KR20120048050A - Recombinant vector for increasing tolerance to paraquat, salt, and drought stresses, uses thereof and transformed plants thereby - Google Patents

Abstract

PURPOSE: A transgenic plant prepared by a recombinant vector for enhancing resistance to paraquat, salt, and drought is provided to cultivate and harvest crops regardless of climate change/disaster. CONSTITUTION: A recombinant vector for enhancing resistance to stress in a plant contains a gene encoding Oriza sativa-derived OsPUB15 having an amino acid sequence of sequence number 2. The stress includes paraquat, salt, or drought. Cells are transformed by the recombinant vector. The cells are Agrobacterium tumefaciens LB4404. A transgenic plant is transformed by the recombinant vector. The plant is monocot plant, Oryza sativa L. A method for enhancing stress resistance in a plant comprises a step of overexpressing the gene in the plant.

Description

Recombinant vector for increasing tolerance to paraquat, salt, and drought stresses, uses about and transformed plants thus}

The present invention relates to a recombinant vector for increasing resistance to stress, the use of the recombinant vector and a transformed plant by the recombinant vector.

Research is being actively conducted to increase crop yields and increase grain yields by increasing the plant's resistance to biological or abiotic stresses. Korean Laid-Open Patent Publication No. 2006-80235 discloses a method for improving stress resistance in plants and methods thereof. In addition, rice (Oryza sativa L.) is used as a staple in many countries including Asia, and as an important model for plant research, many studies have been actively conducted so far, and Korean Patent Publication No. 2008-107497 describes plant stress. OsRDCP1 gene and rice transformed thereby are described which increase resistance.

Meanwhile, the degree of climate change is getting worse and the agricultural environment is greatly threatened. In order to actively cope with these environmental changes, it is necessary to develop plants that are less sensitive to environmental changes.

Therefore, the present inventors have improved the climate adaptability of crops due to subtropical climate change of the Korean Peninsula due to global warming and irregular rainfall, and minimized the damage caused by increasing climate disasters, thereby increasing the crop cultivation area and providing a smooth supply of food. For the expected development of plants, while studying the environmental stress resistance of plants, it was confirmed that plants transformed with the OsPUB15 gene derived from rice have increased resistance to biological or abiotic stresses. Completed.

It is an object of the present invention to provide a recombinant vector comprising the OsPUB15 gene for increasing the plant's resistance to stress such as paraquat, salt, and drought.

Another object of the present invention is to provide a plant with increased resistance to stress transformed with the recombinant vector.

Another object of the present invention is to provide a method of increasing the resistance to stress by overexpressing the gene in plants.

Another object of the present invention is to provide a transgenic plant and its seed having increased stress resistance by the above method.

The present invention relates to a recombinant vector for increasing resistance to paraquats, salts, and droughts containing OsPUB15 gene, the use of the recombinant vector, and a transformed plant by the recombinant vector.

The present inventors first confirmed that the OsPUB15 protein (SEQ ID NO: 2) exhibits high salinity and resistance to drought stress by reducing the amount of Reactive Oxygen Species (ROS) in plants. To provide.

The present invention provides a recombinant vector for increasing resistance to stress, such as paraquat, salt, and drought, of plants, including the OsPUB15 gene.

The recombinant vector of the present invention is a vector that overexpresses OsPUB15 protein in a plant to be transformed, and is preferably a recombinant plant expression vector.

The OsPUB15 gene included in the paraquat, salt, and recombinant vector for increasing tolerance to drought of the present invention includes a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1, and a fragment or variant thereof functionally equivalent.

OsPUB15 gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 as used herein was named according to the nomenclature of [Zeng et al., (2008) Mol Plant 1: 800-815]. OsPUB15 cDNA (SEQ ID NO: 1) is 2475 bp long, which encodes 824 amino acid polypeptides (SEQ ID NO: 2), and also consists of five exons and four introns, as shown in FIG. It has a U-box domain that exhibits Izu enzyme activity and an amidillo repeat domain capable of protein-protein interaction at the C terminus.

As used herein, a "functionally equivalent fragment" with the OsPUB15 gene encodes a polypeptide from which one or more amino acids are deleted or truncated in the amino acid sequence of a wild type OsPUB15 protein consisting of the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide is substantially identical to the wild type OsPUB15 protein. It means a fragment or a portion of the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1, showing an equivalent effect. Such nucleic acid fragments are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, compared to the nucleotide sequences set forth in SEQ ID NO: 1 With 96%, 97%, 98%, 99% or more sequence homology, such nucleic acid fragments can be readily produced by molecular biological methods well known in the art.

As used herein, a "functionally equivalent variant" of an OsPUB15 gene encodes a polypeptide having one or more amino acids modified (eg, substituted, added or deleted) in the amino acid sequence of a wild type OsPUB15 protein consisting of the amino acid sequence of SEQ ID NO: 2, It refers to a variant of the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1, the polypeptide shows an effect substantially equivalent to the wild type OsPUB15 protein. Variants of such nucleic acids are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% compared to the nucleotide sequences set forth in SEQ ID NO: 1 , 96%, 97%, 98%, 99% or more sequence homology, and such variants can be readily produced by molecular biological methods well known in the art.

The term "vector" refers to DNA fragment (s), nucleic acid molecules that are delivered into a cell, which can replicate DNA and be independently reproduced in a host cell. An "expression vector" refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Expression vectors can include one or more origins of replication, promoters, selection markers, enhancers, termination signals, and polyadenylation sequences available in eukaryotic cells. Expression vectors are generally derived from plasmid or viral DNA, or may include elements of both. Thus, expression vectors refer to recombinant DNA or RNA constructs such as plasmids, phages, recombinant viruses or other vectors that result in the expression of cloned DNA upon introduction into an appropriate host cell. Suitable expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic and / or prokaryotic cells and those that remain episomal or that integrate into the host cell genome.

As used herein, any plant expression vector capable of being introduced into a transformed plant to express OsPUB15 protein is available, and such recombinant plant expression vectors can be constructed using conventional techniques known in the art.

Preferred examples of plant expression vectors used in the present invention are Ti-plasmid vectors capable of transferring part of itself, the so-called T-region, to plant cells when present in a suitable host, such as Agrobacterium tumerfaciens. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts, which can produce current plant cells, or new plants that properly insert hybrid DNA into the plant genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce OsPUB15 genes according to the invention into plant hosts are viral vectors such as those derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc., For example, it may be selected from an incomplete plant viral vector. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

Plant expression vectors of the invention also include selection markers, promoters and terminators.

Markers usable in the present invention are typically nucleic acid sequences having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as glyphosate, herbicide resistance genes such as phosphinothricin, kanamycin, G418, bleomycin, hygromycin, chloramphenicol It doesn't happen. Preferably, the Basta resistance (BAR) gene is used.

Promoters usable in the present invention include promoters capable of initiating transcription in plant cells, including CaMV35S, actin, ubiquitin, pEMU, histone promoters, and the like, and preferably CaMV35S promoters.

Terminators usable in the present invention may be terminators commonly used in the art, such as nopalin synthase (NOS) terminator, rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tome Terminator of the octopine gene of agrobacterium tumefaciens, rrnB1 / B2 terminator of Escherichia coli, and the like, and preferably, a NOS terminator is used.

In particular, a preferred recombinant vector in the present invention is a plant expression recombinant vector comprising a BAR gene, a CaMV35S promoter and a North (NOS) terminator, having a herbicide resistance between two T-DNA borders, and in which the OsPUB15 gene is operably linked to the promoter. . The vector according to an embodiment of the present invention is the pCB302 vector described in FIG. 2 (see Plant Mol. Biol. 40: 711-717).

As used herein, operably linked genes and promoters refer to conditions in which nucleic acid sequences are linked to perform their intended function. For example, operably linking a promoter sequence to a desired nucleotide sequence refers to linking the promoter sequence to the desired nucleotide sequence in a manner that enables the promoter sequence to direct transcription of the desired nucleotide sequence.

The recombinant vector may be prepared by recombinant means using recombinant DNA techniques known in the art.

The present invention also provides cells transformed with said paraquats, salts, and recombinant vectors for increased tolerance to drought.

Such recombinant vectors can be introduced into cultured host cells using well known techniques such as infection, transduction, transfection, electroporation and transformation. Representative examples of hosts include bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium cells; And plant cells.

"Plant cells" used for plant transformation are any plant cells available. Plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells. "Plant tissue" refers to a tissue of differentiated or undifferentiated plant, including but not limited to roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used for culture, protoplasts, shoots and callus tissue.

Suitable culture media and conditions for host cells are well known to those skilled in the art. In the present invention, it is preferable to use microorganisms of the genus Agrobacterium for use in plant transformation.

The present invention also provides a transgenic plant with increased resistance to paraquats, salts, and drought, transformed with the recombinant vector described above.

Transformation of a plant by the recombinant vector of the present invention may be performed by any method known in the art for transferring DNA to plants. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both dicotyledonous plants as well as monocotyledonous plants. For example, 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) , Electroporation of protoplasts (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179 -185), Agrobacterium by particle bombardment of various plant elements (DNA or RNA-coated) (Klein TM et al., 1987, Nature 327, 70), invasion of plants or transformation of mature pollen or vesicles Agrobacterium tumefaciens mediated gene transfer ( incomplete ) can be appropriately selected from infection by virus (EP 0 301 316) and the like. Preferably Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

Optimal procedures for the transformation of plants with Agrobacterium vectors vary depending on the type of plant to be transformed. Exemplary methods for Agrobacterium mediated transformation include transformation of explants of embryonic axis, shoot tip, stem or leaf tissue, derived from sterile seedlings and / or small plants. Such transformed plants can be reproduced by sexual reproduction or by cell or tissue culture. Agrobacterium mediated transformation has previously been described for many different kinds of plants and methods for such transformation can be found in literature known in the art.

Agrobacterium strains for plant transformation that can be used in the present invention can be used any strains commonly used. Specifically, in the present invention, Agrobacterium tumefaciens LB4404 ( Agrobacterium tumefaciens LB4404), which is known to have high pathogenicity necessary to penetrate plant samples and transfer foreign genes, may be used. .

Plant transformed in the present invention is used in the present Example ( Oryza Dicotyledonous plants as well as monocotyledonous plants including sativa L. ) can be used without limitation. Preferably, monocotyledonous plants, more preferably rice ( Oryza) sativa L. ) is used.

Plants may be transformed with a recombinant vector comprising the OsPUB15 gene according to the present invention, and then obtained through an asexual propagation method, which is a process of induction of callus, rooting and soil purification according to a conventional method. That is, Agrobacterium transformed with the recombinant vector containing the OsPUB15 gene is supplied to the Kelus-derived fragment.

The invention also provides a method of increasing resistance to stress by over-expressing the OsPUB15 gene in plants. The stress corresponds to paraquat, salt, drought. The method of overexpressing the OsPUB15 gene in a plant may be performed by introducing the OsPUB15 gene into a plant including the OsPUB15 gene or into a plant not containing the OsPUB15 gene. The "overexpression of the gene" means that the OsPUB15 gene is expressed above the level expressed in wild-type plants. As a method of introducing an OsPUB15 gene into a plant, there is a method of transforming a plant using an expression vector containing an OsPUB15 gene which is controlled by a promoter. The promoter is not particularly limited as long as it can overexpress the insertion gene in the plant, such as actin, ubiquitin, and 35S promoter.

The present invention also provides a method for producing a plant having increased resistance to paraquats, salts, and droughts, comprising transforming a plant with the recombinant vector.

The method of transforming a plant with the recombinant vector can be carried out using any of the methods known in the art, as well as the methods mentioned herein above.

Plants transformed with the recombinant vector comprising the OsPUB15 gene of the present invention exhibit increased resistance to paraquats, salts, drought stress, and thus, cultivation and grain harvesting, etc. are advantageous despite climate change / disaster.

Figure 1 shows exons and introns in the nucleotide sequence of the OsPUB15 gene. The OsPUB15 gene has five exons and four introns, and has a ubox and amadillo repeat domain. Gray boxes represent exons and dark gray lines represent introns and untranslation regions (UTRs).
Figure 2 shows a diagram of the vector used for the production of overexpressed plants of OsPUB15 gene and the size and description of the gene in the vector used.
Figure 3 shows the expression analysis results of OsPUB15 gene in abiotic stress conditions.
Figure 4 confirms the E3 ligase enzyme activity of OsPUB15 protein using an antibody specific for MBP.
5 confirms the E3 ligase enzyme activity of OsPUB15 protein using an antibody specific for Ub.
Figure 6 shows the analysis of the expression of the transgenic rice over-expressed OsPUB15 gene by real-time PCR.
Figure 7 shows the phenotype of plants upon treatment with 250 nM paraquat (FIG. 7A), 250 mM NaCl (FIG. 7B), drought (FIG. 7C) for wild type rice and OsPUB15 overexpression.
FIG. 8 shows wild-type rice and over-expression of wild-type rice and OsPUB15 overexpression in 1/2 MS medium for 5 days of treatment (Mock), 250 nM paraquat for 5 days, and over-expression of wild-type rice and over-expression of wild-type rice. The sieve is shown by comparing the amount of hydrogen peroxide (H ₂ O ₂ ) between the experimental groups treated with 250 mM NaCl for 2 hours.
FIG. 9 shows the amount of oxidized protein via DNPH assay at 0, 0.5, 1, 2, 6, and 12 hours after 20 μM paraquat treatment for 7 day old wild type rice and OsPUB15 overexpression.
10 is a schematic diagram showing a cleavage map of a pMAL vector.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only examples to help understanding of the present invention, and the scope of the present invention is not limited thereto.

[ Manufacturing example ]

For hydrogen peroxide, salt, drought stress OsPUB15 Observation of Expression

Seeds of wild rice were grown for one week in a sealed transparent plastic container containing 1/2 MS medium in a growth chamber with 30 ° C. and 40% relative humidity.

Thereafter, 1 mM H ₂ O ₂ in a growth chamber with the same temperature and humidity Transfer the plant grown for 15 days on the solution, 250 mM NaCl solution, and paper towels for 7 days, and then cut the leaves four times, three times at the first 0, 30, 60, and 120 minutes. Was extracted.

After extracting RNA, the leaves were cut into 2 mL tubes, frozen in liquid nitrogen, crushed finely with a mixer (Retsch MM300), and mixed with 1 mL of RNAiso Plus (TaKaRa). Thereafter, the mixture was left at room temperature for 5 minutes, and then 0.1 mL of BCP (Sigma) was added thereto, and the mixture was shaken again and left for 5 minutes at room temperature. The centrifuge was then centrifuged at 13000 rpm for 15 minutes at 4 ° C. and then the supernatant was transferred to a new 1.5 mL tube. 0.5 ml IPA (JUNSEI) was added to the transferred sample, mixed, and then centrifuged in a centrifuge at 13000 rpm for 10 minutes, and the remaining pellet was lightly washed with 70% alcohol.

The pellet was dissolved in 50 µl of distilled water and RNA was quantified to synthesize cDNA using 4 µg of RNA as a template strand. Ubiquitin primers [F2 5′-ACCATACCAGAAGGGAGGA-3 ′ (forward) and R1 5′-CGGGAATTCAGCAAGGAGA-3 ′ (forward)] and OsPUB15 primers [F3 5 ′-GGAATTCAGCAAGGAGAT-3 ′ (forward) and R3 5′-TGCCCTGCATGTTGTACC-3 ′ (reverse)] was used to perform Real-Time PCR (Corbett). Relative expression levels of OsPUB15 were determined based on the expression of ubiquitin (FIG. 3).

As shown in Figure 3, the increase in the expression of OsPUB15 gene was observed under stress conditions such as hydrogen peroxide, salt, drought. It was confirmed that the expression was significantly increased in the case of low temperature and drying. In particular, expression was highest between 1 and 2 hours after stress treatment. OsPUB15 gene was determined to be a gene that functions in relation to external stress, and the following experiment was performed to confirm the function related to external stress of OsPUB15 protein.

[ Example ]

Example  One. OsPUB15  gene Cloning  And production of recombinant vectors for plant expression

OsPUB15 gene was obtained from a rice-based Knowledge-based Oryza Molecular Biological Encyclopedia (KOME) cDNA and PCR was performed using the cDNA plasmid as a template strand.

To clone the OsPUB15 gene, a forward primer (5'-ATGGAAAATTTCTCCCCGAG-3 ') (SEQ ID NO: 3) and a reverse primer (5'-TCATCTCCTTGCTGAATTC-3' (SEQ ID NO: 4)) were prepared. The primers were designed to include a pCB302 vector (binary vector for overexpressing OsPUB15 under the 35S promoter in plants) and a BamHI linker and StuI linker for insertion into pMal-c2 (MBP intrinsic OsPUB15 protein purification vector).

PCR was performed using the primers.

In the ^{GeneAmp ® PCR System 9700 (Applied Biosystems} ) to the template strand of the cDNA of rice using Pfu taq D. Nature 95 ℃ as the primer in the PCR device, 57 ℃ annealing, extension 72 ℃ condition was amplified for 36 cycles. The PCR product thus obtained was attached to a 3 'strand of the amplified strand using Taq pol again and ligated to T-easy vector (Promega) at 14 degrees for 12 hours. The ligation product was mixed with 50 μl of Top10 E. coli competent cells, transferred to a 1.5 ml tube and left on ice for 30 minutes. It was then left in the 37 ° C. oven for 1 minute and then an additional 1 mL of LB liquid medium was added to the tube and left in a 37 ° C. shaking incubator for 1 hour. Subsequently, the resulting colonies were plated in an empicillin resistant LB solid medium and waited for 12 hours to incubate for 12 hours in 1 mL of LB liquid medium.

The cultured cells were centrifuged at 12,000 rpm for 5 minutes at room temperature. The supernatant was then removed, followed by solI (50 mM glucose, 25 mM Tris-Cl PH8.0, 10 mM EDTA), sol II (0.2% NaOH, 1% SDS), and sol III (5 M potassium acetate, glacial The cell wall was disrupted using acetic acid (PH 4.8). After centrifugation at 12,000 rpm for 10 minutes at room temperature, put the supernatant into 1.5 mL new tube, add 95% EtOH and mix, wash the pellet obtained by centrifugation at 12,000 rpm at room temperature for 10 minutes, and then wash it with 70% EtOH. Dissolve in water. (Hereinafter, this process is called "mini-prep")

The recovered DNA was digested with restriction enzymes BamHI and StuI, and then subjected to agarose gel separation by electrophoresis, and cut out a band of OsPUB15 size. The pCB302 vector (pCB302 vector is a PMB (Plant Mol. (See Biol. 40: 711-717), and FIG. 2 for schematic diagram) and ligation were performed at 14 ° C for 12 hours. The ligation product was mixed with 50 μl of Top 10 E. coli competent cells, transferred to a 1.5 ml tube and left on ice for 30 minutes. Subsequently, 1 minute of standing again in a 37 degree oven, 1 ml of LB liquid medium was further added to the tube, and then left in a 37 degree shaking incubator for 1 hour. Subsequently, the resulting colonies were plated in kanamycin resistant LB solid medium and waited 12 hours for miniprep after cell culture in 1 mL of LB liquid medium.

The DNA obtained by miniprep was digested with agarose gel after enzymatic digestion using restriction enzymes BamHI and StuI and the size bands of OsPUB15 and pCB302 vectors were identified. The cloned DNA thus obtained was subjected to sequencing again to select a DNA in which no error occurred. The recombinant vector thus obtained was named pCB302-OsPUB15 vector.

Example  2: preparation of recombinant vector for protein purification and OsPUB15  Protein isolation

Polymerase Chain Reaction (PCR) was performed using the primers for the OsPUB15 gene described in Example 1.

PCR product obtained by amplifying for 36 cycles at 95 ° C., annealing 57 ° C., and extension 72 ° C. using DNA primer as a template strand using Pfu taq in GeneAmp ^® PCR System 9700 (Applied Biosystems) PCR instrument. The aqine was attached to the 3 'strand of the amplified strand using Taq pol again and the ligation to the T-easy vector (Promega) proceeded at 14 ℃ for 12 hours. The advanced ligation product was mixed with 50 µl of Top10 E. coli competent cells, transferred to a 1.5 ml tube, and left on ice for 30 minutes. Subsequently, after 1 minute of standing in a 37 ° C. oven, 1 mL of LB liquid medium was further added to a tube and left in a 37 ° C. shaking incubator for 1 hour. Subsequently, the resulting colonies were plated in an empicillin resistant LB solid medium and waited 12 hours for miniprep after 12 hours of cell culture in 1 mL of LB liquid medium.

DNA obtained from the miniprep was digested with restriction enzymes BamHI and StuI, agarose gel was separated by electrophoresis, and cut out an OsPUB15 size band, and then the ligation with the same enzyme-cut pMAL vector (see FIG. 10) was performed. It proceeded at 14 degreeC for 12 hours. Ligation products are mixed with 50 μl of Top10 E. coli competent cells, transferred to a 1.5 ml tube, left on ice for 30 minutes, then placed in an oven at 37 ° C. for 1 minute, and then additional 1 ml of LB liquid medium is added to the tube. After standing in a 37 ° C. shaking incubator for 1 hour, the cells were plated in an empicillin resistant LB solid medium and waited for 12 hours. The resulting colonies were miniprepped after cell culture in 1 mL of an ampicillin LB liquid medium.

DNAs obtained from the minipreps were digested with agarose gels by electrophoresis after restriction enzymes BamHI and StuI, and then OsPUB15 size bands and pMAL vector size bands were identified. The cloned DNA thus obtained was subjected to sequencing again to select a DNA in which no error occurred. The recombinant vector thus obtained was named pMAL-OsPUB15 vector.

After transforming the selected pMALC2-OsPUB15 vector DNA back into BL21 strain E. coli, the resulting colonies were selected after smearing into an empicillin LB solid medium, and 1 mL LB liquid medium was added to the test tube and grown in a 37 ° C shaking incubator for 12 hours. After subcultured again in 50 mL LB liquid medium, the cells were grown for about 3 hours (absorbance value of 0.5 to 0.6) in a 37 ° C shaking incubator, followed by induction by addition of IPTG 3 mM (Duchefa).

Thereafter, the mixture was raised in a 28 ° C. shaking incubator for 8 hours, centrifuged at 3500 rpm for 5 minutes, and then subjected to sonication twice for 2 minutes by adding 1 mL lysis buffer (Qiagen) to grind the cell walls. After centrifugation at 3500 rpm for 10 minutes, the supernatant was mixed with 500 µl MBP Exellose ^® (Bioprogen) and mixed for 1 hour in a low temperature room at 4 ° C. After 1 hour after spin down, the supernatant was discarded, and 1 mL of washing buffer (Qiagen) was added to spin down, the supernatant was removed, and the washing buffer was exchanged 5 times every 10 minutes. After removing the supernatant at the last time, 200 μl elution buffer (Qiagen) was added, followed by rotation for 30 minutes, mixing, spin down, and the supernatant taken to determine the band of MBP-OsPUB15 on the PAGE gel after protein quantification through a Bradford assay.

Example  3: Construction of Transgenic Cells

The plant expression vector (pCB302-OsPUB15 vector) prepared in Example 1 was extracted.

50 μl of Agrobacterium competent cell (Agrobacterium tumefaciens LB4404) and 2 μg of pCB302-OsPUB15 vector were mixed and placed in a 4 mm cuvette for electroporation. The cuvette was transformed into an electroporator (Bio-rad, USA) and subjected to an electric shock of 300 V and 450 kV. The transformed mixture was placed in 1 mL of YEP liquid medium and left in a shaking incubator for 2 hours, then plated on solid YEP medium (Kanamycin 50 mg / mL) and incubated at 28 ° C. Subsequently, the resulting colonies were grown in 1 mL kanamycin resistant YEP medium for 12 hours, followed by BamHI and StuI restriction enzyme digestion with DNA obtained after miniprep to confirm the size of OsPUB15.

The resulting transformed Agrobacterium was used in the transformation experiment of rice.

Example  4: Construction of Transgenic Plants

Seeds of Dongjin rice in MS solid medium were grown in a growth room at 28 ° C. in a dark condition for about 30 days to generate calli of rice. The resulting callus was mixed with cells grown for 72 hours with Agrobacterium transformed with the pCB302-OsPUB15 vector obtained in Example 3, and left in a 22 ° C. dark growth chamber in MS-Acetosyringone solid medium.

Rinse Agrobacterium-contaminated callus cleanly with tertiary distilled water about five times, and then basta selection of MSD solid medium over primary (phosphinethricin (PPT) 5 mg / L) and secondary (PPT 10 mg / L). Proceed through subculture. Selection took place for four weeks, two weeks each in a 28 ° C. dark growing condition. Transfer the callus to MS-7 (PPT 10 mg / L) solid medium and raise for 10 days in 28 ° C dark condition growth room and divide the divided callus in MS-RI (PPT 10 mg / L) solid medium, regeneration medium for 2 weeks Callus was continued to grow in a 28 ° C. light growing room. Subsequent regeneration of callus to MS solid medium in MS-RII (PPT 10 mg / L) solid medium for 2 weeks at 28 ° C. light growing cell, transfer to MS solid medium and grow in greenhouse at 7 ° C. I grew a plant.

In order to confirm the overexpression of OsPUB15 gene in the obtained regeneration plants, RNA was extracted from the leaves of the regeneration plants. After RNA extraction, the leaves were cut into 2mL tubes, frozen in liquid nitrogen, crushed finely with a mixer (Retsch MM300), and then 1mL of RNAiso Plus (TaKaRa) was added and mixed. After 5 minutes at room temperature, BCP (Sigma) 0.1 mL was added and shaken again, and then left to stand at room temperature for 5 minutes.

The centrifuge was then centrifuged at 4 ° C., 13000 rpm for 15 minutes and the supernatant was transferred to a new 1.5 mL tube. 0.5 ml IPA (JUNSEI) was added to the transferred samples, mixed, and centrifuged again at a centrifuge for 10 minutes at 4 ° C and 13000 rpm. The remaining pellets were lightly washed with 70% alcohol.

The pellet was dissolved in 50 μl of distilled water, followed by RNA quantification to synthesize cDNA using 4 μg of RNA as the template strand, and cDNA as the template strand with ubiquitin primers [F2 (forward) 5′-ACCATACCAGAAGGGAGGA-3 ′ and R1 (reverse) Real-Time PCR (Corbett) was performed with 5′-CGGGAATTCAGCAAGGAGA-3 ′] and OsPUB15 primers [F3 (forward) 5′- GGAATTCAGCAAGGAGAT-3 ′ and R3 (reverse) 5′-TGCCCTGCATGTTGTACC-3 ′]. Relative expression levels of OsPUB15 were determined based on the expression of ubiquitin. As a control, mRNA expression levels of OsPUB15 and ubiquitin in wild-type plants were confirmed by the same method as above. As a result, it was observed that the expression increased especially in 22 of 36 plants (Fig. 6).

[ Experimental Example ]

Experimental Example  One: OsPUB15  Enzyme Activity Analysis of Proteins

The ubiquitination assay of OsPUB15 protein isolated in Example 2 was performed.

Specifically, in order to analyze the enzymatic activity of OsPUB15 protein, human UBC5c (Sigma) and Arabidopsis UBA1 (At2g30110) were used as sources of E1 and E2, respectively.

OsPUB15 protein was constructed by fusion to maltose binding protein (MBP), so 200 ng hUBC5c (Sigma), AtUBA1 100 ng, 40 mM Tris-Cl (Sigma) pH 7.5, 5 mM MgCl ₂ in vitro using MBP-OsPUB15 protein (Sigma), 2 mM ATP (Sigma), 2 mM dithiothreitol (DTT) (Sigma), 300 ng / ml ubiquitin (Sigma), and 1 µg of MBP-OsPUB15 protein were reacted at room temperature for 1 hour using E3 ligase. . After the reaction, the MBP-OsPUB15 mixture was boiled in 100 ° C. water for 5 minutes and then placed on ice. After cooling, the mixture was hung on PAGE, and then Western blot was performed using MBP antibody and Ub antibody to detect signals.

As shown in FIG. 4, OsPUB15 cloned into pMAL-C2 vector was fused to MBP (maltose binding protein), and E1, E2, and Ubiquitin were detected when Western blot was performed with MBP antibody. Ladder-shaped bands appeared only when they existed.

The same result was obtained when Western blot was performed with Ubiquitin antibody (FIG. 5). It was found that the MBP-OsPUB15 protein was autoubiquitated. Therefore, it was observed that OsPUB15 protein has E3 ligase enzyme activity.

Experimental Example  2: stress ( Paraquat Resistance to salts, salts and droughts)

(1) sample growth

For stress treatment, the transgenic plants prepared in Example 4 were grown to third generation and used for the following experiments. The conditions for growing rice plants were the same until the stress treatment described below.

Add agar to 1/2 MS salt medium (MS salt including vitamin 2.2g, sucrose 30g per L) to make a solid medium, add 250 nM paraquat and 100mM NaCl as needed in the experiments below. Brown rice seeds were grown in a plant growth room which was automatically adjusted to 40% relative humidity, 30 degrees, 18 hours light condition and 6 hours dark condition.

After growing the brown rice seeds in the growth room for 7 days, each growth size was measured or used for the next stress treatment.

(2) stress ( Paraquat Treatment, salt, drought)

① On paraquat  Tolerability

The wild type brown rice seeds grown for 7 days above were grown in 1/2 MS salt solid medium containing 250 nM paraquat for 1 week, and the length of each rice paddy was measured (FIG. 7A).

As shown in FIG. 7A, four OsPUB15 overexpressing plants (Ox14, Ox17, Ox22) were grown larger than four wild-type rice under paraquat treatment conditions.

② Check for resistance to salt

The salt treatment was observed for each time period after submerging the young rice grown in the solid medium for 7 days in 250 mM NaCl solution, respectively, and the recovery of the rice was observed by transferring to NaCl-free water (Fig. 7B).

Wild type rice and OsPUB15 overexpressing plants grown for 7 days were placed in 250 mM NaCl for 24 hours and then transferred to water for 72 hours. As shown in FIG. 7B, it was confirmed that four wild-type rice (WT) cells did not have new leaves, whereas the four overexpression (Ox17) four new leaves appeared.

③ Confirmation of drought tolerance

Drought treatment was performed after 45 days of 25 ° C, 55% relative humidity, 14 hours of light condition and 10 hours of dark condition. Drought treatment was performed, and water was again observed to recover rice (FIG. 7C).

As shown in FIG. 7C, wild type (WT) rice showed a bleaching phenomenon and rapidly withered, whereas OsPUB15 overexpressing plant (Ox17) withered the leaf tip and showed green leaves. Under the conditions of re-supply of water after 3 days of drought treatment, wild-type rice also failed to recover and bleached with leaves, while OsPUB15 overexpressed leaves were dried but still greened throughout the leaves.

(3) Determination of hydrogen peroxide in plants during stress treatment

The wild-type rice and overexpression samples treated with each stress above were ground in a grinder using liquid nitrogen and pulverized, and then 0.5 ml of hydrogen peroxide extract buffer solution (0.05 mM sodium phosphate buffer, pH 7.4) per 0.1 g of sample was added. Put it in and mix well. At 13000 rpm for 10 minutes and then centrifuged for 50 μl of the supernatant was allowed to stand at room temperature for 1 hour dark conditions in the mixing ^{Amplex ® Red reagent / HRP working solution} (Invitrogen) in 50 μl. Then, the electrons were excited at a wavelength of 530 nm in the absorption spectrum, and the wavelength was measured at 560 nm in the emission spectrum.

Wild-type rice and overexpression treated with wild type (WT) rice and OsPUB15 overexpressing plants (Ox14, Ox17, Ox22) for 5 days in 1/2 MS medium, 250 nM paraquat for 5 days The 7-day-old wild-type rice and overexpression were shown by comparing the amount of hydrogen peroxide (H ₂ O ₂ ) between the experimental group treated with 250 mM NaCl for 2 hours (FIG. 8). OsPUB15 overexpression The amount of hydrogen peroxide in the plant was confirmed to be less than the wild type. Thus, it was found that OsPUB15 overexpressing plants were more resistant to stress.

(4) Determination of oxidized protein in plants during stress treatment

Samples of wild-type rice and overexpressing samples grown for 7 days were sampled at 0, 0.5, 1, 2, 6, and 12 hours after 20 μm paraquat treatment, ground in a grinder using liquid nitrogen, and then ground into powder 0.3. 1 ml of extract solution (10 mM NaCl, 10 mM MgCl ₂ , 5 mM EDTA, 10 mM β-mercaptoethanol, 1 mM PMSF, and 25 mM Tric-HCl, pH 7.5) was added and mixed well.

After centrifugation at 13000 rpm for 10 minutes, 5 μl of the supernatant was used to quantify the protein through the Bradford assay. Subsequently, transfer 1 mg of protein into each of the two tubes and add 1 mL of 2 N HCl in one and 1 mL of 2 N HCl with 10 mM 2,4-dinitirophenylhydrazine (DNPH) in another. It was.

After standing at room temperature for about an hour, 10% trichloroacetic acid was added and left for an additional hour on ice. The tube was centrifuged at 13000 rpm for 15 minutes and the precipitate remaining in the tube was washed with 1: 1 ethanol: ethyl acetate.

The precipitate was dissolved in 20 mM sodium phosphate solution containing 6 M guanidine-HCl. The absorbance of the solution in which the precipitate was dissolved was measured in a spectrum of 370 nm. The amount of oxidized protein was calculated by subtracting the absorbance obtained by 2 N HCl from the absorbance obtained by 2 N HCl-DNPH, and multiplying by the extinction coefficient 22,000. As time passed, it was confirmed that the amount of oxidized protein in wild-type plants was further increased compared to OsPUB15 overexpression. OsPUB15 overexpression was found to show increased resistance to stress.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Recombinant vector for increasing tolerance to paraquat, salt,          and drought stresses, uses approximately and transformed plants <130> P10-041-KHU <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 2475 <212> DNA <213> Oryza sativa <220> <221> gene (222) (1) .. (2475) <223> OsPUB15 cDNA <400> 1 atggaaaatt tctccccgag aaccctgctc aatagtatct tgcgtatcac tgtcttaacc 60 tccgatggct ctactgcaag gcccaagccc attcagaagt actgccaaaa tgtgtgtgat 120 atctcaagca ttgtgagccc tctcatagag gatctatgtg agtctcctga agagcaactc 180 aatgaggtgt taagggagct tggcactgct attaacagag cttcagggct tattgggaac 240 tggcaacaga caaccagcaa aatatatttt atatggcaga ttgaatcagt aatctcagat 300 atccagggat gttctctaca gctgtgccag cttgttaact ctctattacc ttctttgact 360 ggccgtgcat gcacatgtat tgagaaactc caagacataa attatgaaaa catgtttgat 420 ctggtaaagg agtcttcatt ggagctagtt gagacggaca caacaagtcc tgagaatctg 480 tcgagactat ctagttcatt gagtttgtca actaacctgg aattttacat ggaagctgtt 540 tcccttgaga atctcagagc aagggcaatg cggagtgaga accgtgaaga aatggatctg 600 gctgacaaga tgatccccct ggtcaactat atgcatgacc accttctgag ggaaacacaa 660 ctgcttagca tcaatggggt gcccattcct gcagattttt gctgcccgct gtccctagag 720 ctgatgtcag atcctgttat tgtagcatct ggtcagacat atgagcgggt ttatatcaag 780 ttatggcttg atgagggttt tactatctgc ccgaagacac gccaaagact tggtcactcc 840 aatttaattc caaattacac cgtgaaagct ttgatagcta attggtgcga atcacacaac 900 attaggcttc ctgatcctat gaaatccttg aaattgaact tccctttggc tgcgtctgct 960 ctccaggatt cgagcaccac aggaagcagc cctctacatc ctactgtcgc tgctaagggt 1020 aatattcctg ggtccccgga agctgacctt tatatgagaa gcttgaatag agcatctcct 1080 ccacacagtg tagtccatca gaattctcat gcgcatgtga accgtgctgg tcatgaagcc 1140 tccattaagc aatcttcaga aaatgctaat ggttctgcat cagatgtttc aaggttatct 1200 cttgcaggtt ctgaaacaag agagtctagt ctggaagaaa gaaatgctgg ttctatcggt 1260 caaacttcag aacagtcaat tgaggaagca tttcaagcat ctaatttgga cagggattca 1320 catgaccatg tgggtagttc ttcggtgaat ggtagccttc caaatagcgg tcaacttgat 1380 gcagaatgtg acaatgggcc aagcgaaagg acaaattaca gtagtgatgc atctggagaa 1440 gttacagatg ggccttcagc atcttctgct cctcagaggg agcatctaat cccttctaga 1500 ttggctgatg ttcgtagtag aggccaattt gttcggcgac catctgaaag gggtttcccc 1560 agaataatat cttcctcatc catggataca cggagtgatc tttccgccat cgagaatcag 1620 gtccgcaagt tagttgatga tttaagaagt gattctgtag atgttcaaag atcagcgaca 1680 tcagatatcc gccttttagc taagcacaac atggagaaca ggatcatcat tgcaaactgt 1740 ggagctataa acttgctggt tggtcttctt cattcgccag attccaaaac ccaagagcat 1800 gccgtgacag cccttctgaa tttgtcaatc aatgataata ataagattgc cattgcaaat 1860 gctgatgctg ttgaccccct catccatgtc cttgagactg ggaaccctga agccaaggag 1920 aattcagcgg ctacattatt cagtctctcg gttattgaag aaaacaaagt gaggattgga 1980 agatccggtg ccatcaaacc tctcgtcgac ctactaggaa atgggacccc tcgaggaaag 2040 aaagatgcag ctactgcatt gtttaattta tccatattac atgagaacaa ggcgcgtatt 2100 gtgcaggctg acgctgtgaa gtacctagtt gaacttatgg accctgctgc tggaatggtt 2160 gacaaagctg tggctgtttt ggcaaacctt gctaccatac cagaagggag gacagcaatt 2220 ggtcaagcgc gtggtattcc agcccttgtt gaagttgttg aactcggttc agcaaggggg 2280 aaggaaaatg cggctgcagc attgcttcag ctatgtacaa acagcagcag attttgcagt 2340 atagttcttc aagagggtgc tgtgcctcct ctagttgcat tgtcacagtc aggcacgcca 2400 cgggcaagag agaaggcaca ggctcttctc agctactttc gcagccaaag gcacgggaat 2460 tcagcaagga gatga 2475 <210> 2 <211> 824 <212> PRT <213> Oryza sativa <220> <221> PEPTIDE (222) (1) .. (824) <223> OsPUB15 protein <400> 2 Met Glu Asn Phe Ser Pro Arg Thr Leu Leu Asn Ser Ile Leu Arg Ile   1 5 10 15 Thr Val Leu Thr Ser Asp Gly Ser Thr Ala Arg Pro Lys Pro Ile Gln              20 25 30 Lys Tyr Cys Gln Asn Val Cys Asp Ile Ser Ser Ile Val Ser Pro Leu          35 40 45 Ile Glu Asp Leu Cys Glu Ser Pro Glu Glu Gln Leu Asn Glu Val Leu      50 55 60 Arg Glu Leu Gly Thr Ala Ile Asn Arg Ala Ser Gly Leu Ile Gly Asn  65 70 75 80 Trp Gln Gln Thr Thr Ser Lys Ile Tyr Phe Ile Trp Gln Ile Glu Ser                  85 90 95 Val Ile Ser Asp Ile Gln Gly Cys Ser Leu Gln Leu Cys Gln Leu Val             100 105 110 Asn Ser Leu Leu Pro Ser Leu Thr Gly Arg Ala Cys Thr Cys Ile Glu         115 120 125 Lys Leu Gln Asp Ile Asn Tyr Glu Asn Met Phe Asp Leu Val Lys Glu     130 135 140 Ser Ser Leu Glu Leu Val Glu Thr Asp Thr Thr Ser Pro Glu Asn Leu 145 150 155 160 Ser Arg Leu Ser Ser Ser Leu Ser Leu Ser Thr Asn Leu Glu Phe Tyr                 165 170 175 Met Glu Ala Val Ser Leu Glu Asn Leu Arg Ala Arg Ala Met Arg Ser             180 185 190 Glu Asn Arg Glu Glu Met Asp Leu Ala Asp Lys Met Ile Pro Leu Val         195 200 205 Asn Tyr Met His Asp His Leu Leu Arg Glu Thr Gln Leu Leu Ser Ile     210 215 220 Asn Gly Val Pro Ile Pro Ala Asp Phe Cys Cys Pro Leu Ser Leu Glu 225 230 235 240 Leu Met Ser Asp Pro Val Ile Val Ala Ser Gly Gln Thr Tyr Glu Arg                 245 250 255 Val Tyr Ile Lys Leu Trp Leu Asp Glu Gly Phe Thr Ile Cys Pro Lys             260 265 270 Thr Arg Gln Arg Leu Gly His Ser Asn Leu Ile Pro Asn Tyr Thr Val         275 280 285 Lys Ala Leu Ile Ala Asn Trp Cys Glu Ser His Asn Ile Arg Leu Pro     290 295 300 Asp Pro Met Lys Ser Leu Lys Leu Asn Phe Pro Leu Ala Ala Ser Ala 305 310 315 320 Leu Gln Asp Ser Ser Thr Thr Gly Ser Ser Pro Leu His Pro Thr Val                 325 330 335 Ala Ala Lys Gly Asn Ile Pro Gly Ser Pro Glu Ala Asp Leu Tyr Met             340 345 350 Arg Ser Leu Asn Arg Ala Ser Pro Pro His Ser Val Val His Gln Asn         355 360 365 Ser His Ala His Val Asn Arg Ala Gly His Glu Ala Ser Ile Lys Gln     370 375 380 Ser Ser Glu Asn Ala Asn Gly Ser Ala Ser Asp Val Ser Arg Leu Ser 385 390 395 400 Leu Ala Gly Ser Glu Thr Arg Glu Ser Ser Leu Glu Glu Arg Asn Ala                 405 410 415 Gly Ser Ile Gly Gln Thr Ser Glu Gln Ser Ile Glu Glu Ala Phe Gln             420 425 430 Ala Ser Asn Leu Asp Arg Asp Ser His Asp His Val Gly Ser Ser Ser         435 440 445 Val Asn Gly Ser Leu Pro Asn Ser Gly Gln Leu Asp Ala Glu Cys Asp     450 455 460 Asn Gly Pro Ser Glu Arg Thr Asn Tyr Ser Ser Asp Ala Ser Gly Glu 465 470 475 480 Val Thr Asp Gly Pro Ser Ala Ser Ser Ala Pro Gln Arg Glu His Leu                 485 490 495 Ile Pro Ser Arg Leu Ala Asp Val Arg Ser Arg Gly Gln Phe Val Arg             500 505 510 Arg Pro Ser Glu Arg Gly Phe Pro Arg Ile Ile Ser Ser Ser Ser Met         515 520 525 Asp Thr Arg Ser Asp Leu Ser Ala Ile Glu Asn Gln Val Arg Lys Leu     530 535 540 Val Asp Asp Leu Arg Ser Asp Ser Val Asp Val Gln Arg Ser Ala Thr 545 550 555 560 Ser Asp Ile Arg Leu Leu Ala Lys His Asn Met Glu Asn Arg Ile Ile                 565 570 575 Ile Ala Asn Cys Gly Ala Ile Asn Leu Leu Val Gly Leu Leu His Ser             580 585 590 Pro Asp Ser Lys Thr Gln Glu His Ala Val Thr Ala Leu Leu Asn Leu         595 600 605 Ser Ile Asn Asp Asn Asn Lys Ile Ala Ile Ala Asn Ala Asp Ala Val     610 615 620 Asp Pro Leu Ile His Val Leu Glu Thr Gly Asn Pro Glu Ala Lys Glu 625 630 635 640 Asn Ser Ala Ala Thr Leu Phe Ser Leu Ser Val Ile Glu Glu Asn Lys                 645 650 655 Val Arg Ile Gly Arg Ser Gly Ala Ile Lys Pro Leu Val Asp Leu Leu             660 665 670 Gly Asn Gly Thr Pro Arg Gly Lys Lys Asp Ala Ala Thr Ala Leu Phe         675 680 685 Asn Leu Ser Ile Leu His Glu Asn Lys Ala Arg Ile Val Gln Ala Asp     690 695 700 Ala Val Lys Tyr Leu Val Glu Leu Met Asp Pro Ala Ala Gly Met Val 705 710 715 720 Asp Lys Ala Val Ala Val Leu Ala Asn Leu Ala Thr Ile Pro Glu Gly                 725 730 735 Arg Thr Ala Ile Gly Gln Ala Arg Gly Ile Pro Ala Leu Val Glu Val             740 745 750 Val Glu Leu Gly Ser Ala Arg Gly Lys Glu Asn Ala Ala Ala Ala Leu         755 760 765 Leu Gln Leu Cys Thr Asn Ser Ser Arg Phe Cys Ser Ile Val Leu Gln     770 775 780 Glu Gly Ala Val Pro Pro Leu Val Ala Leu Ser Gln Ser Gly Thr Pro 785 790 795 800 Arg Ala Arg Glu Lys Ala Gln Ala Leu Leu Ser Tyr Phe Arg Ser Gln                 805 810 815 Arg His Gly Asn Ser Ala Arg Arg             820 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for cloning OsPUB15 gene (forward) <400> 3 atggaaaatt tctccccgag 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for cloning OsPUB15 gene (reverse) <400> 4 tcatctcctt gctgaattc 19

Claims (11)

A recombinant vector for increasing resistance to stress of a plant, comprising a gene encoding an OsPUB15 protein derived from rice consisting of an amino acid sequence represented by SEQ ID NO: 2. The recombinant vector of claim 1, wherein the stress is at least one selected from paraquat, salt, and drought. The recombinant vector according to claim 1, wherein a gene encoding OsPUB15 protein derived from rice consisting of the amino acid sequence represented by SEQ ID NO: 2 is inserted into the pCB302 vector described in FIG. 2. A transformed cell transformed with the recombinant vector according to claim 1. The transformed cell of claim 4, wherein the cell is a microorganism of the genus Agrobacterium. The transformed cell of claim 5, wherein the microorganism of the genus Agrobacterium is Agrobacterium tumefaciens LB4404. A transformed plant transformed by the recombinant vector of claim 1. The transgenic plant of claim 7, wherein the plant is a monocotyledonous plant. The method of claim 8, wherein the monocotyledonous plant Oryza sativa L. ). A method of increasing plant resistance to at least one stress selected from paraquats, salts and droughts by overexpressing in a plant a gene encoding an OsPUB15 protein derived from rice consisting of the amino acid sequence represented by SEQ ID NO: 2. The method of claim 10, wherein the overexpression of the gene encoding the OsPUB15 protein derived from rice in the plant is transformed into a plant with a recombinant vector comprising a gene encoding the OsPUB15 protein derived from rice consisting of the amino acid sequence represented by SEQ ID NO: 2 And expressing said introduced gene in a plant.

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