KR20130009279A - Method for enhancing drought stress resistance of plant using lea gene - Google Patents

Abstract

PURPOSE: A method for improving drought stress resistance of a plant is provided to enable a transgenic plant to grow into a drought resistant race and to stably supply agricultural products. CONSTITUTION: A method for improving drought resistance of a plant comprises a step of overexpressing a protein with an amino acid sequence of sequence number 2 and improving drought resistance. A method for overexpressing the protein comprises: a step of transforming a plant with a recombinant expression vector containing a promoter and a gene encoding the protein or a step of transducing the gene into a plant cell. The gene has a base sequence of sequence number 1. The promoter is inducible RD29A promoter. The plant includes potato, barley, corn, wheat, rye, grass, sugar cane, or onion. The recombinant expression vector is pBINRD29A vector.

Description

Method for enhancing drought stress resistance of plant using LEA gene}

The present invention relates to a method for improving the dry resistance of a plant, a method for producing a plant having improved dry resistance, and a plant having improved dry resistance produced by such a method.

Since plants are very mobile, their ability to avoid the many types of environmental stresses that face their survival process is significantly lower than that of animals, so most plants have a variety of defenses that are self-resistant to stresses such as low temperature, salt, and drying. Have a small Recently, research on the response to the stress or defense mechanism of the plant is being actively conducted, many genes related to the stress of the plant have been searched and its function is revealed. Genes that are resistant to plant stress, as found in these studies, can ultimately be used as a useful gene source for imparting resistance to environmental stresses on plants and crops.

Accordingly, the problem to be solved by the present invention is a method of improving the dry resistance of a plant using a dry resistance gene, a method of producing a plant having improved dry resistance using such a gene, and a trait improved by drying resistance produced by the method. It is to provide a conversion plant.

In order to solve the above problems, the present invention is a method of improving the dry resistance of plants by increasing the intracellular level of the protein having the amino acid sequence represented by SEQ ID NO: 2, more preferably the amino acid represented by SEQ ID NO: 2 Provided is a method of overexpressing a protein having a sequence to enhance the drying resistance of the plant.

The present invention also provides a method for producing a dry resistant plant, characterized in that the plant is transformed into a recombinant expression vector in which a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 is linked to a promoter.

The present invention also provides a recombinant expression vector linking a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 to a promoter, and a transformant having improved dry resistance by transformation with the recombinant expression vector.

Below. The present invention will be described in detail.

Proteins having an amino acid sequence represented by SEQ ID NO: 2 (hereinafter, also referred to as LEA protein) include functional equivalents thereof. The term "functional equivalent" is at least 70%, preferably 80% or more, more preferably 90% or more, and still more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid. Preferably, it refers to a protein having a sequence homology of 95% or more, and having a substantially homogeneous physiological activity with a protein having an amino acid sequence represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" means activity that enhances resistance to drying in plants. Substitution of the amino acid is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in the wild type are as follows; (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids is preferably located at portions not directly involved in the bioactivity of the LEA protein. The functional equivalent range also includes protein derivatives in which some chemical structures of the protein are modified while maintaining the basic backbone of the LEA protein and its physiological activity. For example, fusion proteins made by fusion with other proteins, such as GFP, while maintaining structural alterations and physiological activities to alter the stability, shelf life, volatility or solubility of the proteins of the present invention. In addition, polypeptides having a physiological activity substantially equivalent to that of the wild type LEA protein as fragments of the protein having the amino acid sequence represented by SEQ ID NO: 2 form another preferred group of functional equivalents of the wild type LEA protein. The "fragment" is an amino acid sequence corresponding to a part of a protein, and has a common origin element, structure, and mechanism of action within the scope of the present invention, and exists in the wild type, or is cleaved using a protease. Or chemically cleaved.

The base sequence of the gene encoding the LEA protein may include all gDNA, cDNA and RNA. The base sequence includes both base sequences encoding wild type proteins and their functional equivalents and sequences that hybridize with these sequences under high stringent conditions. High-tension conditions are as described in known literature (Molecular Cloning, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 1989). Preferably, the LEA according to the present invention The gene encoding the protein may include the nucleotide sequence represented by SEQ ID NO: 1 and sequence variants thereof. Specifically, the sequence variant is a base having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the base sequence represented by SEQ ID NO: 1 Sequences may be included.

As used herein, the term “homology” is intended to indicate a degree of similarity to the amino acid sequence of a wild type protein or a nucleotide sequence encoding the same, and the amino acid sequence or nucleotide sequence of the present invention and the percentage or more as described above. Include sequences with identical sequences. This homology comparison can be performed by using a comparison program that is easy to see with the naked eye. Commercially available computer programs can calculate the percent homology between two or more sequences, and the percent homology can be calculated for adjacent sequences.

The "intracellular level" refers to the amount present in a cell, which can be controlled by various methods known to those skilled in the art. For example, intracellular levels can be regulated through, but not limited to, regulation at the transcriptional stage or at the post-transcriptional stage. Modulation at the transcriptional stage is a method for enhancing the expression of a gene known to those skilled in the art, for example, a method for enhancing the expression of the gene by preparing a recombinant expression vector linking a target gene or a variant thereof to a promoter or the target gene. Or a method of inserting an expression control sequence such that the expression of the gene is enhanced around the variant thereof. Modulation at the post-transcriptional stage is a method for enhancing protein expression known to those of skill in the art, for example, to enhance the stability of mRNA transcribed into a target gene or variant thereof, to enhance the stability of the target protein, or It can be carried out by a method for enhancing the activity of the protein.

Preferably, in the present invention, the increase in the intracellular level of the protein of SEQ ID NO: 2 or a functional equivalent thereof may be performed by a method of increasing the expression of the gene encoding the protein. For this increase, a method known to those skilled in the art can be used. For example, a recombinant expression vector is prepared by linking a gene having a amino acid sequence represented by SEQ ID NO: 2 to a promoter or a gene encoding a functional equivalent thereof. By transformation, expression can be enhanced.

As used herein, 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, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in the wild-type form of the cell in either the sense or antisense form. Recombinant cells can also express genes found in wild-type cells, but the genes are modified and reintroduced into cells by artificial means. The term "vector" in the present invention is used when referring to DNA fragment (s), nucleic acid molecules that are delivered into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. As used herein, the term “recombinant expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable base sequence necessary for expressing the coding sequence operably linked in a particular host organism. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. The recombinant vector is preferably a recombinant plant expression vector. Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 116,718 B1) is currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced which properly insert hybrid DNA into the genome of the plant. Preferred forms of Ti-plasmid vectors are so-called binary vectors. 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 can be advantageous, especially when it is difficult to properly transform a plant host. Suitable recombinant expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers (promoter), and are prepared as desired. Can be. The recombinant expression vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable recombinant expression vector, a replication origin. The selection marker is a nucleotide sequence having properties that can be selected by a conventional chemical method, which corresponds to all genes that can distinguish the transformed cells from non-transformed cells. For example, there are herbicide resistance genes such as glyphosate or phosphinotricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol It is not limited to this.

In the recombinant expression vector according to the present invention, the promoter may be, for example, but not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. As used herein, the term "promoter" refers to a genome that regulates the expression of a nucleic acid sequence operably linked in a given host cell, in which one nucleic acid fragment is operably linked to another. Refers to a nucleic acid fragment that is affected by its function or expression by another nucleic acid fragment. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. A "plant promoter" is a promoter capable of initiating transcription in plant cells. The promoter may be a promoter (constitutive promoter) to induce the expression of the target gene at all times at all times or an inducible promoter (inducible promoter) to induce the expression of the target gene at a specific position, time, and conditions, Examples include RD29A promoter, SV40 promoter, CMV promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108: 193-199, 1991; Monahan et al., Gene Therapy, 7: 24-30, 2000), CaMV 35S promoter (Odell et al., Nature 313: 810-812, 1985), Rsyn7 promoter (US patent application Ser. No. 08 / 991,601), rice actin promoter (McElroy et al., Plant Cell, 2: 163-171 , 1990), ubiquitin promoters (Christensen et al., Plant Mol. Biol. 12: 619-632, 1989), ALS promoters (US Patent Application No. 08 / 409,297), and the like. In addition, U.S. Patent Nos. 5,608,149; The promoters disclosed in 5,608,144 5,604,121 5,569,597 5,466,785, 5,399,680 5,268,463, 5,608,142, and the like can all be used. Preferably, an environmental stress inducible promoter, more preferably a dry stress inducible promoter, may be used, such as the RD29A promoter. The terminator can use a conventional terminator, and examples thereof include nopalin synthase (NOS), potato α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens) There are terminators of octopine genes, but the present invention is not limited thereto. Terminator regions are known to increase the certainty and efficiency of transcription in plant cells.

Preferably, the recombinant expression vector provided by the present invention is operably linked to a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 or a gene having a nucleotide sequence represented by SEQ ID NO: 1 downstream of a promoter. , More preferably, a gene having a nucleotide sequence represented by SEQ ID NO: 1, or preferably, the promoter is an inducible RD29A promoter.

Most preferably, the recombinant expression vector provided by the present invention is the pBINRD29A vector shown in FIG. 2.

The present invention provides a transformant having improved dry resistance transformed with the recombinant expression vector.

Transformation in the present invention can be carried out by a variety of transformation techniques known to those skilled in the art. Preferably microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion (PEG) mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method), Agrobacteria spraying method (Agrobacteria spraying method) can be used.

In addition, transformants include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Staphylococcus and Agrobacterium tumefaciens is possible, but is not limited to this.

In addition, the transformant may be a plant or a plant cell, but is not limited thereto. Plants include whole plants, parts of plants, callus, plant tissues, plant cells and plant seeds. The plant may be a plant such as potato, barley, corn, wheat, rye, grass, sugar cane, orchardgrass, corn, and onion. In one embodiment of the present invention, the recombinant expression vector was transformed into Agrobacterium tumefaciens, and inoculated to potato to prepare a transformant.

The present inventors prepared a recombinant expression vector comprising a LEA gene having a nucleotide sequence represented by SEQ ID NO: 1 and transformed it into potatoes to increase the level of intracellular LEA expression. As a result of investigating the characteristics of the overexpressing transformant of LEA, it was confirmed that the plant can be resistant to dry stress by overexpressing the LEA gene (see FIG. 5).

Plants transformed with genes resistant to dry stress according to the present invention can be grown as dry resistant varieties, from which it is possible to supply stable agricultural products without being affected by productivity even under sudden climate change such as global warming.

Figure 1 shows the LEA gene expression according to the stress treatment.
Figure 2 shows the pBINRD29A vector for potato transformation.
Figure 3 shows a StLEA gene overexpressing transformant with the StLEA gene inserted.
Figure 4 is an analysis of the expression of the LEA gene by RT-PCR of the LEA gene transformant.
Figure 5 shows the drying resistance of the potato transformant inserted LEA gene.

Hereinafter, preferred examples and comparative examples are presented to aid in understanding the present invention. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1-1. Sequence analysis of potato-derived StLEA gene

Example 1-1. StLEA Gene Cloning

Total RNA was extracted from the leaves of potato ( Solanum tuberosum) and first-strand cDNA was synthesized using Sprint RT Complete-OligodT (Clontech).

To amplify the LEA gene by RT-PCR, sense primer LEA-S: 5'-ATGGCTCGTTTTTTCTCTAACTCT-3 '(SEQ ID NO: 3) and antisense primer LEA-AS: 5'-TCAGTTGCTTCCAGATCTGTTCTTC-3' (SEQ ID NO: 4) Synthesis was carried out in a PCR reaction solution with rTaq polymerase (Takara) and amplified gene using Applied Biosystem 9700. For RT-PCR, after 10 minutes of denaturation at 95 ° C. for 1 minute, 56 ° C. for 1 minute, and 72 ° C. for 2 minutes, the mixture was treated at 72 ° C. for 10 minutes for expansion. After the PCR reaction, the amplified gene was electrophoresed in 1% agarose, and the band estimated as the LEA gene was cut out from the agarose gel, purified using a QIAquick gel extraction kit, and used for cloning.

Example 1-2. Cloning to Topo 2.1 vector

Clonal PCR was performed by cloning the gene obtained in Example 1-1 into a Topo 2.1 vector (Invitrogen, TOPO TA Cloning kit) and plating white colonies obtained by plating onto X-gal-added LB medium. As a result, the plasmid of the colonies where the band was confirmed was extracted and used for sequencing.

Example 1-3. StLEA gene identification by sequencing

After sequencing using the ABI 3700 sequencer, it was confirmed that the StLEA gene by BLAST search of NCBI.

Example 2 Analysis of Expression Pattern by RT-PCR after Stress Treatment in order to Analyze Expression Characteristics of LEA Gene

 MS (Murashige & Skoog) medium and under 0.1mM ABA, low temperature (4 ° C), salt (250mM NaCl), dry (20% PEG) stress conditions, 30 min, 3 hrs, 12hrs, 24hrs, 48hrs, 5-day The expression of the LEA gene in the case of sustained stress was confirmed. RT-PCR reaction was performed under the same conditions as in Example 1.

As a result, under dry stress conditions (lane 5 in FIG. 1), the expression level gradually increased up to 12 hours after PEG treatment, but the expression amount decreased slightly at 48 hours, but the expression level was maintained again at 5 days. Through these results, the inventors have found that the LEA gene has resistance to dry stress.

Example 3. Construction of Dry Resistance LEA Gene Transformant

Example  3-1. LEA  Production of vector for transformation of gene

Binary vector was completed by connecting the LEA gene isolated from potato to the pBINRD29A vector in which the RD29A promoter isolated from Arabidopsis was inserted into the commercial vector of pBIN121 (FIG. 2).

Example  3-2. Agrobacterium Agrobacterium ) Transformation

To transform the binary vector with the LEA gene into Agrobacterium (LBA4404), repeat the freeze and thaw 2-3 times, and then transformed by heat shock method at 37 ° C overnight, then YEP medium (Yeast 10g, NaCl 5g, peptone 10g, Agar 15g / 1L) and confirmed the colony. Colonies confirmed to be transformed by colony PCR are AB medium (AB buffer (K ₂ HPO ₄ 60g, NaH ₂ PO ₄ 20g / 1L), AB Salts (NH ₄ Cl 60g, MgSO ₄ ㆍ 7H) in order to transform into potatoes ₂ O 6g, and cultured at 3g KCl, CaCl ₂ and _{2H 2 O 0.265g, FeSO 4 .7H} 2 O 50mg / 1L), Glucose 5g / 1L).

Example  3-3. Potato Transformation with Agrobacterium

In order to obtain potato transformants, the potato slices grown in MS medium for 2 days and the Agrobacterium transformed genes grown in YEP medium were incubated together in MS liquid medium for 30 minutes, followed by MS solid medium containing cefotaxime and Kanamycine. Transferred to and incubated for 7 days. It was transferred to callus induction medium and cultured for 7 days, and then cultured in fresh MS medium to which 1/5 Kanamycine was added for 7 days. To induce shooting, transfer to a new medium every two weeks, repeated two times, and after shooting (shooting) when the rooting was subcultured to secure the StLEA gene overexpressing transformant inserted StLEA gene.

Example  4. LEA  Confirmation of Dry Resistance of Gene Overexpressing Transformants

In order to confirm that the LEA gene is an overexpressing transformant, the presence or absence of the LEA gene of the transformant was confirmed by RT-PCR (FIG. 4).

After dry stress was applied to about 7 lines which were confirmed to be an overexpressing transformant of the LEA gene, it was confirmed whether the LEA gene overexpressing transformant had dry resistance. More specifically, after three weeks of subculture in a 22 ℃ culture room and purified for 3-5 days, and then re-cultivated in a greenhouse for 3 weeks to grow about 20 cm and experimented, enough water to horticulture pots in a greenhouse of about 25 ℃ Given the weight of about 300-350g and began to dry, the pot weight weighed about 150g (about 3 weeks after the drying process) to check the degree of withering to confirm the resistance.

As a result, the edible potato 'Sumi' and 'vector (pBINRD29A) -inserted potato, which are known to be highly resistant to stress, withered after three weeks of drying, but the transformant with the LEA gene survived and showed dry resistance (Fig. 5 left). In addition, when water was supplied again after 3 weeks of drying treatment, the transformants containing the Sumi and the vector (pBINRD29A) inserted did not survive, whereas the transformants containing the LEA gene survived again. (Figure 5 right).

<110> Rural development administration <120> Method for enhancing drought stress resistance of plant using LEA          gene <130> P11-088 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 297 <212> DNA <213> solanum tuberosum <400> 1 atggctcgtt ttttctctaa ctctaagatt ctctctgctt tcgtctccga ctccgtttct 60 gcttttctta gcaggcgtgg atatgcggcg gcatcacaag gtgcagtctc cggtgtagcg 120 aaaggaggag tgccaaggag caacgtgatg atgcaaaaaa gtggagagga atccgtgaag 180 acttcatggg tgccagatcc agttaccggt tactacaggc cggaaggaca agcaaatgag 240 attgacgctg ctgaactccg gaagatgctg ttgaagaaca gatctggaag caactga 297 <210> 2 <211> 98 <212> PRT <213> solanum tuberosum <400> 2 Met Ala Arg Phe Phe Ser Asn Ser Lys Ile Leu Ser Ala Phe Val Ser   1 5 10 15 Asp Ser Val Ser Ala Phe Leu Ser Arg Arg Gly Tyr Ala Ala Ala Ser              20 25 30 Gln Gly Ala Val Ser Gly Val Ala Lys Gly Gly Val Pro Arg Ser Asn          35 40 45 Val Met Met Gln Lys Ser Gly Glu Glu Ser Val Lys Thr Ser Trp Val      50 55 60 Pro Asp Pro Val Thr Gly Tyr Tyr Arg Pro Glu Gly Gln Ala Asn Glu  65 70 75 80 Ile Asp Ala Ala Glu Leu Arg Lys Met Leu Leu Lys Asn Arg Ser Gly                  85 90 95 Ser Asn         <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sense primer LEA-S <400> 3 atggctcgtt ttttctctaa ctc 23 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> antisense primer LEA-AS <400> 4 tcagttgctt ccagatctgt tcttc 25

Claims (10)

A method of improving the dry resistance of a plant by increasing the intracellular level of a protein having an amino acid sequence represented by SEQ ID NO: 2. The method of claim 1, wherein the protein having the amino acid sequence represented by SEQ ID NO: 2 is overexpressed to improve the drying resistance of the plant. The method of claim 2, wherein the protein is overexpressed by a method of transforming a plant with a recombinant expression vector linking a gene encoding the protein to a promoter, or introducing a gene encoding the protein into a plant cell. How is characterized by the thing. The method of claim 1, wherein the gene encoding the protein has a nucleotide sequence represented by SEQ ID NO: 1. 4. The method of claim 3, wherein the promoter is an inducible RD29A promoter. The method of claim 1, wherein the plant is potato, barley, corn, wheat, rye, grass, sugar cane, orchardgrass, corn, or onion. A method for producing a dry resistant plant, characterized by transforming a plant into a recombinant expression vector in which a promoter is linked to a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2. A recombinant expression vector linking a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 to a promoter. The recombinant expression vector of claim 8, wherein the recombinant expression vector is a pBINRD29A vector shown in FIG. 2. A transformant having improved dry resistance by transforming with the recombinant expression vector of claim 8.

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