KR101630840B1 - Eshsp16.9 gene and uses thereof - Google Patents

Abstract

The present invention relates to an abiotic stress tolerance-related EsHsp16.9 ( Elymus sibiricus L. Heat shock protein 16.9) protein or a gene encoding it and a method for promoting tolerance to abiotic stress of a plant. The Eshsp16.9 gene of the present invention increases expression in a variety of abiotic stress environments such as salts, famines, or heavy metals. Thus, the Eshsp16.9 gene can be used to develop transgenic plants that are resistant to abiotic stress by being used for molecular sowing of grasses.

Description

EsHsp16.9 gene and its use {Eshsp16.9 gene and uses thereof}

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

As the plant grows, it is exposed to various environmental stresses, and the quantity and quality of the plants are remarkably deteriorated by such environmental stress. Development of disaster tolerant crops is an indispensable condition in order to prevent the decrease in yields due to such environmental factors and to maximize productivity.

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. Smallest HSPs with molecular weights of 15-30 kDa represent the largest amount and type of stress-induced proteins in plants.

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

Elymus Siberian L. is a perennial, caespitose and self-polluting weed, which is widely distributed in northern Asia, northern Europe, Japan and parts of North America (Ma et al., 2012). In addition, it has strong environmental adaptability due to resistance to famine and cold stress, and is therefore used as a feed resource in Central Asia and northern China (Lu, 1993; Yan et al., 2007). Environmental stress caused by global warming has long been considered a major problem in agriculture. Changes in temperature, water deficit and soil contamination have significantly reduced the productivity of crops in arable land (Ahuja et al., 2010; Araus et al., 2002). Siberian wild-lye shows a strong environmental adaptability, so it may be used as a feed crop with excellent stress tolerance, but its gene has not been studied yet.

It is an object of the present invention to provide a composition comprising a heat shock protein which is active in various abiotic environmental stresses or a gene coding therefor.

Another object to be solved by the present invention is to provide a method for transforming the gene into a plant to enhance tolerance to abiotic environmental stress.

The present inventors have made intensive studies to overcome the problems of the prior arts, and as a result, the present inventors have surprisingly found that the Eshsp16.9 gene of the present invention is involved in abiotic stress resistance of plants, thereby completing the present invention.

In the present invention, Eshsp16.9 cDNA clones were isolated by differential screening of cDNA libraries obtained from Siberian wild rye grass ( Elymus sibiricus L.). This cloned gene was derived from an abiotic stress-resistance reaction and was expressed in E. coli, which is associated with an increase in resistance to salt, heavy metals, or famine stress.

The present invention provides a composition for promoting abiotic stress tolerance of a plant comprising EsHsp16.9 ( Elymus sibiricus L. Heat shock protein 16.9) protein represented by the amino acid sequence of SEQ ID NO: 2. In addition, the present invention provides a composition for promoting abiotic stress tolerance of a plant comprising a gene represented by the nucleotide sequence of SEQ ID NO: 1.

The composition of the present invention comprises the Eshsp16.9 protein or a gene coding therefor as an active ingredient. The composition comprising the gene represented by the nucleotide sequence of SEQ ID NO: 1 can enhance the abiotic stress tolerance of the plant by transforming the Eshsp16.9 gene into the plant.

The range of the EsHsp16.9 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 of a plant.

The invention also includes fragments, derivatives and analogues of the EsHsp16.9 protein. As used herein, the terms "fragments", "derivatives" and "analogs" refer to polypeptides that possess substantially the same biological function or activity as the EsHsp16.9 polypeptides of the present 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 a DNA or RNA encoding EsHsp16.9 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 Eshsp16.9 gene of the present invention is derived from Siberian wild rye grass ( Elymus sibiricus L.). However, an embodiment of the present invention is directed to a method of screening for high homology (e.g., greater than 60%, i.e., 70%, 80%, 85%) with Siberian wild rye grass ( Elymus sibiricus L.) Eshsp16.9 gene %, 90%, 95%, even 98% sequence identity), as well as other genes from other plants. Methods for sequencing, and means for determining sequence identity or homology (e.g., BLAST) are well known in the art.

In addition, 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 Eshsp16.9 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 Eshsp16.9 protein may 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 containing Eshsp16.9 protein-encoding DNA sequences 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 a pKBS1-1 vector, a pBI101 vector, or a pCAMBIA vector, but is not limited thereto.

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 is preferably 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 or the like. 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 a plant cell and overexpressing the EsHsp16.9 gene.

The plant to be transfected may be a conventional plant, not particularly limited to its kind, and preferably a plant such as orchardgrass, tall fescue, perennial ryegrass, creeping bent grass, timothy , Italian Ryegrass, Kentucky Bluegrass, Red Top, Reed Canary Grass, Bermuda Grass, Tepgrass, 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, salt, famine, or heavy metal stress. The heavy metal may preferably be arsenic, but is not limited thereto.

In addition, the present invention provides a transgenic plant or a seed thereof which is over-expressed by EsHsp16.9 gene and has been enhanced in resistance to abiotic stress.

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 Eshsp16.9 gene derived from the Siberian wild rye grass ( Elymus sibiricus L. sibiricus L.) of the present invention has an increased expression in various abiotic stress environments such as high temperature, salt, famine, or heavy metals . Thus, the Eshsp16.9 gene can be used to develop transgenic plants that are resistant to abiotic stress by being used for molecular sowing of grasses.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the gene sequences and amino acid sequences of Siberian wild-lys small Hsp 16.9 (EsHsp 16.9).
Figure 2 shows a phylogenetic analysis of Eshsp16.9 with Arabidopsis genes coding for small Hsps. The small Hsp genes of Arabidopsis include AT1G53540 (NCBI accession number, ABD57522, Protein name, AT17.6C class I), AT3G46230 (AEE78138; At17.4 class I), AT5G59720 (AED97223; 1 class I), AT1G07400 (AEE28120, At17.8 class I), AT1G59860 (US57519, At17.6A class I), AT2G29500 (US57517, At17.6B class I), AT4G10250 (AAO44068; At22), AT5G37670 (AED94218, At17 AT5G12030 (AED91752, At17), AT4G25200 (BAH19967; At23.6), AT5G51440 (AED96081), AT4G27670 (AEE85379; At21), AT1G52560 (AAR92344; At26.5), AT5G54660 (ABL66753, At21.7 class V) AT5G12020 (AED91751, At17.6 class II), AT1G54050 (AEE33041, At17.3 class III), AT4G21870 (ABF59020, At15.4 class IV), AT3G22530 (AEE76650), AT1G06460 (ABF74713), AT5G04890 (AED90800) and AT3G10680 (AEE74942).
Figure 3 compares the amino acid sequence of Eshsp16.9 with the small Hsps of other plant species to compare the Eshsp16.9 protein with the Hordeum vulgare (Hsp17; ADW78607), Zea mays (Hsp16.9 class I; NP_001150783) , Triticum monococcum (Hsp17; CAM96547), Aegilops longissima (Hsp16.9; CAM96537), Setaria italic (Hsp16.9 class I; XP_004968026), Oryza sativa (Hsp; AAB39856) , Homologues of the medicago truncatula (Hsp18.2 class I; XP_003619703), tobacco ( Nicotiana tabacum ) (Hsp 3B class I; ADK36668) and chickpea ( Cicer arietinum ) (Hsp8.5 class I; XP_004505085) It is sorted.
Fig. 4 shows the result of phylogeny analysis of Eshsp16.9 with the homology of other plants.
FIG. 5 shows the results of Northern blot analysis of the expression level of Eshsp16.9 RNA when high temperature, low temperature, salt, heavy metals (copper, cadmium, arsenic), methyl viologen, hydrogen peroxide and famine stress were added to plants.
Figure 6 shows the expression of the recombinant Eshsp16.9 protein. The Eshsp16.9 gene was induced with either 0 or 0.2 mM IPTG. Total extract (T), supernatant (S), and pellet (P) fractions were separated by 10% SDS-PAGE. The molecular marker (M) is indicated on the left.
Figure 7 shows the purification of the Eshsp16.9 protein. The supernatant (S) was purified by affinity chromatography using glutathione Sepharose 4B resin and eluted with reduced glutathione.
Figure 8 shows the molecular chaperone activity of the recombinant Eshsp16.9 protein. Bovine serum albumin (BSA) or Orchardgrass Hsp70 (DgHsp70) was used as a negative or positive control, respectively.
Figure 9 shows the results of an analysis of the stress tolerance of an Escherichia coli system transformed with Eshsp16.9.
10 is a schematic diagram showing a plant expression vector (pCAMBIA3300-Eshsp16.9) for expressing Eshsp16.9 gene.
FIG. 11 is a photograph showing the resistance to arsenic stress by expressing vector and Eshsp16.9 protein in tobacco plants.

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 Eshsp16.9 gene

Seeds of Siberian wild railgras used in this example were obtained from Ulaanbaatar, Mongolia. The plant material of Siberian Wild Ryegrass was grown in the grass feed section of the National Livestock College. The seeds were seeded 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). Using a GeneFishing ^TM DEG kit (Seegene), a 344 bp partial cDNA fragment of EsHsp 16.9 was isolated using ACP-based PCR as described in Lee et al. (2012).

Nucleic acids, amino acid sequences, and conserved domains were analyzed using NCBI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and Bioedit (version 7.2.3) software (Hall, 1999) Respectively. The isoelectric point (pI) and molecular weight of Eshsp 16.9 were calculated using the computer pI / Mw tool of ExPASy (http://web.expasy.org/compute_pi/).

Using DEG analysis for gene-fishing technique, several cDNA fragments were obtained from Siberian wild-type mRNA exposed to high temperature stressor. The amplified fragments were eluted and separated and sequenced into pTOPO vector. Through Blast Search, one segment is the barley ( Hordeum vulgare ) Homologous with 17 kDa class I small Hsp. Full length cDNA produces a 456 bp open reading frame encoding a 151 amino acid with a conserved alpha-crystallin domain between 45 and 136 amino acids. The small Hsps is characterized by a conserved alpha -crystalline domain consisting of about 100 amino acids and has an N-terminal arm that is disordered and a short, flexible C-terminal extension. Using the computer pI / Mw tool, the molecular weights of calculated isoelectric point (pI) and DEG66 are expected to be 5.53 and 16,923 Da, respectively. The full length cDNA sequence was deposited on GenBank with accession number KM025035.

Example 2: Molecular genetic analysis of Eshsp16.9 gene

In order to clarify which class Eshsp16.9 belongs to, we carried out a phylogenetic analysis with Arabidopsis small Hsps as shown in Fig. The homology of Eshsp16.9 from the amino acid sequence of small Hsps in arabinic acid and other plant species was retrieved from the GenBank ^™ 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/).

Eshsp16.9 showed high sequence homology (70.9%) with AtHsp18.1 belonging to the cytoplasmic class I small Hsp, and shares 60-70% sequence homology with other cytoplasmic class I small Hsp of Arabidopsis. In addition, Eshsp16.9 was compared to other cytoplasmic classes I small Hsps present in other plants (Figure 3). Sequence homology showed that Eshsp16.9 was close to the class I small Hsps of wheat (96%) or barley (94.7%). In addition, Eshsp16.9 showed a high similarity of more than 80% in the monocots and 70% in the dicotyledons (Fig. 4).

Example 3: Expression of Eshsp16.9 gene by abiotic stress treatment

(4 ° C), salt (300 mM NaCl), copper (CuSO ₄ , 500 μM), and cadmium (CdCl ₂ , respectively) were added to 4-week-old Siberian wild- , 500 μM), arsenic (Na ₂ HAsO ₄ .7H ₂ O, 500 μM), methyl viologen (MV, 1 mM) or hydrogen peroxide (H ₂ O ₂ , 10 mM). 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. For methyl viologen and hydrogen peroxide treatment, leaf slices were floated on water containing MV or hydrogen peroxide at the above concentrations. The stressed plants were harvested, cooled with liquid nitrogen and stored at -80 ° C until use.

Northern blot analysis

To investigate transcriptional expression of the Eshsp16.9 gene in response to various abiotic stresses, total RNA was isolated from each stressed leaf with TRIzol reagent (Qiagen, CA, USA). The extracted total RNA (10 g) was fractionated by electrophoresis on 1.2% agarose gel containing formaldehyde and transferred to a Hybond-N ⁺ nylon membrane (Amersham, Little Chalfont, UK). Hybridization overnight at 65 in a blot of the PCR-amplified gene Eshsp16.9 ³² P- labeled probe ⁽³² P-labeled probe), followed by performing a Northern blot analysis.

As shown in Figure 5, the amount of Eshsp16.9 mRNA rapidly and dramatically increased in a high temperature stress environment and remained high until 36 hours after treatment. On the other hand, expression of Eshsp16.9 was rarely detected under low temperature, salt, copper, or cadmium treatment. Arsenic stress caused a slight increase in Eshsp16.9, and methylvioligen (MV) and hydrogen peroxide causing oxidative stress induced a massive increase in Eshsp16.9 mRNA. Familial stress also increased the expression of Eshsp16.9 after 36 hours of water withdrawal. These results indicate that Eshsp16.9 plays a major role in oxidative stresses such as high temperature, famine, heavy metals, or MV and hydrogen peroxide.

Example 4 Expression and Purification of Recombinant Eshsp16.9 Protein

For the expression of the recombinant Eshsp16.9 protein with glutathione S- transferase (GST) and 6x-histidine-tag (6x-His tag), Eshsp16.9 was added to the pET-41a plasmid Lt; / RTI > The plasmid was transformed into E. coli . BL21 (DE3) cells, and single colonies containing the plasmids were grown overnight at 37 占 폚. Cultures were inoculated into fresh LB liquid medium and cultured to an OD600 of 0.6-0.8. Recombinant proteins 0.2 mM isopropyl-1-thio -β- _D - was induced by addition of galactosyl-pyrano side (isopropyl-1-thio-β- D -galactopyranoside, IPTG) were cultured for 4 hours after the cells. After harvesting by centrifugation, cells were resuspended in phosphate buffered saline (PBS) and chilled at -80 ° C until use. After thawing, resuspended cells were cultured on ice for 20 minutes in the presence of 1% Triton X-100 and degraded by sonication. After centrifugation, the supernatant was treated with a Glutathione Sepharose 4B affinity column. The GST-fused Eshsp16.9 recombinant protein was eluted with 10 mM reduced glutathione and then dialyzed against 40 mM HEPES (pH 7.5) buffer.

For expression conditions of the recombinant Eshsp16.9 protein, cells containing Eshsp16.9 were incubated at various temperatures, such as 18, 26, and 37 ° C, and various IPTG concentrations, such as 0, 0.2 mM, and 1 mM, Lt; / RTI > A vast amount of recombinant Eshsp16.9 protein was generated at 37 ° C for 4 hours after 0.2 mM IPTG induction. As shown in Figure 6, the expected size of the recombinant Eshsp16.9 (43.8 kDa) was highly expressed in the whole fraction after induction of IPTG, but not in the absence of IPTG (0 mM). Furthermore, the recombinant Eshsp16.9 protein was similarly expressed in both supernatant (S) and pellet (P) fractions (Figure 6). The supernatant fractions were used to purify through Glutathione Excellose resin, elute through reduced glutathione (GSH), and on 10% SDS-PAGE. As shown in Figure 7, a vast amount of recombinant Eshsp16.9 protein was purified.

Example 5: Analysis of Holder chaperone in vitro

Chaperone activity was tested by measuring the ability of the recombinant GST-fused Eshsp16.9 protein to reduce thermal aggregation of arabicidal malate dehydrogenase (MDH, EC 1.1.1.37). MDH agglutination was assayed in 40 mM HEPES (pH 7.5) by measuring the absorbance at 340 nm using a Beckman DU-80 spectrophotometer for a thermostatic cell holder assembly at 45 ° C. In the presence or absence of GST-fused Eshsp16.9.

Small Hsps are known as molecular chaperones that prevent irreversible aggregation of substrates under high temperature stress conditions. To elucidate the functional chaperone properties of Eshsp 16.9, malate dehydrogenase (MDH), one of the best model substrates for chaperone analysis, was denatured under thermal coagulation conditions at 45 ° C and observed for 15 min.

In the absence of the Eshsp16.9 protein, MDH rapidly denatured to 10 minutes under high temperature denaturation conditions, and the curve then became nearly flat for 5 minutes indicating that the MDH was completely denatured. However, MDH denaturation was strongly and dose-dependently protected in the presence of Eshsp16.9 and Eshsp16.9, a 5-molar concentration higher than MDH, was almost 90% protected against MDH denaturation. The light scattering values of Eshsp16.9 without MDH did not increase at high temperature denaturation conditions, indicating that the Eshsp16.9 protein is a high temperature stability protein (Figure 8).

Example 6: Growth inhibition assay against abiotic stress in Escherichia coli

Resistance to abiotic stress of Eshsp16.9 protein, which increases transcriptional activity when stress is applied, was measured. To this end, a prokaryotic system using E. coli growth inhibition assay was applied.

E. coli cells containing the empty vector (pET-41a) and Eshsp16.9 (pET-41a: Eshsp16.9) were cultured overnight at 37 DEG C and inoculated with fresh LB liquid medium at a 1: 100 volume ratio (cell: medium). The cells were continuously cultured to an OD600 of 0.6. After induction of IPTG (0.5 mM), the cells were immediately treated with various stresses such as NaCl (400 mM), arsenic (As, 0.1 mM), polyethylene glycol (3%) and high temperature (48 ° C). Cell growth was measured with a spectrophotometer (OD600) at each hour for 4 hours. The results averaged six independent experiments.

Cells containing the vector (pET41a, control) or Eshsp16.9 (pET41a: Eshsp16.9) were over-expressed via IPTG as shown in Figure 9a. And cells were diluted to show the same cell optical density (OD) prior to abiotic stress treatment. Diluted cells were immediately exposed to salt (NaCl), arsenic (As), polyethylene glycol (PEG), or high temperature (45 ° C) stress and cell growth was monitored hourly for 4 hours.

In a stress-free environment, cells containing the vector or Eshsp16.9 grew similarly during 4 hour incubation (Figure 9a). However, the cell growth of the vector was significantly inhibited in the presence of various stresses (Fig. 9b-e).

On the other hand, cells containing Eshsp16.9 grew more in the presence of salt, arsenic, and PEG than cells containing the vector (Fig. 9b-d). Unexpectedly, however, at high temperature stress, the growth of Eshsp16.9 cells was inhibited similarly to vector growth (Fig. 9e). These results indicate that overexpression of Eshsp16.9 in E. coli increases resistance to salt, arsenic, or PEG stress.

Example 7: Resistance measurement of heavy metals (arsenic) according to expression of EsHsp16.9 protein using model plant (tobacco)

The transformation techniques used in this example are described by Dane K. Fisher and Mark J. Guiltyinan. "Plant Molecular Biology Reporter" pp. 278-289, 1995. < / RTI > The plant transformation vector pCAMBIA3300-EsHsp16.9 system was used under the control of the CaMV 35S promoter to transform EsHsp16.9 gene into model plants (Fig. 10).

In order to analyze the tolerance of EsHsp16.9 transformed tobacco (Tg-1) plants to arsenic, transformed tobacco plants were germinated on MS medium supplemented with 300 M arsenic (As) After the cultivation, the growth of the plants was examined. As a result, the growth of the non-transformed plant (NT) was greatly suppressed and killed in the treatment with arsenic, but the transformed tobacco (Tg-1) transformed with EsHsp16.9 was grown.

From these results, it was confirmed that the expression of the transformed EsHsp16.9 protein enhances the resistance of the plant to arsenic stress.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> Eshsp16.9 gene and uses thereof <130> P14-236 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 456 <212> DNA <213> EsHsp16.9 (Elymus sibiricus L. Heat shock protein 16.9) <400> 1 atgtcgatcg tgcggcggag caacgtcttc gacccattcg ctgacctctg ggccgacccc 60 ttcgacacct tccgctccat cgttccggcg atctcaggca gcaacagcga gacggctgcg 120 ttcgccaacg cccgggtgga ctggaaggag acgccagagg cgcacgtctt taaggccgac 180 ctccccggcg tgaagaaaga ggaggtcaaa gttgaggtgg aggacggcaa cgtgctcgtc 240 gtcagcggtg agcgcactaa ggagaaggag gacaagaacg acaagtggca ccgcgtggag 300 cgaagcagcg gcaagttcga gcggcgcttc cgcctgccgg aggacgccaa ggtggaggag 360 gtcaaggctg ggctggagaa cggcgtgctc actgtcaccg tgcccaaggc agaggtcaag 420 aagcccgagg tgaaggccat cgagatctcc ggctga 456 <210> 2 <211> 151 <212> PRT <213> EsHsp16.9 (Elymus sibiricus L. Heat shock protein 16.9) <400> 2 Met Ser Ile Val Arg Arg Ser Asn Val Phe Asp Pro Phe Ala Asp Leu   1 5 10 15 Trp Ala Asp Pro Phe Asp Thr Phe Arg Ser Ile Val Pro Ala Ile Ser              20 25 30 Gly Ser Asn Ser Glu Thr Ala Ala Phe Ala Asn Ala Arg Val Asp Trp          35 40 45 Lys Glu Thr Pro Glu Ala His Val Phe Lys Ala Asp Leu Pro Gly Val      50 55 60 Lys Lys Glu Glu Val Lys Val Glu Val Glu Asp Gly Asn Val Leu Val  65 70 75 80 Val Ser Gly Glu Arg Thr Lys Glu Lys Glu Asp Lys Asn Asp Lys Trp                  85 90 95 His Arg Val Glu Arg Ser Ser Gly Lys Phe Glu Arg Arg Phe Arg Leu             100 105 110 Pro Glu Asp Ala Lys Val Glu Glu Val Lys Ala Gly Leu Glu Asn Gly         115 120 125 Val Leu Thr Val Thr Val Pro Lys Ala Glu Val Lys Lys Pro Glu Val     130 135 140 Lys Ala Ile Glu Ile Ser Gly 145 150

Claims (7)

An EsHsp16.9 ( Elymus sibiricus L. Heat shock protein 16.9) protein represented by the amino acid sequence of SEQ ID NO: 2,
Salt, famine, and heavy metal. A 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 EsHsp16.9 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 EsHsp16.9 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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