KR101183112B1 - Mshsp23.3 gene from Medicago sativa and uses thereof - Google Patents

Abstract

The present invention relates to Mshsp23.3 ( Medicago sativa) related to abiotic stress resistance from alfalfa. Heat shock protein23.3) protein, gene encoding the protein, recombinant vector containing the gene, host cell transformed with the recombinant vector, and the recombinant vector are transformed into tobacco plant cells to abiotic stress of the tobacco plant A method for enhancing resistance, a transformed tobacco plant and seed thereof having enhanced abiotic stress resistance produced by the method, a method for producing a non-biological stress resistant transformed tobacco plant using the recombinant vector, and a tobacco comprising the gene A composition for enhancing the abiotic stress tolerance of plants.

Description

Mshsp23.3 gene from Medicago sativa and uses approximately}

The present invention relates to alfalfa-derived Mshsp23.3 protein and a gene encoding the same, and more particularly, to abiphalic stress-resistant Mshsp23.3-related Mshsp23.3 ( Medicago sativa). Heat shock protein23.3) protein, gene encoding the protein, recombinant vector containing the gene, host cell transformed with the recombinant vector, and the recombinant vector are transformed into tobacco plant cells to abiotic stress of the tobacco plant A method for enhancing resistance, a transformed tobacco plant and seed thereof having enhanced abiotic stress resistance produced by the method, a method for producing a non-biological stress resistant transformed tobacco plant using the recombinant vector, and a tobacco comprising the gene A composition for enhancing the abiotic stress tolerance of plants.

As the plants grow, they are exposed to various environmental stresses, and the yield and quality of the plants are significantly reduced by these environmental stresses. The development of disaster-resistant crops is an indispensable condition in order to prevent yield decreases and to maximize productivity.

Stress is abiotic stress caused by other organisms and abiotic stress caused by changes in the physical or chemical environment. It is very important to understand the ability of plants to adapt to environmental stress at the molecular level and to cultivate varieties that can maintain and improve the productivity of plants even under environmental stress conditions through molecular breeding. It is emerging as an alternative to overcome the limitations of disaster resistant crop development.

Recently, research on abiotic stresses has been actively conducted in plants, and various kinds of abiotic stress-inducing proteins, namely heat shock protein (HSP), have been identified and analyzed, and most of them function as molecular chaperones. (Chen et al, 1994, Biol Chem 269: 13216-13223; Vierling, 1991, Plant Mol Biol 42: 579-620; Waters et al, 1996, J Exp Bot 47: 325-338).

Among them, small HSPs, which are plant-specific HSPs, have been actively researched to separate small HSPs that function in various organs in cells (Chen et al, 1994, Biol Chem 269: 13216-13223; LaFayette et al, 1996, Plant Mol Biol 30: 159-169, Lee et al, 1995, J Biol Chem 270: 10432-10438; Osteryoung et al, 1994, J Biol Chem 269: 28676-28682; Vierling et al, 1988 , EMBO J 7: 575-581), transgenic plants have been reported to exhibit partial high temperature resistance following the introduction of these stress resistant genes (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 damaging factors in 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 degeneration and folding of the denatured protein at high temperatures to prevent irreversible aggregation of the protein, thereby obtaining resistance to stress. The largest amounts and types of proteins induced by stress in plants are small HSPs with a molecular weight of 15-30 kDa.

In the present invention, alfalfa ( Medicago) Mshsp23.3 cDNA clones were isolated by differential screening of cDNA libraries obtained from sativa L.). The cloned gene was induced in an abiotic stress resistant response and was expressed in Escherichia coli and found to be associated with increased resistance to high temperature, salt and heavy metal stress. Accordingly, the present inventors have made intensive studies to overcome the problems of the prior art, and as a result, have found that the Mshsp23.3 gene of the present invention is involved in plant abiotic stress resistance, thereby completing the present invention.

Korea Patent Registration No. 10-0599211 discloses abiotic stress resistance gene is disclosed that the Oslti32, is a stress tolerance gene OsMSRPK1 disclosed in Korea Patent Registration No. 10-0781075 call.

The present invention is derived by the request as described above, the present inventors have completed the present invention by confirming that the Mshsp23 .3 gene of alfalfa-derived up-regulation under abiotic stress treatment.

In order to solve the above problems, the present invention Alfalfa ( Medicago sativa ) -related abiotic stress tolerance Mshsp23.3 ( Medicago sativa Heat shock protein23.3) provides protein.

The present invention also provides a gene encoding the Mshsp23.3 protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a host cell transformed with the recombinant vector.

The present invention also provides a method of enhancing the abiotic stress resistance of tobacco plants by transforming the recombinant vector into tobacco plant cells.

In addition, the present invention provides a transformed tobacco plant and seed thereof having enhanced abiotic stress tolerance produced by the above method.

The present invention also provides a method for producing a non-biological stress resistant transformed tobacco plant using the recombinant vector.

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The alfalfa-derived Mshsp23.3 gene of the present invention has increased expression under the treatment of various abiotic stresses such as high temperature, salt and arsenic. Therefore, the Mshsp23.3 gene may be useful for developing transgenic plants that are highly resistant to abiotic stress.

Figure 1 shows the cDNA sequence of the Mshsp23.3 gene isolated from alfalfa.
Figure 2 shows the amino acid sequence deduced from the cDNA nucleotide sequence of the Mshsp23.3 gene of FIG.
Figure 3 is to isolate the whole RNA from alfalfa leaf tissues treated with high temperature, low temperature, salt and heavy metal (copper, cadmium and arsenic) stress to examine the expression of Mshsp23.3 gene isolated from alfalfa and performed a northern blot analysis Results are shown.
Figure 4 shows a photograph analyzed by protein electrophoresis (SDS-PAGE) by expressing a large amount of recombinant protein containing the entire Mshsp23.3 gene isolated from alfalfa in E. coli.
Figure 5 shows a graph comparing the analysis of resistance to high temperature stress in E. coli and E. coli expressing Mshsp23.3 protein.
Figure 6 shows a graph of comparative analysis of resistance to salt stress in E. coli and E. coli expressing Mshsp23.3 protein.
7 shows a graph comparing the resistance to arsenic stress in E. coli expressing the vector E. coli and Mshsp23.3 protein.
8 shows a schematic diagram showing a plant expression vector (pMshsp23.3) for expressing the Mshsp23.3 gene of alfalfa of the present invention.
9 is a comparison of resistance to high temperature, salt and arsenic stress of transformants (Tg-1, Tg-2) and non-transformers (NT) of Mshsp23.3 gene isolated from alfalfa in tobacco plants as model plants Show the analyzed picture.

In order to achieve the object of the present invention, the present invention is composed of the amino acid sequence represented by SEQ ID NO: 2, alfalfa ( Medicago sativa ) -related abiotic stress tolerance Mshsp23.3 ( Medicago sativa Heat shock protein23.3) provides protein.

The range of Mshsp23.3 proteins according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 isolated from alfalfa and functional equivalents of the proteins. 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 homogeneous physiological activity" means activity that enhances the abiotic stress tolerance of a plant.

The invention also includes fragments, derivatives and analogues of the Mshsp23.3 protein. As used herein, the terms “fragment”, “derivative” and “analogue” refer to a polypeptide having biological function or activity substantially the same as the Mshsp23.3 polypeptide of the invention. Fragments, derivatives, and analogs of the present invention comprise (i) polypeptides substituted with one or more conservative or nonconservative amino acid residues (preferably conservative amino acid residues), wherein the substituted amino acid residues are encoded by a genetic code. Or (ii) a polypeptide having substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of a polypeptide, such as polyethylene glycol) A polypeptide derived from a bound mature polypeptide, or (iv) an additional amino acid sequence (eg, a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) and It may be a polypeptide derived from said polypeptide bound. Such fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

The present invention also provides a gene encoding the Mshsp23.3 protein. The gene of the present invention may be DNA or RNA encoding Mshsp23.3 protein. DNA includes cDNA, genomic DNA or artificial synthetic DNA. DNA can be single stranded or double stranded. The DNA may be a coding strand or a non-coding strand.

 Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

Polynucleotides encoding a mature polypeptide represented by SEQ ID NO: 2 include a coding sequence encoding only a mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences encoding 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 invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequences as described herein, or fragments, analogs, and derivatives thereof. Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitutional variants, deletional variants, and insertional variants. As is known in the art, an allelic variant is an alternative to a polynucleotide, which may comprise one or more substituted, deleted or inserted nucleotides and which does not result in a substantial functional change in the polypeptide encoded by the variant Do not.

In addition, the present invention provides a poly-hybridized hybridization with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with a nucleotide sequence of SEQ ID NO. It relates to nucleotides. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" are (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 represented by SEQ ID NO: 2.

In accordance with an embodiment of the invention, it should be understood that the Mshsp23 .3 gene is preferably alfalfa derived. However, embodiments of the present invention are highly homologous to the alfalfa Mshsp23 .3 gene (eg, at least 60%, ie 70%, 80%, 85%, 90%, 95%, even 98% sequence identity). And 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.

The present invention also provides a recombinant vector comprising the gene according to the invention Mshsp23 .3.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. 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 Mshsp23.3 protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, 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 Mshsp23.3 protein-encoding DNA sequence and appropriate transcriptional / translational control signals can be constructed by methods well known to those of skill in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. 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. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the 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, and a pCAMBIA vector.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

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

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Octopine gene terminator, 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 of the present invention. The host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains.

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

The method of carrying the vector of the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation 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 transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. Method is 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), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. 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 EP A 120 516 and U.S. Pat. No. 4,940,838.

The present invention also provides a method for enhancing the abiotic stress resistance of tobacco plants, comprising the step of transforming the recombinant vector to tobacco plant cells overexpressing the Mshsp23.3 gene. Preferably, the abiotic stress may be, but is not limited to, high temperature, salts or heavy metals. In addition, the heavy metal is preferably arsenic, but is not limited thereto.

In addition, the present invention provides a transformed tobacco plant and seed thereof having enhanced abiotic stress tolerance produced by the above method.

In addition, the present invention comprises the steps of transforming tobacco plant cells with the recombinant vector; And

It provides a method for producing a non-biological stress-resistant transformed tobacco plant comprising the step of regenerating the plant from the transformed tobacco plant cells.

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

Transformed tobacco plant cells should be re-differentiated 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 present invention also provides a composition for enhancing the abiotic abiotic stress resistance of tobacco plants, comprising the Mshsp23 .3 gene of the invention. In the composition of the present invention, the Mshsp23 .3 gene may be composed of the nucleotide sequence of SEQ ID NO: 1. The compositions of the present invention as an active ingredient comprising a Mshsp23 .3 gene of alfalfa origin, which is transformed by the Mshsp23 .3 gene in tobacco plants and to promote the abiotic stress tolerance of the tobacco plant.

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

Example  One: Alfalfa  Induced by high temperature stress Mshsp23 .3 nucleotide and amino acid sequence analysis of genes

Poly (A +) mRNA was extracted from stress and unstressed leaf tissues of alfalfa grown over 3 weeks in plant growth, and fragments expressed only by high temperature stress according to the manufacturer's recommendation method using PCR Select cDNA Subtraction (Clontech). CDNA was obtained. The obtained cDNA was cloned into the subcloning vector pCR2.1-TOPO vector (invitrogen), plasmid DNA was isolated using a Plasmid purification kit (COSMO), and sequencing was performed by Cosmojintech.

Comparative analysis of sequences was performed by the DNASIS and BLAST programs provided in the NCBI, GenBank, EMBL and SwissProt databases. Based on the nucleotide sequence results, the entire nucleotide sequence was secured through RACE PCR. As a result, it was confirmed that the cDNA of the Mshsp23.3 gene induced by high temperature stress was composed of a total of 630 bp sequences as shown in FIG. 1. 209 amino acids were deduced by the nucleotide sequence of FIG. 1, and the results are shown in FIG. 2. The base sequence and deduced amino acid sequence was found to be a new gene induced by abiotic stress by database search.

Example  2: high temperature, low temperature, salt and heavy metal stress treatment Mshsp23 .3 gene expression investigation

Alfalfa grown for 3 weeks or longer on plant growth was subjected to high temperature (42 ° C), low temperature (4 ° C), salt (300mM NaCl) and 300μM heavy metals (copper, cadmium and arsenic) stress treatment at different times, and then the leaf tissues were sampled. Total RNA was isolated using TRIzol (invitrogen). The whole RNA was electrophoresed in a 1.2% formaldehyde gel, and then the gel was transferred to a nylon membrane (Amersham) with 20 × SSC solution (3 M NaCl, 0.3 M Sodium citrate) and radioisotope [α Northern blot analysis was performed using 630 bp full-length cDNA of the Mshsp23.3 gene labeled with -32P] dCTP as a probe.

As a result, it was confirmed that Mshsp23.3 transcript strongly induced after 12 hours of high temperature and salt stress treatment in alfalfa leaf tissues, and the amount of transcript was decreased after 12 hours of low temperature stress treatment (FIG. 3). In addition, it was confirmed that Mshsp23.3 transcript strongly induced 6 hours after arsenic treatment, in addition to the amount of transcript weakly expressed by copper and cadmium (Fig. 3).

Example  3: E. coli expression Vector  Expression of Recombinant Fusion Proteins

In order to determine the expression of the protein expressed in E. coli, pET28a (Invitrogen) in the E. coli expression vector was used. 5'-Mshsp23.3 primer (5'-ATGGCTTCTTCTGTTGCAGTC-3 '; SEQ ID NO: 3) and 3'-Mshsp23.3 primer (5'-CTACTCAACATTGACATTAATCACATC-3) using the Mshsp23.3 gene cDNA isolated in Example 1 After PCR amplification with SEQ ID NO: 4), the generated PCR product was PCR cloned into the pET28a vector to confirm whether the C-terminal fusion of His-tag was performed by DNA sequencing.

To express the alfalfa Mshsp23.3 gene in Escherichia coli, the transfer vector pET28a-Mshsp23.3 was inserted into E. coli BL21 (DE3) pLysS cells at a temperature shock (42 ° C). Inserted Escherichia coli cells were incubated for 1 hour in LB (Luria-Bertani) liquid medium containing 75 μg / mL of Duchafa, and then plated in LB-Amp solid medium for at least 16 hours at 37 ° C. The cultured cells were cultured to OD 0.6 (1.0 × 10 ⁶ cells) in LB-Amp 50 mL liquid medium, and then divided into groups without addition of 1 mM IPTG and groups added thereto, and then cultured at 28 ° C. for 3 hours. . After incubation, the cells were collected by centrifugation at 3,000 rpm for 5 minutes, and then 1.7 mL of 50 mM Tris-HCl (pH 8.0) buffer was added and the cells were disrupted by using a cell crusher. It was then centrifuged at 5,000 rpm for 5 minutes. After centrifugation, the supernatant was separated and again centrifuged at 12,000 rpm for 10 minutes, and the water-soluble protein was isolated to perform 12% SDS-protein electrophoresis (Laemmli, Nature, vol 227, p680-685, 1970) to perform recombinant fusion His Mshsp23.3 protein expression was analyzed (FIG. 4).

As a result of all the above, His-Mshsp23.3 protein was efficiently induced in Escherichia coli through the His-tag fusion protein expression system, and abiotic resistance investigation could be performed later.

Example  4: Mshsp23 .3 High Temperature Resistance Investigation of Proteins

In order to measure the high temperature resistance of alfalfa-derived Mshsp23.3 protein, E. coli with the vector inserted and E. coli expressing Mshsp23.3 protein were added to OD 0.6 in LB-Amp 75 mL liquid medium as in the method of Yeh et al. (2002). After incubation, 1 mM IPTG was added and incubated at 28 ° C. for 2 hours. Each E. coli induced by IPTG was treated at 55 ° C. for 0, 5, 10, 15, 30, 45 and 60 minutes to analyze the viability of E. coli (FIG. 5).

As a result, E. coli and Mshsp23.3-incorporated Escherichia coli showed survival rates of 43% and 78%, respectively. In the group treated with high temperature stress for 30 minutes, E. coli and Vector E. coli introduced with Mshsp23.3 showed survival rates of 19% and 39%, respectively. In addition, E. coli introduced with vector Escherichia coli and Mshsp23.3 showed 1% and 18% survival, respectively, in the group treated with high temperature stress for 60 minutes. Therefore, E. coli expressing His-Mshsp23.3 was found to have a much higher survival rate. These results indicate that Mshsp23.3 protein expression enhances the resistance of E. coli to high temperature stress.

Example  5: Mshsp23 .3 investigation of salt and arsenic resistance of proteins

To determine the salt and arsenic resistance of alfalfa-derived Mshsp23.3 protein, a salt was added to each of the vector Escherichia coli and the E. coli expressing Mshsp23.3 protein at IPTG-induced concentrations of 1, 3, 5, and 7 mM. After treatment for minutes, the survival rate of Escherichia coli was analyzed (FIG. 6). In addition, arsenic was added in 0.01, 0.1, 1, 5, and 10 mM concentrations in the same manner, and then treated for 30 minutes to analyze the survival rate of E. coli (FIG. 7). As a result, it was found that the reduction of survival rate by the addition of salt and arsenic was more prominent in E. coli expressing Mshsp23.3 than in E. coli. Thus, it was found that Mshsp23.3 protein expression enhances the stress resistance of E. coli against salt and arsenic stress.

Example  6: Mshsp23 .3 Investigation of Tolerance of Tobacco Plants to High Temperature, Salt and Arsenic Stress by Protein Expression

The transformation techniques applied are described in Dane K. Fisher and Mark J. Guiltinan. "Plant Molecular Biology Reporter" pp. 278-289, 1995, which is based on the Agrobacterium gene delivery system. In an embodiment of the present invention, we used the plant transformation vector pCAMBIA1300-Mshsp23.3 system under the control of the CaMV 35S promoter (FIG. 8).

MS medium treated at high temperature (52 ° C.) for 30 minutes to analyze resistance to high temperature, salt and arsenic of transformed tobacco plants (Tg-1, Tg-2) using Mshsp23 .3 gene, 250 The transformed tobacco plants were incubated for 3 weeks in MS medium supplemented with mM NaCl or 300 μM arsenic, respectively. After incubation, the growth of the tobacco plants was examined. After high temperature treatment, all non-transformed (NT) plants were killed, but Mshsp23.3 plants showed stronger resistance than non-transformed (NT) plants. In addition, growth of non-transformed (NT) plants in the salt or arsenic-treated medium was significantly inhibited, but Mshsp23.3 plants were slightly inhibited, showing differences between lines (FIG. 9). These results show that the expression of Mshsp23.3 protein enhances the resistance of tobacco plants to high temperature, salt and arsenic stress.

In the above described and illustrated with respect to the preferred embodiment of the present invention, the present invention is not limited to the above specific embodiment, those skilled in the art various modifications without departing from the gist of the present invention It will be understood that such modifications are within the scope of the claims of the present invention.

Attach an electronic file to a sequence list

Claims (11)

Medicago , consisting of the amino acid sequence of SEQ ID NO: 2 sativa ) -related abiotic stress tolerance Mshsp23.3 ( Medicago sativa Heat shock protein23.3) protein. A gene encoding the protein of claim 1. 3. The gene according to claim 2, wherein the gene consists of the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 4. The method of claim 4, wherein the recombinant vector of claim 4 is transformed into tobacco plant cells, thereby over-expressing the Mshsp23.3 gene. delete A transgenic tobacco plant with enhanced high temperature, salt, or heavy metal stress resistance prepared by the method of claim 6. Seeds of plants according to claim 8. Transforming tobacco plant cells with the recombinant vector of claim 4; And
A method for producing a high temperature, salt, or heavy metal stress resistant transformed tobacco plant comprising the step of regenerating a plant from the transformed tobacco plant cells. delete

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