat shock protein (HSP) Waters et al., 1996, J Exp Bot 47: 325-338), which has been shown to function as a protein (Chen et al, 1994, Biol Chem 269: 13216-13223; Vierling, 1991; Plant Mol Biol 42: 579-620; .

Particularly, studies on small HSPs (low molecular weight HSPs), which are plant-specific HSPs, have been actively conducted and small HSPs functioning in various organs in cells have been isolated (Chen et al., 1994, Biol Chem 269: 13216-13223; Osteryoung et al., 1994, J Biol Chem 269: 28676-28682; Vierling et al, 1988; Plant et al., 1996; Plant Mol Biol 30: 159-169; Lee et al, 1995; J Biol Chem 270: 10432-10438; , EMBO J 7: 575-581), and transgenic plants showing partial high temperature tolerance due to the introduction of these stress tolerance genes have been reported (Lee et al, 1996, Plant J 8: 603-612; Waters et al, 1996, J Exp Bot 47: 325-338).

Abiotic environmental stress is one of the most critical damages to plants. Under stress, the expression of most Housekeeping genes is stopped and the gene expression system necessary for HSP synthesis is activated to synthesize HSPs having various molecular weights. These HSPs help prevent protein denaturation at high temperatures and folding of denatured proteins, thereby preventing irreversible aggregation of the proteins and thereby acquiring resistance to stress.

Korean Patent No. 10-1183112 discloses the Mshsp23.3 gene derived from alfalfa, which is an abiotic stress tolerance gene.

It is an object of the present invention to provide a composition comprising a protein that is active in various abiotic environmental stresses or a gene encoding the same.

Another object to be solved by the present invention is to provide a method for enhancing resistance to abiotic stress by transforming the gene.

The inventors of the present invention have made intensive researches for discovering novel genes related to abiotic stress tolerance and have found that the DNAJ-like gene derived from alfalfa ( medicago sativa ) of the present invention is involved in abiotic stress resistance of plants Thus, the present invention has been completed.

In the present invention, DNAJ-like cDNA clones were isolated by differential screening of cDNA libraries obtained from alfalfa. This cloned gene showed a difference in expression of genes during high temperature, low temperature, salt, drying, and heavy metal stress treatment in abiotic stress tolerance reaction, which is induced by abiotic stress, .

The present invention provides a composition for promoting abiotic stress tolerance of a plant comprising the MsDNAJ protein represented by the amino acid sequence of SEQ ID NO: 2. Also, the present invention provides a composition for promoting abiotic stress tolerance of a plant comprising the MsDNAJ gene represented by the nucleotide sequence of SEQ ID NO: 1.

The composition of the present invention comprises an MsDNAJ protein or a gene encoding the MsDNAJ protein as an active ingredient. The composition comprising the gene represented by the nucleotide sequence of SEQ ID NO: 1 can increase the abiotic stress tolerance of the microorganism or the plant by transforming the MsDNAJ gene.

The range of the MsDNAJ protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous bioactivity" means an activity promoting abiotic stress tolerance.

The invention also includes fragments, derivatives and analogues of the MsDNAJ protein. As used herein, the terms "fragments", "derivatives" and "analogs" refer to polypeptides that possess substantially the same biological function or activity as the MsDNAJ polypeptides of the invention. The fragments, derivatives, and analogs of the present invention may be used in conjunction with (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted (the substituted amino acid residues are encoded (Ii) a polypeptide having a substituent (s) at one or more amino acid residues, (iii) binding to another compound (a compound capable of extending the half-life of the polypeptide, for example, polyethylene glycol) (Iv) a polypeptide derived from a mature polypeptide that is coupled to an additional amino acid sequence (e. G., A leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) Lt; / RTI > polypeptide. Such fragments, derivatives and analogs as defined in the present invention are well known to those skilled in the art.

The gene of the present invention may be a gene represented by SEQ ID NO: 1 or DNA or RNA encoding MsDNAJ protein. DNA includes cDNA, genomic DNA, or artificial synthetic DNA. The DNA may be single stranded or double stranded. The DNA may be a coding strand or a non-coding strand.

A polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 encodes only a mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences coding for noncoding sequences.

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides comprising the same amino acid sequences as above, or fragments, analogs and derivatives thereof. Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. The nucleotide mutant includes a substitution mutant, a deletion mutant, and an insertion mutant. As is known in the art, allelic variants are alternatives to polynucleotides, which may include one or more substituted, deleted or inserted nucleotides, and do not result in substantial functional changes in the polypeptide encoded by the variant.

The present invention also relates to a polynucleotide which hybridizes with a nucleotide sequence of SEQ ID NO: 1 described above and a nucleotide sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with the nucleotide sequence of SEQ ID NO: 1 described above Lt; / RTI > nucleotides. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" means (1) hybridization and washing under lower ionic strength and higher temperature such as 0.2 x SSC, 0.1% SDS, 60 ° C; Or (2) in the presence of a denaturant such as 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll and 42 ° C; Or (3) at least 80%, preferably at least 90%, more preferably at least 95%. In addition, the biological function and activity of the polypeptide encoded by the hybridizable polynucleotide is the same as the biological function and activity of the mature polypeptide of SEQ ID NO: 2.

Preferably, the MsDNAJ gene of the present invention is derived from alfalfa. However, embodiments of the present invention have high homology (e.g., greater than 60%, i.e., 70%, 80%, 85%, 90%, 95%, even 98% sequence identity) to the alfalfa -derived MsDNAJ gene But also other genes derived from other plants. Methods for sequencing, and means for determining sequence identity or homology (e.g., BLAST) are well known in the art.

Also, the present invention provides a composition for promoting abiotic stress tolerance of a plant, which comprises a recombinant vector comprising a gene represented by the nucleotide sequence of SEQ ID NO: 1 or a gene encoding the MsDNAJ protein. The term "recombinant " as used herein refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

In the present invention, the polynucleotide sequence encoding the MsDNAJ protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors comprising the MsDNAJ protein-encoding DNA sequence and appropriate transcription / translation control signals can be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to a plant cell when present in a suitable host, such as Agrobacterium tumefaciens. Another type of Ti-plasmid vector (see European Patent Registration No. 0116718) is currently used to transfer hybrid DNA sequences to plant cells, or to protoplasts in which new plants capable of properly inserting hybrid DNA into the genome of a plant can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in European Patent No. 0120516 and US Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly. Preferably, the recombinant vector may be, but is not limited to, a pKBS1-1 vector, a pBI101 vector, a pCAMBIA vector, or a pET41a (+) vector.

The expression vector may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. For example, herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, and the like. Antibiotic resistance genes, but are not limited thereto.

In the recombinant vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, SWPA2 or a histone promoter But is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

In the recombinant vector of the present invention, a conventional terminator can be used, for example, nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, or Agrobacterium and the terminator of the Octopine gene of Tumefaciens, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a host cell transformed with the recombinant vector. As stabilize the vector of the invention in prokaryotic host cells capable of continuous cloning and expression may also take advantage of any host cells known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E.coli W3110, Bacillus subtilis, Bacillus strains such as Bacillus Chuo ringen sheath, and Salmonella typhimurium, Serratia Marseille CL and And various enterococci such as Pseudomonas species and strains.

When the vector of the present invention is transformed into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, human cells (for example, Chinese hamster ovary, W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells. The host cell may preferably be a microorganism or a plant cell.

The method of delivering the vector of the present invention into a host cell can be carried out by the CaCl ₂ method, one method (Hanahan, D., J. MoI. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell, , And an electric drilling method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

Transformation of a plant means any method of transferring DNA to a plant. Such transfection methods do not necessarily have a regeneration or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), the infiltration of plants or the transformation of mature pollen or vesicles into Agrobacterium tumefaciens Infection by viruses (non-integrative) in virus-mediated gene transfer (European Patent No. 0301316) and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in European Patent Publication No. 120516 and US Patent No. 4,940,838.

In addition, the present invention provides a method for enhancing tolerance to abiotic stress of a plant, comprising the step of transfecting the recombinant vector into cells and overexpressing the MsDNAJ gene.

The object to be transfected may be any conventional microorganism or plant, but is not limited to its kind. Preferably, the plant is selected from the group consisting of orchard grass, tall fescue, perennial rice, creeping bent grass, Timothy, Italian Ryegrass, Kentucky Bluegrass, Red Top, Reed Canary Grass, Bermuda Grass, or Bahia Grass; Alfalfa, buzzfoot trefoil, white clover, or red clover; Or a feed crop such as corn, sorghum, barley, rye, and the like, but is not limited thereto.

The abiotic stress may include, but is not limited to, high temperature, low temperature, salt, drying, or heavy metal stress. In addition, the heavy metal may preferably be copper, cadmium, or arsenic.

In addition, the present invention provides a transgenic plant or a seed thereof, wherein the MsDNAJ gene is overexpressed and the resistance against abiotic stress is enhanced.

Also, the present invention provides a method for producing a plant cell, comprising the steps of: transforming a plant cell with the recombinant vector; And regenerating the plant from the transformed plant cell. The present invention also provides a method for producing a transgenic plant having enhanced resistance to abiotic stress.

The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tyumeo Pacific Enschede may be mediated by (Agrobacterium tumefiaciens). In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any method known in the art can be used to regenerate transgenic plants from transformed plant cells.

Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989, Momillan, N. Y.).

The alfalfa-derived MsDNAJ gene of the present invention has an increased expression in various abiotic stress environments such as high temperature, low temperature, salt, drying, or heavy metals. Thus, the MsDNAJ gene can be used to develop transgenic plants which are used in molecular breeding and have strong abiotic stress tolerance.

Fig. 1 shows the nucleotide sequence and amino acid sequence of MsDNAJ gene derived from alfalfa.
Figure 2 shows the amino acid sequence of MsDNAJ from alfalfa in comparison with the DNAJ amino acid sequence from Medicago truncatula , Cicer arietinum , Glycine max , Phaseolus vulgaris , and Vitis vinifera .
Fig. 3 shows the results of phylogenetic analysis of alfalfa-derived MsDNAJ protein with Medicago truncatula , Cicer arietinum , Glycine max , Phaseolus vulgaris , and grape ( Vitis vinifera ).
FIG. 4 shows the Northern blot analysis of the expression of the MsDNAJ gene according to various stress conditions and time.
Figure 5 shows the constructs of the pCAMBIA1300 vector.
Figure 6 is a linear representation of the final structure map in which MsDNAJ was transformed into the pCAMBIA1300 vector.
FIG. 7 shows the results of comparing the growth of plants transformed with MsDNAJ in a salt, dry, or heavy metal stress environment with the control group.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1: Sequence and amino acid sequence analysis of MsDNAJ gene

The seeds of alfalfa used in this example were obtained from National Livestock Academy as a vernal variety. The alfalfa seeds were planted in pots containing a horiculture nursery medium (Biomedia) and grown in a growth chamber at 25 ° C for 14 hours at a light intensity of 400 μmol m ^-2 s ^-1 .

4 weeks old oil plants were exposed for 6 hours under high temperature of 42 ℃ in the growth phase, and the control group was exposed to room temperature at 25 ℃. After each treatment, leaves were harvested and frozen by liquid nitrogen. Total RNA was isolated from treated and control plant leaves using TRIzol reagent (Qiagen, CA, USA).

GeneFishing ^TM DEG kit (purchased from Seegene) was used to obtain cDNA fragments expressed only by high temperature stress at 42 ° C. according to the manufacturer's recommendation method and Lee et al. (2012). The obtained cDNA was cloned into a subcloning vector, pCR2.1-TOPO vector (purchased from Invitrogen), and the plasmid DNA was isolated using a plasmid purification kit (purchased from COSMO) TEC.

Nucleic acid, amino acid sequences, and conserved domains are reported in NCBI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and Bioedit (version 7.2.3) software (Hall, 1999) Lt; / RTI > Based on the nucleotide sequence, the entire nucleotide sequence was obtained by RACE PCR.

As a result, the cDNA of the MsDNAJ gene induced by high temperature stress was composed of 498 bp nucleotide sequence as shown in Fig. 1, and 166 amino acids were deduced by the nucleotide sequence shown in Fig. The nucleotide sequence and deduced amino acid sequence have been found to be novel genes induced by abiotic stress by database search.

Example 2 Molecular Genetic Analysis of MsDNAJ Gene

In order to clarify to which class MsDNAJ belongs, as shown in Fig. 2, DNAJ from Medicago truncatula , Cicer arietinum , Glycine max , Phaseolus vulgaris , Vitis vinifera , Amino acid sequence and phylogenetic analysis were performed. Other plant-derived DNAJ proteins were searched from the GenBank ^TM database after searching for similarity using the blastp in NCBI-BLAST. Multiple sequence alignments were performed using ClustalX2 (Larkin et al., 2007) and GeneDoc (version 2.7) software (Nicholas and Nicholas, 1997).

The scheme was constructed using the algorithm from the ClustalW2 program on EMBL-EBI ( http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/ ). MsDNAJ showed high similarity to DNAJ of the legume ( Medicago truncatula ) (Fig. 3).

Example 3: Expression of MsDNAJ gene according to environmental conditions

To analyze the expression of MsDNAJ, 4-week-old alfalfa plants were treated with high temperature (42 캜), low temperature (4 캜), salt (300 mM NaCl) or dry (irrigation stop) stress, ₄ , 500 μM), cadmium (CdCl ₂ , 500 μM), and arsenic (Na ₂ HAsO ₄ .7H ₂ O, 500 μM). For high or low temperature treatments, the oil plants were transferred to a temperature-controlled growth phase at 42 ° C or 4 ° C. For the treatment of salts or heavy metals, the oil plants were irrigated with water containing the above concentrations of salts or heavy metals. The stressed plants were harvested, cooled with liquid nitrogen and stored at -80 ° C until use.

Northern blot analysis

To investigate the transcriptional expression of MsDNAJ gene in response to various abiotic stresses, total RNA was isolated using TRIzol (purchased from Invitrogen) by sampling the stressed leaf tissue with time. The separated total RNA was electrophoresed on 1.2% formaldehyde gel, and the gel was transferred to a nylon membrane (purchased from Amersham) with a 20 x SSC solution (3 M NaCl, 0.3 M sodium citrate), and the radioactive isotope Northern blot analysis was performed using the full-length cDNA of the MsDNAJ gene labeled with [[alpha] -32P] dCTP as a probe.

In the alfalfa leaf tissues, strong induction of MsDNAJ transcript was observed 12 hours after the high temperature and low temperature stress treatment, and the transcriptional body was decreased from 24 hours after the high temperature and low temperature stress treatment. In the case of salt and dry treatment, the MsDNAJ transcript was found to be gradually decreased from 1 hour after the stress treatment. In addition, in the alfalfa root tissue, the transcriptional body gradually decreased after treatment with arsenic, copper, or cadmium (Fig. 4).

These results indicate that MsDNAJ plays a major role in tolerance to abiotic stresses such as high temperature, low temperature, salt, drying, or heavy metals.

Example 4: Resistance investigation using model plant tobacco for high temperature, low temperature, salt, drying, or heavy metal stress according to expression of MsDNAJ protein

The transformation technique used in this example is described by Dane K. Fisher and Mark J. Guiltyinan. "Plant Molecular Biology Reporter" pp. 278-289, 1995). ≪ / RTI > To transform the MsDNAJ gene into model plants, the plant transformation vector pCAMBIA1300-MsDNAJ system was used under the control of the CaMV 35S promoter (FIGS. 5 and 6).

In order to analyze resistance to salt, dry, or heavy metal stress of the transformed plants using the MsDNAJ gene, the transformed plants were cultured in MS medium supplemented with 250 mM NaCl, 20% PEG, or 300 μM arsenic, Lt; / RTI > After the cultivation, the growth of the plant was examined (Fig. 7).

In the medium treated with salt, there was a slight difference between the lines but the growth of the non - transformant was suppressed and tended to decline, but the growth of the pre - plant was less inhibited. In the medium treated with 20% PEG, the transformed plants were less inhibited in leaf growth than non-transformants. In addition, all of the non - transgenic plants died after treatment with arsenic, but the transgenic plants showed more resistance than the non - transgenic plants.

From these results, it was found that the expression of MsDNAJ protein promotes plant resistance to salt, dryness, or arsenic stress.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, And such modified embodiments are within the scope of the claims of the present invention.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> DNAJ like protein from medicago sativa and uses thereof <130> P14-282 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 498 <212> DNA <213> MsDNAJ from Medicago sativa L. <400> 1 atgctttctt caatatctct cccttcttct ctctacgctg ccggtaactt caccggcacc 60 gctgtgacac caccgccgtg ccgtgtcaga tcgaagccga tagtcatcac ctgcagcgct 120 actgcaaccg cggaagctcg ctctgcatgg acagaacaac aaagaccttc atatcttaac 180 atgaactctt cttctcactc ttctccagca tcactttacc agattctcgg tatatcagcg 240 ggtgcttcta atcaagaaat caaggcagcc taccggcgac ttgctagagt ctgccatcct 300 gatgtggcgg cgattgatcg gaaaaactca tcggcggatg atttcatgaa gattcaatct 360 gcatactcta cactctccga tccagataaa cgtgctaact atgatcggag tcttttccgg 420 caacaacggc ggccgttgtc gtcgatggtg tcttccggct actcgagtcg gaaatgggaa 480 acggatcagt gttggtag 498 <210> 2 <211> 165 <212> PRT <213> MsDNAJ from Medicago sativa L. <400> 2 Met Leu Ser Ser Ile Ser Leu Pro Ser Ser Leu Tyr Ala Ala Gly Asn   1 5 10 15 Phe Thr Gly Thr Ala Val Thr Pro Pro Pro Cys Arg Val Val Ser Ser              20 25 30 Pro Ile Val Ile Thr Cys Ser Ala Thr Ala Thr Ala Glu Ala Arg Ser          35 40 45 Ala Trp Thr Glu Gln Gln Arg Pro Ser Tyr Leu Asn Met Asn Ser Ser      50 55 60 Ser His Ser Ser Ala Ser Leu Tyr Gln Ile Leu Gly Ser Ser Ala  65 70 75 80 Gly Ala Ser Asn Gln Glu Ile Lys Ala Ala Tyr Arg Arg Leu Ala Arg                  85 90 95 Val Cys His Pro Asp Val Ala Ala Ile Asp Arg Lys Asn Ser Ser Ala             100 105 110 Asp Asp Phe Met Lys Ile Gln Ser Ala Tyr Ser Thr Leu Ser Asp Pro         115 120 125 Asp Lys Arg Ala Asn Tyr Asp Arg Ser Leu Phe Arg Gln Gln Arg Arg     130 135 140 Pro Leu Ser Ser Met Val Ser Ser Gly Tyr Ser Ser Arg Lys Trp Glu 145 150 155 160 Thr Asp Gln Cys Trp                 165

Claims (7)

A MsDNAJ protein represented by the amino acid sequence of SEQ ID NO: 2,
Salt, famine, and heavy metal. A MsDNAJ gene represented by the nucleotide sequence of SEQ ID NO: 1,
Salt, famine, and heavy metal. A recombinant vector comprising the gene of claim 2 or a gene encoding the protein of claim 1,
Salt, famine, and heavy metal. A host cell transformed with a recombinant vector comprising the gene of claim 2 or a gene encoding the protein of claim 1. Transfecting a plant cell with a recombinant vector comprising the gene of claim 2 or a gene encoding the protein of claim 1, and overexpressing the MsDNAJ gene,
Salt, famine, and heavy metals. A recombinant vector comprising the gene of claim 2 or a gene encoding the protein of claim 1 is transformed into a plant cell to overexpress the MsDNAJ gene,
Wherein the stress tolerance of the transgenic plant is enhanced by at least one of the group consisting of a salt, a famine, and a heavy metal. Transforming a plant cell with a recombinant vector comprising the gene of claim 2 or a gene encoding the protein of claim 1; And regenerating the plant from the transformed plant cell.
Wherein the tolerance to stress is enhanced by a method selected from the group consisting of a salt, a famine, and a heavy metal.

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