KR100599824B1 - A Novel Stress Resistance Protein Originated from Soybean a Gene Enconding the Same and Use Thereof - Google Patents

Abstract

The present invention relates to a low temperature and / or salt resistant protein derived from soybean, a gene encoding the protein, a recombinant vector containing the gene, a microorganism transformed with the recombinant vector, and a low temperature and / or salt resistance transfected with the gene. It is about a plant.

The stress (low temperature and salt) resistance genes according to the invention are useful for preparing stress resistant transgenic plants as well as for research.

Soybean, low temperature, salt, resistant, stress, transgenic plant

Description

A Novel Stress Resistance Protein Originated from Soybean, a Gene Enconding the Same, and Use Thereof}             

1 shows Northern blot results of total RNA isolated from soybeans treated with low temperature and various stresses.

Figure 2 shows the construction of a recombinant vector containing a low temperature resistant gene according to the present invention.

3 is a map of a recombinant transition vector containing a low temperature resistant gene according to the present invention.

Figure 4 shows the results of electrophoresis (SDS-PAGE) of cold-resistant protein expressed in transformed Escherichia coli.

5 is a photograph of purified recombinant low temperature resistant protein expressed in transformed E. coli using affinity chromatography.

Figure 6 is a result of comparing the survival rate of E. coli (SLTI66) containing a low temperature resistant gene and E. coli (vector) that does not contain at a very low temperature.

7 is a result of comparing and comparing the survival rate of E. coli (SLTI66) containing a resistance gene and E. coli (vector) not included in the OD value according to the concentration of salt.

The present invention relates to a low temperature and / or salt resistant protein derived from soybean, a gene encoding the protein, a recombinant vector containing the gene, a microorganism transformed with the recombinant vector, and a low temperature and / or salt resistance transfected with the gene. It is about a plant.

Soybeans are grown in a variety of climates around the world. Among such climatic environmental conditions, it is known that it is most affected by temperature. Especially, it is known that it is due to low temperature. So in evolution, plants have increased their resistance by creating materials that adapt to their environment with their survival strategies to overcome their environmental factors. According to the main research, the effect of low temperature in the plant is known to be water deficiency phenomenon as the intracellular water escapes out of the cell due to the imbalance of the moisture potential by freezing between the plasma and the cell membrane. This affects the mechanical breakdown of tissues due to freezing of cells and the metabolism of cells due to the imbalance of moisture, resulting in the loss of plant growth and the death of tissues.

However, plants that are resistant to low temperatures are known to continue normal metabolism by preventing intracellular water deficiency by reducing the mechanical breakdown caused by freezing of cells and increasing the ability to maintain intracellular water potential. There are many known DNAs that produce these resistant proteins, and their variety is also known. Examples of such DNA are cold inducible (KIN), low-temperature induced (LTI), cold regulated (COR), early responsive to dehydration (ERD), and responsive to dehydration (RD). Is known. In addition, lea (late embryogenesis abundant) protein, found in cotton and then in various plant species, is composed of hydrophilic amino acids and is known to be induced in large amounts early in stress. It is also known to protect cells at the cell membrane site by inducing a large amount due to lack of moisture. However, there are few reports of cold stress resistant proteins in soybeans.

Therefore, the present inventors isolated stress-resistant novel genes from the cDNA gene library of fragments induced by soybean stress (low temperature, salt) through RACE PCR technique and expressed the genes in recombinant E. coli, and then purified the recombinant protein. Assaying resistance to low temperature and salt in E. coli confirmed that the gene is resistant to low temperature and salt stress, and the present invention was completed.

The main object of the present invention is to provide a soybean-derived low temperature stress resistance gene and its protein.                         

Another object of the present invention is to provide a recombinant vector containing the low temperature resistance gene and a microorganism transformed with the recombinant vector.

Still another object of the present invention is to provide a stress resistant plant transfected with the gene.

In order to achieve the above object, the present invention provides a low temperature and / or salt resistant protein having an amino acid sequence of SEQ ID NO: 2 and a gene encoding the same ( Gmslti66 ). In the present invention, the gene may be characterized by having a nucleotide sequence of SEQ ID NO: 1, the gene may be characterized in that for encoding a low temperature and / or salt resistance protein.

The present invention also provides a recombinant vector containing the gene and a transformed microorganism obtained by introducing the recombinant vector into a host cell selected from the group consisting of bacteria, yeast and fungi.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli DH5α, E. col JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, E. coli B21, and the like. It includes. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the aforementioned E. coli , strains of the genus Agrobacterium, such as Agrobacterium A4, bacilli , such as Bacillus subtilis , Salmonella typhimurium or Serratia marghesen another variety of enteric bacteria and Pseudomonas (Pseudomonas) in strains such as marcescens) may be used as host cells.

Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841, 1982) can also be used for transformation of these cells.

As the vector used for overexpression of the gene according to the present invention, an expression vector known in the art may be used, and it is preferable to use a pET family vector (Novagen). When the cloning is performed using the pET family vector, histidine groups are bound to the ends of the expressed protein, and thus the protein can be effectively purified. In order to separate the expressed protein from the cloned gene, a general method known in the art may be used, and specifically, it may be separated by a chromatographic method using Ni-NTA His-binding resin (Novagen). . In the present invention, the recombinant vector may be characterized in that the pET-SLTI66, the host cell may be characterized in that E. coli or Agrobacterium.

The present invention also provides a method for producing a low temperature and / or salt resistant protein having the amino acid sequence of SEQ ID NO: 2, characterized in that the culture of the transformed microorganism. More specifically, the present invention provides a method of controlling the expression of (a) a DNA sequence encoding a low temperature resistant protein having the amino acid sequence of SEQ ID NO: 2 operatively linked to the DNA sequence to regulate its expression Inserting into a vector comprising an expression control sequence; (b) transforming the host with a recombinant expression vector formed therefrom; (c) culturing the resulting transformants under medium and conditions appropriate for causing the DNA sequence to be expressed; (d) recovering the substantially pure low temperature resistant protein from the culture, the method for producing a low temperature and / or salt resistant protein.

The expression “expression control sequence” in the present invention means a DNA sequence essential for the expression of a coding sequence operably linked in a particular host organism. Such regulatory sequences include promoters for performing transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. For example, suitable control sequences for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells include promoters, polyadenylation signals, and enhancers. The factor that most influences the amount of gene expression in the plasmid is the promoter. As the promoter for high expression, an SRα promoter, a promoter derived from cytomegalovirus, and the like are preferably used. To express the DNA sequences of the invention, any of a wide variety of expression control sequences can be used in the vector. Useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter regions of phage lambda, fd code protein Regulatory region of, promoter for 3-phosphoglycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoter of yeast alpha-crossing system and gene expression of prokaryotic or eukaryotic cells or viruses Other sequences known to modulate and various combinations thereof. The T7 promoter can be usefully used to express the proteins of the invention in E. coli.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

As used herein, the term “expression vector” generally refers to a fragment of DNA that is generally double stranded as a recombinant carrier into which fragments of heterologous DNA have been inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once the expression vector is in a host cell, it can replicate independently of the host chromosomal DNA and several copies of the vector and the inserted (heterologous) DNA thereof can be produced.

As is well known in the art, to raise the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the host cell is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.

Host cells transformed or transfected with the expression vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. As used herein, the term "transfection" means that the expression vector is accepted by the host cell whether or not any coding sequence is actually expressed.

Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be taken into account because the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting expression control sequences, several factors must be considered. For example, the relative strength of the sequence, the controllability, and the compatibility with the DNA sequences of the present invention should be considered, particularly with regard to possible secondary structures. Single cell hosts may be selected from a host for the selected vector, the toxicity of the product encoded by the DNA sequence of the invention, the secretory properties, the ability to accurately fold the protein, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention from the host. It should be selected in consideration of factors such as the ease of purification. Within the scope of these variables, one skilled in the art can select a variety of vector / expression control sequence / host combinations capable of expressing the DNA sequences of the invention in fermentation or large scale animal culture. As a screening method when cloning the cDNA of a protein according to the present invention by expression cloning, a binding method (binding method), a panning method, a film emulsion method and the like can be applied.

By "substantially pure" in the definition of the present invention is meant that the protein according to the invention and the DAN sequence encoding it are substantially free of other proteins derived from bacteria.

The present invention also provides low temperature and / or salt resistant plants transfected with the gene. Transfection of plants can be achieved by conventional methods using Agrobacterium, viral vectors and the like. For example, after transforming the microorganism of the genus Agrobacterium with a recombinant vector containing a gene according to the present invention, the transformed Agrobacterium microorganisms can be infected by tissues of the target plant, etc. to obtain a transfected plant. . More specifically, (a) pre-culture the explant of the plant (preplant), and then transfected by co-culture with the transformed Agrobacterium; (b) culturing the transfected explants in a callus induction medium to obtain callus; And (c) cutting the obtained callus, and culturing it in shoot induction medium to form shoots, thereby producing a transfected plant.

In the present invention, the term "explant" refers to a slice of tissue cut out of a plant, and includes cotyledon or hypocotyl. The explants of the plant used in the method of the present invention may be cotyledons or hypocotyls, it is more preferable to use the cotyledons obtained by germinating in MS medium after disinfecting and washing the seeds of the plants.

Transfection target plants usable in the present invention include, but are not limited to, tobacco, tomatoes, peppers, beans, rice, corn, and the like. In addition, even if the plant used for transformation is a sexually propagating plant, it will be apparent to those skilled in the art that it can be repeatedly reproduced by tissue culture or the like.

The gene revealed by the present invention is a DNA having a characteristic of lea DNA, which is induced in a large amount by low temperature and salt stress. Unlike so far, it is revealed in recombinant E. coli that exhibits strong resistance to low temperature or other stress in plants. It is industrially useful for producing stress resistant plants by applying them to practically other plants.

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

Example 1: Nucleotide sequence and amino acid sequence analysis of soybean-derived stress resistance gene

Poly (A +) mRNA was extracted from stressed and unstressed leaves in soybeans grown for 3 weeks or longer, and then cDNA expressed only by stress (cold, salt) using PCR-Select cDNA Subtraction (Clontech). Secured. The obtained cDNA was cloned into pCR2.1 (Invitrogen), which is a subcloning vector, and plasmid DNA was isolated using a plasmid purification kit (purchased by NucleoGen). Sequencing was performed by requesting macrogen. Based on the sequencing results, the entire nucleotide sequence was analyzed by RACE PCR. As a result, the cDNA of the stress resistance gene according to the present invention was composed of a total of 270 bp nucleotide sequence (SEQ ID NO: 1), and deduced it, it was composed of 90 amino acids (SEQ ID NO: 2). As a result of comparison and investigation through DNASIS and BLAST programs provided by NCBI, GenBank, EMBL and SwissProt databases, the nucleotide sequence and amino acid sequence were found to be a new gene for low temperature resistance ( Gmslti66 ).

Example 2: Expression of Low Temperature Resistance Genes

Soybeans grown for more than 3 weeks in growth were subjected to various stresses, hormones, and seedlings on the 3rd and 7th days, and the total RNA was separated by HOT phenol method. The whole RNA was electrophoresed on 1.0% formaldehyde gel, and then the gel was transferred to a nylon membrane (Schleicher & Schuell) with 20 × SSC solution (3M NaCl, 0.3M sodium citrate) and radioisotope. Northern blot analysis was performed using a low temperature resistance gene (270 bp cDNA) labeled with [α- ³² P] dCTP as a probe.

As a result, transcripts at low temperature (5 ° C) 24 hours (LT 24hr), salt 6 hours (NaCl 6hr), drought 24 hours (Dr 24hr), and ABA 6 hours (ABA 6hr) for hormone treatment in leaves. The transcripts were confirmed only in the cotyledon of seedling as tissue specificity (FIG. 1). From this result, it was confirmed that the low temperature resistance gene according to the present invention is expressed in some stress and hormone (ABA) and tissue-specific.

Example 3: Expression of Stress Resistant Proteins in Recombinant Escherichia Coli

In order to determine whether E. coli is resistant to low temperature or salt concentration, the low temperature resistant gene isolated in Example 1 was inserted into pET42a (Novagen) of the E. coli expression vector. The gene cDNA and the expression vector pET42a were treated with restriction enzymes Eco Ri and Hind III, respectively, and then ligated to construct a recombinant transfer vector pET-SLTI66 (FIGS. 2 and 3). The recombinant transfer vector has a characteristic of inducing expression of recombinant fusion protein expression under the control of the T7 promoter and IPTG.

In order to express the low temperature resistant gene of soybean in E. coli, transformed E. coli was prepared by inserting 5 μg of the recombinant transfer vector pET-SLTI66 into 50 μg of E. coli BL21 at a temperature shock (42 ° C.). The transformed Escherichia coli was cultured in LB (Luria-Bertani) liquid medium containing 50 µg / ml of kanamycin for 1 hour, and then plated in LB-Kana solid medium and cultured at 37 ° C. for 16 hours or more. The resulting bacteria were incubated to OD 0.6 (1.0 × 10 ⁶ cells) in LB-Kana 50ml liquid medium, 0.5mM IPTG was added and incubated for 3 hours at 28 ℃.

After incubation, cells were collected by centrifugation at 3000 rpm for 5 minutes, and then 1.7 ml of 50 mM Tris-HCl (pH 8.0) buffer was added. The cells were disrupted using a cell crusher and centrifuged at 5000 rpm for 5 minutes. After centrifugation, the supernatant was separated and again centrifuged at 12,000 rpm for 10 minutes to separate the water-soluble and non-water-soluble proteins, followed by 12% SDS-protein electrophoresis (Laemmli, Nature, 227: 680, 1970) to perform recombinant fusion. Cold resistant protein expression was analyzed (FIG. 4 and FIG. 5). As a result, as shown in Figure 5, about 43kDa recombinant fusion protein bands could be confirmed. Lanes 4 to 6 represent total protein, water soluble protein and non water soluble protein, respectively.

Example 4 Determination of Low Temperature Resistance and Salt Resistance in Recombinant Escherichia Coli Containing a Low Temperature Resistance Gene

Whether the recombinant fusion protein expressed in Example 3 is resistant to low temperature and salt in E. coli was analyzed and compared with the control. Escherichia coli expressing a large amount of the stress-resistant protein and a control group expressing only the vector were incubated with stress at each cryogenic and NaCl concentration.

Cryogenic was quenched with liquid nitrogen and completely dissolved at room temperature (25 ° C.). Using this method, three quenching and dissolving cycles were repeated, followed by culturing at a constant rate in solid medium. In the case of Escherichia coli expressing a large amount of stress-resistant genes, the survival rate was 40-50% higher than that of the control (FIG. 6).

In addition, the experiment for the resistance of the salt was incubated for 0-10 hours with the concentration of salt (NaCl) in 3%, 5% in a liquid medium to check the OD value for each time. As a result, when more than 3% of the salt is present, the survival rate was significantly higher than that of the control (Fig. 7).

From this result, it was confirmed that the new stress resistance gene isolated from soybean is functionally resistant to low temperature and salt.

As described in detail above, the present invention provides a low temperature and salt resistant protein, a gene encoding the protein, a recombinant vector containing the gene, a microorganism transformed with the recombinant vector and a low temperature and salt resistant plant transfected with the gene. It provides the effect. In addition, the stress resistant gene according to the present invention is useful for producing stress resistant plants not only for research but also industrially.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (12)

A low temperature and / or salt resistant protein having the amino acid sequence of SEQ ID NO: 2. A gene encoding the amino acid sequence of SEQ ID NO. The gene according to claim 2, wherein the gene has a nucleotide sequence of SEQ ID NO: 1. The gene according to claim 2 or 3, wherein the gene encodes a low temperature and / or salt resistant protein. Recombinant vector containing the gene of claim 2. The recombinant vector according to claim 5, which is pET-SLTI66. A transformed microorganism obtained by introducing the recombinant vector of claim 5 into a host cell selected from the group consisting of bacteria, yeast and fungi. 8. The transformed microorganism according to claim 7, wherein the host cell is Escherichia coli or Agrobacterium. A method for producing the low temperature and / or salt resistant protein according to claim 1, wherein the microorganism of claim 7 is cultured. 10. The method of claim 9, wherein (a) one or more expression modulators that operably linked to a DNA sequence encoding a cold resistant protein having the amino acid sequence of SEQ ID NO: 2 are operatively linked to the DNA sequence. Inserting into a vector comprising an expression control sequence; (b) transforming the host with a recombinant expression vector formed therefrom; (c) culturing the resulting transformants under medium and conditions appropriate for causing the DNA sequence to be expressed; And (d) recovering the substantially pure low temperature resistant protein from the culture. delete delete

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