KR101258079B1 - BrCIPK3 gene from Brassica rapa and uses thereof - Google Patents

Abstract

The invention cabbage ( Brassica) rapa ) -derived abiotic stress resistance-related BrCIPK3 (Brassica rapa CBL-calcium interacting protein kinase 3) protein, a gene encoding the protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, and A method of increasing the abiotic stress resistance of a plant by transforming a recombinant vector into a plant cell, a method of producing a transgenic plant having increased abiotic stress resistance using the recombinant vector, and abiotic produced by the method The present invention relates to a composition for increasing abiotic stress resistance of a transgenic plant having increased stress resistance, a seed thereof, and a plant comprising the BrCIPK3 gene.

Description

ChrCIPK3 gene from Chinese cabbage and its use {BrCIPK3 gene from Brassica rapa and uses approximately}

The present invention relates to a BrCIPK3 gene derived from cabbage and its use, and more particularly to cabbage ( Brassica) rapa ) -derived abiotic stress resistance-related BrCIPK3 (Brassica rapa CBL-calcium interacting protein kinase 3) protein, a gene encoding the protein, a recombinant vector containing the gene, a host transformed with the recombinant vector Cells, a method of transforming the recombinant vector into a plant cell to increase the abiotic stress resistance of the plant, a method of producing a transgenic plant having increased abiotic stress resistance using the recombinant vector, the method produced by the method The present invention relates to a transgenic plant having increased abiotic stress resistance, a seed thereof, and a composition for increasing abiotic stress resistance of a plant comprising the BrCIPK3 gene.

Abiotic stresses, including salt and aluminum toxicity, are the most important issues limiting rice production. Development of crops that are resistant to stress can play an important role in increasing yield. Plants respond to these stresses by activating many signaling pathways that often involve various protein kinases, including calcium interacting protein kinases that are able to defend against or regulate the stress.

In plants, many Ca21-sensitive protein kinases have been reported to be involved in the stress response. The protein kinase is a calcium dependent protein kinase (Mori et al., 2006, PLoS Biolology 4: e327) and SnRK (Suc non-fermentation-related kinase) (Guo et al., 2002, Plant Cell 16: 435-449) Include. In Arabidopsis, CIPK (calcineurin B-like [CBL] protein interaction protein kinase) (Mahajan et al., 2006, Federation of European Biochemical Societies Journal 273: 907-925) or SOS2 (SALT OVERLY) associated with AtCBL / SOS3-like There are 38 SnRKs (Hrabak et al., 2003, Plant Physiology 132: 666-680), including the SnRK3 subfamily called SENSITIVE2) -like protein kinase (PKS).

 It is of fundamental importance in biology to understand the mechanism by which plants sense signals from the environment and deliver them to cellular machinery to activate adaptive responses. Thus, overexpression of the CIPK3 (CBL-calcium interacting protein kinase 3) gene may be very useful for the survival of rice against stress.

Korean Patent No. 10-0803174 discloses a method for imparting plant stress resistance and a plant obtained thereby, and Korean Patent No. 10-0990333 discloses a method for enhancing flame resistance of a plant using a NHX gene derived from barley. It is.

The present invention is derived from the above demands, the inventors of the Chinese cabbage ( Brassica The present invention was completed by confirming that rice plants transformed with the rapa ) -derived BrCIPK3 gene exhibit resistance to abiotic stresses such as salt, aluminum, low temperature and oxidative stress.

In order to solve the above problems, the present invention is Chinese cabbage ( Brassica) Promote abiotic stress resistance from rapa ) BrCIPK3 (Brassica rapa CBL-calcium interacting protein kinase 3) protein.

The present invention also provides a gene encoding the protein.

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

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

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

The present invention also provides a method for producing a transgenic plant having increased abiotic stress resistance using the recombinant vector.

The present invention also provides a transgenic plant with increased abiotic stress tolerance produced by the above method and its seeds.

The present invention also provides a composition for increasing abiotic stress resistance of plants comprising the BrCIPK3 gene.

Chinese cabbage of the present invention (Brassica Rapa ) -derived BrCIPK3 gene may be useful for developing transgenic plants that are resistant to abiotic stresses such as salt, aluminum, low temperature and oxidative stress.

1 is a Chinese cabbage ( Brassica) rapa ) shows a schematic diagram of the binary Ti plasmid pFLCIII containing BrCIPK3 full-length cDNA.
2 shows BrCIPK3 . The full length cDNA sequence (A), its phylogenetic tree (B) using the associated species selected from BLAST analysis, and the amino acid sequence (C) of BrCIPK3 are shown.
3 shows the mRNA expression (C) in different tissues of Organ mRNA expression (A), determine the BrCIPK3 and hpt gene inserted in rice plant (B), and the transformed rice plants of BrCIPK3 gene in Chinese cabbage plant.
Lane descriptions in Panel A: Lane 1 (seed), 2 (pistil), 3 (surgery), 4 (eye), 5 (petal), 6 (calyx), 7 (petal), 8 (leaf), 9 (stem) ), 10 (root)
Lane description in panel B: lanes M (DNA ladder), 1-6 (transformed rice ( BrCIPK3 )), 7 (wild-type rice), 8 (plasmid DNA)
Lane description in panel C: Lane 1 (seedling), 2 (leaf), 3 (leaf), 4 (stem), 5 (root), 6 (seed)
4 shows the effect (A) of aluminum toxicity (1,000 μM) on freshly germinated transgenic rice ( BrCIPK3 ) and high quality rice (wild type) after 7 days of exposure, and aluminum resistance and growth of roots and shoots (B). .
Figure 5 shows the mRNA expression (A) and free proline content (B) over time after 48 hours treatment with 130 mM NaCl.
FIG. 6 shows the degree of salting of rice plants against salt treatment when treated with 130 mM salt after two weeks of growth of wild type high quality rice, Brcipk3 transformed rice, and susceptible and resistant varieties.
Figure 7 shows germination analysis (A) of transformed rice ( BrCIPK3 ) and wild type Gopumbyeo under salt treatment, and the survival rate (%) (B) of germinated seed between transformed rice ( BrCIPK3 ) and high rice. . All experiments were repeated three times with at least 30 plants.
8 shows the test results of hyumyeonseong BrCIPK3 mutant compared to wild-type rice gopum. The test was performed three times with at least 30 seeds per test.
Figure 9 shows the results of mRNA expression analysis of BrCIPK3 gene under cold treatment of wild-type high-quality rice and BrCIPK3 transformed rice for two weeks.
Figure 10 shows the results of analyzing the free proline content of the wild type high-quality rice and BrCIPK3 transformed rice under cold treatment for 2 weeks.
Figure 11 shows the results of analyzing the soluble sugar content of the wild type high-quality rice and BrCIPK3 transformed rice under cold treatment for 2 weeks.
12 is a result of examining the phenotype of the rice plants after growing the high-quality rice and BrCIPK3 gene-transformed rice for 2 weeks at 15 ℃ water temperature in the cold-resistant black packaging of Chuncheon branch of the National Institute of Food Science.
Figure 13 shows the results of the change in the height of the rice plants after growing the high-quality rice and BrCIPK3 gene-transformed rice at 15 ℃ water temperature in the cold-resistant black packaging of Chuncheon branch of the National Institute of Food Science.
FIG. 14 shows the results of examining the phenotypes of rice plants in response to oxidative stress after treatment with wild type high- grade rice, BrCIPK3 gene-transformed rice, and susceptible and resistant rice for 2 weeks in Yoshida nutrient solution containing 20% PEG6000.

In order to achieve the object of the present invention, the present invention consists of an amino acid sequence of SEQ ID NO: 2, Chinese cabbage ( Brassica rapa ) -derived abiotic stress resistance related BrCIPK3 (Brassica rapa CBL-calcium interacting protein kinase 3) protein.

The range of BrCIPK3 protein according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 isolated from cabbage and functional equivalents of the proteins. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" means activity that enhances the abiotic stress tolerance of a plant.

The invention also includes fragments, derivatives and analogues of BrCIPK3 protein. As used herein, the terms “fragment”, “derivative” and “analogue” refer to a polypeptide having biological function or activity substantially the same as the BrCIPK3 polypeptide of the invention. Fragments, derivatives, and analogs of the present invention comprise (i) polypeptides substituted with one or more conservative or nonconservative amino acid residues (preferably conservative amino acid residues), wherein the substituted amino acid residues are encoded by a genetic code. Or (ii) a polypeptide having substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of a polypeptide, such as polyethylene glycol) A polypeptide derived from a bound mature polypeptide, or (iv) an additional amino acid sequence (eg, a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) and It may be a polypeptide derived from said polypeptide bound. Such fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

The present invention also provides a gene encoding the BrCIPK3 protein. The gene of the present invention may be DNA or RNA encoding BrCIPK3 protein. DNA includes cDNA, genomic DNA or artificial synthetic DNA. DNA can be single stranded or double stranded. The DNA may be a coding strand or a non-coding strand.

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

Polynucleotides encoding a mature polypeptide represented by SEQ ID NO: 2 include a coding sequence encoding only a mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences encoding noncoding sequences.

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.

The invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequences as described herein, or fragments, analogs, and derivatives thereof. Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitutional variants, deletional variants, and insertional variants. As is known in the art, an allelic variant is an alternative to a polynucleotide, which may comprise one or more substituted, deleted or inserted nucleotides and which does not result in a substantial functional change in the polypeptide encoded by the variant Do not.

In addition, the present invention provides a poly-hybridized hybridization with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with a nucleotide sequence of SEQ ID NO. It relates to nucleotides. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" are (1) hybridization and washing under lower ionic strength and higher temperature such as 0.2 x SSC, 0.1% SDS, 60 ° C; Or (2) in the presence of a denaturant such as 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll and 42 ° C; Or (3) at least 80%, preferably at least 90%, more preferably at least 95%. In addition, the biological function and activity of the polypeptide encoded by the hybridizable polynucleotide is the same as the biological function and activity of the mature polypeptide represented by SEQ ID NO: 2.

According to embodiments of the invention, it should be understood that the BrCIPK3 gene is preferably derived from cabbage. However, embodiments of the invention have high homology (eg, 60% or more, ie 70%, 80%, 85%, 90%, 95%, even 98% sequence identity) with the cabbage BrCIPK3 gene and other It also includes other genes derived from plants. Sequencing methods and means for determining sequence identity or homology (eg BLAST) are well known in the art.

The present invention also provides a recombinant vector comprising the BrCIPK3 gene according to the present invention.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

In the present invention, the polynucleotide sequence encoding BrCIPK3 protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors comprising BrCIPK3 protein-encoding DNA sequences and appropriate transcriptional / translational control signals can be constructed by methods well known to those of skill in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

Preferred examples of recombinant vectors of the invention are Ti-plasmid vectors capable of transferring part of themselves, the so-called T-DNA region, to plant cells when present in a suitable host, such as Agrobacterium tumerfaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

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

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

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Octopine gene terminator, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a host cell transformed with the recombinant vector of the present invention. Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

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

The method of carrying the vector of the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

The present invention also provides a method of increasing the abiotic stress resistance of a plant comprising the step of transforming the recombinant vector into plant cells, overexpressing the BrCIPK3 gene. Preferably, the abiotic stress may be, but is not limited to, salt, aluminum, low temperature or oxidative stress. The low temperature may be preferably 4 ~ 15 ℃.

The present invention also comprises the steps of transforming plant cells with the recombinant vector; And

It provides a method for producing a transgenic plant with increased abiotic stress resistance comprising the step of regenerating the plant from the transformed plant cells.

In the method of the present invention, the abiotic stress may preferably be, but is not limited to, salt, aluminum, low temperature or oxidative stress. The low temperature may be preferably 4 ~ 15 ℃.

The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tyumeo Pacific Enschede may be mediated by (Agrobacterium tumefiaciens). In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989, Momillan, N. Y.).

The present invention also provides a transgenic plant with increased abiotic stress tolerance produced by the above method. The plant may be a monocotyledonous plant, but is not limited thereto. Preferably, the monocotyledonous plant is rice.

The monocotyledonous plants include Alismataceae, Hydrocharitaceae, Juncaginaceae, Schuchzeriaceae, Pomomotontonaceae, Najadaceae, Zosteraceae, Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae, Iridaceae, Burmanniaceae, Juncaceae, Rootaceae Commelinaceae, Eriocaulaceae, Panaxaceae, Gramineae, Poaceae, Araceae, Lemnaceae, Spaghaniaceae, Typhaceae, Cyperaceae, Cyperaceae (Musaceae), Zingiberaceae, Cannaceae or Orchidaceae (Orchidaceae), but is not limited thereto.

The present invention also provides a composition for increasing abiotic stress resistance of plants comprising the BrCIPK3 gene of the present invention. In the composition of the present invention, the BrCIPK3 gene may be composed of the nucleotide sequence of SEQ ID NO: 1. The composition of the present invention comprises a BrCIPK3 gene derived from Chinese cabbage as an active ingredient, and is capable of enhancing the abiotic stress resistance of the plant by transforming the gene BrCIPK3 into the plant. The plant and abiotic stresses are as described above.

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

Materials and methods

Plant material and transformation

Old rice ( Oryza sativa L. Gopumbyeo) was used for transformation and provided in wild type. The rice was grown in open field, and the seeds were harvested and washed. Mature rice seeds of japonica rice cultivar Gopumbyeo were screened to remove healthy seeds. The peeled seeds were sterilized with 70% ethanol for 1 minute and then washed with sterile water. Seeds were re-sterilized for 15 minutes with 2.5% sodium hypochlorite containing 1 drop of Tween 20 per 50 ml and then washed 5 times with sterile water. This step was repeated once more without Tween 20. The sterilized seeds were inoculated in N6D medium solidified with 0.4% Gelrite and incubated at 32 ° C. for 5 days under continuous light conditions.

Agrobacterium Agrobacterium ) infection

Chinese cabbage rapa ) Binary Ti plasmid pFLCIII containing full-length cDNA of cDNA library-derived calcium interacting protein kinase 3 gene Agrobacterium (Agrobacterium) having (1) Strain EHA105 contains 50 mg / L kanamycin sulfate and was incubated under dark conditions for 3 days at 28 ° C. in AB medium solidified to 1.5% agar. Agrobacterium cultures were scraped from the plate and suspended in AAM medium to make OD ₆₅₀ about 0.3. The precultured seeds were inverted into the Agrobacterium suspension by gently inverting the tube for 10 minutes and then dried using sterile filter paper to remove excess bacteria. The seeds were transferred to sterile filter paper wetted with 0.5 ml AAM medium in 2N6-AS medium solidified with 0.4% Gelrite.

Selection of transformed newsletter

After 3 days co-culture at 25 ° C., dark conditions, the seeds were washed 5 times with sterile water and then at least 5 with sterile water containing 500 mg / L carbenicillin to remove Agrobacterium. Washed twice. The seeds were rapidly dried using sterile filter paper and incubated under continuous light conditions at 32 ° C. for 2 weeks in N6D medium containing 50 mg / L hygromycin and 400 mg / L carbenicillin. Proliferating callus from scutellum was transferred to RE-III medium. The callus derived carrier was transferred to RF medium to induce roots. After that, the seedlings that were grown were transferred to the greenhouse.

DNA  extraction

Lyophilized rice leaf tissue was floured after treatment with liquid nitrogen to transfer 40 mg to a 1.5 ml tube. 1 ml of preheated CTAB (cetyl trimethylammonium bromide) extraction buffer was added. The tube was then vortexed to homogenize the buffer and tissue. Samples were incubated at 65 ° C. for 90 minutes with gentle continuous stirring. The sample was cooled down for 10 minutes at room temperature. 500 μl of chloroform: isoamyl alcohol (24: 1) was added and the sample was stirred continuously for 10 minutes at room temperature. Samples were centrifuged at 35,000 rpm for 10 minutes. The upper layer containing water was transferred to a new tube and treated for 30 minutes with 5 μl RNAse at 37 ° C. Thereafter, 1/2 volume of 100% isopropanol was added and mixed gently. The sample was then centrifuged for 30 minutes at 35000 rpm to form pellets at the bottom of the tube. The supernatant was discarded and 1 ml of 75% ethanol was added to wash the pellets. Ethanol was discarded and the pellet dried until ethanol was completely evaporated. DNA pellet was resuspended in 100 μl of distilled water. Relative purity and concentration of the extracted DNA was measured using NanoDrop-1000.

Genome PCR  analysis

PCR analysis hpt (hygromycin phosphotransferase) HPT-Fw for confirming the transfection of genes (GGA TTT CGG CTC CAA TGT CCT GAC GGA; SEQ ID NO: 3), and HPT-Rv (CTT CTA CAC AGC CAT CGG TCC AGA; SEQ ID NO: 4 ) primer, and cabbage (Brassica rapa ) pBigs_SfiI-Fw (TAT TCG GAG AGG GTA CGT ATT TTT AC; SEQ ID NO: 5) and pBigs_sfiI-Rv (GCA ACA GGA TTC AAT CTT AAG AAA CT; SEQ ID NO: 6 to confirm the import of full-length cDNA from the BrCIPK3 gene Primers were used to perform different transgenic generations.

PCR amplification was performed with 1 μl genomic DNA, 0.5 μl each primer (diluted at 10 μM concentration), 2 μl 10 × PCR buffer (CosmoGeneTech), 2 μl 10 mM dNTP (CosmoGeneTech), and 0.1 μl Taq polymer. A total of 20 μl containing polymerase was performed. The PCR profile used was 1 cycle performed at 94 ° C. for 3 minutes, followed by 35 cycles of denaturation, annealing and stretching steps for 30 seconds at 94 ° C., 45 seconds at 55 ° C. and 45 seconds at 72 ° C., and Last stretch step at 72 ° C. for 15 minutes. PCR was performed on PTC-100 and 220 thermocycler (MJ Research). The PCR product was analyzed by staining with ethidium bromide after agarose gel electrophoresis.

RNA  extraction

Total RNA was extracted from the leaves of each transgenic plant incubated for 2 weeks after transfer to the soil. Leaf samples were powdered using a mortar after liquid nitrogen treatment. RNeasy Plant Mini Kit (QIAGEN) was used according to the manufacturer's instructions. Relative purity and concentration of the extracted RNA was measured using NanoDrop-1000.

cDNA  Synthetic and RT - PCR

The first strand cDNA was synthesized in a total volume of 20 μl from the oligo (dT) primer and each RNA preparation (1 μg / reaction) using the SuperScript II RT cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions.

The specific sequences of the primer pairs used for semiquantitative RT-PCR were CIPK3-Fw (5'-ATC GGA AGC AAG TCA AGC G-3 '; SEQ ID NO: 7) and CIPK3-Rv (5'-CTC CAT CGT AGC CTC CGT CAT-3 ′; SEQ ID NO: 8). Actin primers were used as loading controls and internal controls for standardization of quantitative RT-PCR responses.

Semiquantitative RT-PCR was performed using 2 μl of template cDNA per 20 μl. The reaction was carried out for 5 minutes at 94 ° C. followed by 45 cycles consisting of 30 seconds at 94 ° C., 30 seconds at 58 ° C. and 30 seconds at 72 ° C., and the last extension step for 8 minutes at 72 ° C. CosmoGeneTech Taq was used. RT-PCR was performed on PTC-100 and 220 thermocycler (MJ Research). The RT-PCR product was analyzed by agarose gel electrophoresis and stained with ethidium bromide.

BrCIPK3  Gene expression

Rice culture technology

Rice ( Oryza sativa L. cv. Gopumbyeo seeds were soaked in 1% sodium hypochlorite for 15 minutes, washed with sterile water for 30 minutes until the sodium hypochlorite was completely gone, and then immersed in sterile water. Rice seeds germinated at 32 ° C. under dark conditions. Microbial contamination was kept to a minimum by changing sterile water daily.

Salinity Analysis

Seedlings 2-3 weeks old were floated with styrofoam and treated with 130 mM sodium chloride mixed with nutrient solution (Yoshida et al., 1976, International Rice research Institute, Los Banos, Laguna, Philippines. P61). Sampling of leaves was performed at 0, 12, 36 and 48 hours after salt treatment for RT-PCR analysis and enzyme analysis (free-proline and sugar). Final determination of viable plant numbers was performed 2 weeks after recovery from stress.

Germination Analysis

Wild type and Brcipk3 Thirty seeds from each of the mutants were treated threefold with 130 and 150 mM sodium chloride and incubated at 37 ° C. for 2 days. The plate was then transferred to 28 ° C. under long conditions. Germination (radical development) was measured after 7 days.

Aluminum toxicity analysis

Wild type and Brcipk3 Thirty seeds from each mutant were germinated by triple treatment of 1,000 μM / L of aluminum chloride in an optimized Magnavaca nutrient solution. At the same time, control plants using the same sample source were incubated using Yoshida (1967) nutrient solution. Root length, relative root length and shoot shoots of germinated seedlings were measured after 2 weeks.

Dormancy  analysis( Dormancy Assay )

Wild type and Brcipk3 Thirty seeds from each of the mutants were treated in triplicates in sterile tissue culture test plates and incubated at 37 ° C. for 2 days. Germination was evaluated after 7 days.

Free proline and sugar analysis ( Free proline and Sugar assays )

Four-leaf stage transgenic plants were used for biochemical analysis. The free proline content in the leaves was determined according to the method of Troll and Lindsley (1955, Journal of Biological Chemistry 215: 655-660). About 50 mg of leaves were homogenized in 1 mL of sulfosalicylic acid (3%) and the homogenization mixture was centrifuged at 4 ° C. for 15 minutes at 13,000 rpm. The extract (200 μl) was transferred to a new tube and mixed with 200 μl of ninhydrin solution (2.4 g of glacial acetic acid and 0.1 g of ninhydrin dissolved in 1.6 mL of 6-ortho-phosphoric acid) and 200 μl of acetic acid It was. The reaction mixture was treated in a constant temperature water bath at 100 ° C. for 30 minutes and then at 4 ° C. for 30 minutes. 400 μl of toluene was then added to the leaf extract and mixed thoroughly. Finally, 120 μl of toluene phase was removed for absorbance measurements (520 nm) using a spectrophotometer (Beckman). Free proline is represented using the following formula:

[(Μg proline / ml × ml toluene) /115.5 μg / μmole] / [(g sample) / 5] = μmole proline / g raw weight.

For sugar content analysis, total soluble sugars in the leaves were determined by modified phenolsulfuric acid method (Dubois, 1956). About 0.1 g of leaves were homogenized in 8 mL of distilled water, and then treated twice in a constant temperature water bath at 100 ° C. for 30 minutes. The extract (about 500 μl) was transferred to a new tube and 1.5 mL of distilled water, 1 mL of 9% (v / v) phenol, and 5 mL of sulfuric acid were added and held at room temperature for 30 minutes. Absorbance was measured at 485 nm using a spectrophotometer (Beckman).

Example  One: BrCIPK3 of Battlefield cDNA  Sequence analysis and phylogenetic tree

pBigs_SfiI primer and a positive BrCIPK3 Genomic DNA of the transformant containing the gene was used to identify the complete sequence. Actual sequencing was performed by CosmoGeneTech company and the results are shown in Figure 2A. As a result of sequencing , the full-length cDNA sequence of BrCIPK3 gene was 1,982 bp in total consisting of 216 bp 5'-UTR (untranslated region), 1,509 bp coding region and 257 bp 3'-UTR. The coding region (ORF) encodes a polypeptide of 502 amino acids and the expected molecular weight of the protein is 74.06 kDa. The sequence showed 23.9-71.2% sequence identity with the species of association as shown in FIG. 2B. Phylogenetic analysis of BrCIPK3 was performed on Arabidopsis Lyrata ), Eastern ( Vigna unguiculata ), Arabidopsis ( Arabodpsis) thaliana ) and Triticum eastivum . Surprisingly, rice ( Oryza sativa ) and other related species formed another group that did not belong to the same group containing BrCIPK3.

Example  2: BrCIPK3  Gene Insertion and Overexpression in Other Sites of Transgenic Rice

As a result of analyzing the mRNA expression of BrCIPK3 gene for each region of the Chinese cabbage ( Brassica rapa ) plant, it showed the highest expression in pistil, stem and calyx as shown in Fig. 3A. Showed very weak expression but not at petals, leaves and roots.

BrCIPK3 Using genomic DNA of transgenic rice, we confirmed the presence of BrCIPK3 gene in the rice genome as shown in FIG. 3B. To analyze the function of BrCIPK3 in plant calcium signaling, expression patterns of the genes were examined by RT-PCR. We found that the expression of BrCIPK3 is greatly regulated. MRNA expression in the transformed rice of BrCIPK3 gene was high in seedlings and leaves and relatively high in roots and seeds as shown in FIG. 3C, but mRNA was expressed at very low levels in vines and stems. It became.

Example 3: Resistance to Aluminum Toxicity of Transgenic Rice Containing BrCIPK3 Gene

To test the resistance of rice containing the BrCIPK3 gene to aluminum toxicity, we germinated seeds using a cylinder tube floated with styrofoam containing a net supporting germinating seeds under 1,000 μM of aluminum chloride. . Shoot and root growth as well as relative root growth are parameters for measuring resistance / tolerance (Magnavaca et al., 2010, Martinus Nijhoff, Dordrecht, Netherlands, pp 255-265). In the present invention, the Brcipk3 transformed rice lineage showed higher shoot and root growth compared to wild type pulp . In addition, the relative root length also increased equally. This indicated that the Brcipk3 transgenic rice line was resistant to high concentrations of aluminum chloride (FIG. 4).

Example 4: Salinity Analysis and Germination

Stress adaptation in plants depends on changes in molecular processes, in particular gene expression, which initiates the realignment of physiology and metabolism. To determine how the Brcipk3 transgenic rice line reacts under physiological saline conditions, we included the Yoshida nutrient solution (Yoshida et al., 1976, International Rice research Institute, Los Banos, Laguna, Philippines.p61). As a result of analyzing mRNA expression by treatment with 130 mM sodium chloride, mRNA expression was observed in BrCIPK3 transgenic rice from 12 hours after treatment as shown in FIG. 5A, and increased linearly up to 42 hours, but was weakly expressed in wild-type high quality rice. In addition, the free proline content did not differ from 0 to 12 hours when rice plants were treated with 130 mM sodium chloride, but the Brcipk3 transgenic rice line was significantly higher than 24 hours (FIG. 5B). The experimental results indicate that Brcipk3 gene is involved in abiotic stress.

As the plant phenotype after the brCIPK3 gene-transformed rice was grown for 2 weeks and treated with 130 mM sodium chloride, as shown in FIG. 6, the damage symptom appeared in Dongjin rice and wild-type rice plant susceptible to salt treatment. However, BrCIPK3 transgenic rice was resistant to Hwayoung rice, which is known to be resistant to 130 mM salt treatment. Meanwhile, as a result of analyzing the germination rate and the survival rate of 2 week old seedlings when treated with 130 mM sodium chloride in Yoshida nutrient solution, transgenic plants containing Brcipk3 gene showed remarkable resistance compared to wild type as shown in FIG. 7. High survival was observed in Brcipk3 transgenic rice. Germination rate was 0% in wild type compared to Brcipk3 transgenic rice line (70.66%). The same results were also shown in the experiment using two week old seedlings (FIG. 7). In FIG. 7, labeled “control” is rice that was not treated with 130 mM sodium chloride.

Example 5: Dormancy test

Pre-harvest sprouting (PHS) causes grain quality degradation, limits the application of end use, and causes significant financial losses for farmers and food processors. To determine the effect of the Brcipk3 gene on this feature, Brcipk3 Transformed rice and wild type were treated in triplicates in sterile tissue culture test plates and incubated at 37 ° C. for 2 days. As shown in Figure 8, the germination rate (40-68%) of the transformed rice lineage was lower than the wild type (100%). Since the samples were collected at the same time, we assumed that the Brcipk3 gene was involved in dormancy. The mechanism of response of the Brcipk3 gene to PHS should be investigated.

Example  6: Low temperature stress resistance Cold tolerance )

After b-transformed rice and wild-type rice grown at 4 ° C for 2 weeks, mRNA expression was analyzed. As shown in FIG. 9, the BrCIPK3 gene was not expressed in wild-type rice, but BrCIPK3 was not expressed at all. The transgenic rice showed high mRNA expression at 6 hours after treatment, and the expression level was linearly increased up to 42 hours, which was extremely high at 42 hours. As for the free amino acids, the free proline and the soluble sugar content began to increase after 6 hours of 4 ° C. cold treatment, and significantly increased after 24 hours, and significantly increased after 48 hours, as shown in FIGS. 10 and 11. Showed. It is thought that the free amino acid content increased rapidly by the BrCIPK3 gene introduced and resistance to low temperature stress was caused by the increase of these free proline and soluble sugar content.

Biomass, chlorophyll content, water content and SES score (standard evaluation system for cold tolerance) were investigated after growing wild type high- grade rice and BrCIPK3 gene-transformed rice for 2 weeks at 15 ℃ water temperature in Chuncheon branch of National Food Science Institute. As shown in Table 1 showed resistance to low temperatures in the BrCIPK3 transgenic rice. Table 1 shows the results of investigating the biomass, chlorophyll content, water content, and SES score of rice plants after growing the high-quality rice and BrCIPK3 gene-transformed rice for 2 weeks at 15 ℃ water temperature in the cold-resistant black packaging at CNU . .

Although there was no significant difference in plant phenotype between wild type high- grade rice and BrCIPK3 transgenic rice, which were known to have moderate resistance at low temperatures in the resistance test of cold-resistant assay packaging (Fig. 12), they showed a similar SES score (4.5). BrCIPK3 gene transgenic rice 1313701-1 except for the 13700-3 and are found to be resistant to low temperatures indicated the SES score 4.25. In addition, the height of grass height was not increased in wild type high quality rice after 2 weeks of cold treatment in cold-resistant black packaging. However, most of the BrCIPK3 transgenic rices showed higher growth rate than high quality rice, especially transgenic rice 13713. At -2, 13710-1, and 13709-2, the height increased by 5 to 21 times (Fig. 13). Chlorophyll content was not different between wild-type and transgenic rice, and biomass showed a difference between wild-type and transgenic rice, which increased in the transgenic rice strains 13709-2, 13710-1, 13713-2, etc. One).

Example 7 Oxidative Stress Tolerance

Wild type high-quality rice and rice transformed with BrCIPK3 gene were treated for 2 weeks in Yoshida nutrient solution containing 20% PEG6000 (Yoshida et al., 1976, International Rice research Institute, Los Banos, Laguna, Philippines. P61). Later, after examining the phenotype of rice plants in response to oxidative stress, as shown in FIG. 14, susceptible gaya rice showed damage symptoms and many leaves died. However, BrCIPK3 transgenic rice was known to be resistant to 20% PEG6000. Resistant as the field rice.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> BrCIPK3 gene from Brassica rapa and uses approximately <130> PN11226 <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 1982 <212> DNA <213> Brassica rapa <400> 1 acacaaacaa caacaacatt acattttaca ttctacaact acatctagag gccaaatcgg 60 ccatcacggc ttcttcttca cacatcaaat cctctcttga ttcttcctct tcagtttagc 120 ttcttcattc taagcttgtc ttgatttgga gtaacctttg ttttggaggt tcttagcatt 180 tggtggaacc tttctaaatc tggtgatttg gtgaaaatga atcggaggca gcaagtcaag 240 cgtagagtag gtaagtatga agtgggaagg acaattgggg aaggaacttt tgctaaagtc 300 aagtttgcaa gaaactctga aactggagaa cctgtagccc tcaagattct tgataaagag 360 aaagtcctca agcataagat gtctgaacag atcagaagag agatagctac tatgaagttg 420 attaagcatc caaatgttgt tcaattgtat gaggtgatgg caagcaagac aaagatattt 480 atcatcttgg agtatgttac aggaggagaa ctctttgata agattgtgaa tgatgggagg 540 atgaaagaag atgaggctcg taggtatttt caacagcttg tgcatgctgt ggactactgt 600 catagcagag gtgtttacca tagagatctc aagcctgaga atttgctatt ggatgcatat 660 ggaaacctca agatctcaga ttttggatta agtgctttgt ctcagcaagt cagggatgat 720 ggactccttc atacatcgtg tggaactcca aactacgttg ctcctgaggt tcttaatgac 780 ggaggctacg atggagcaac agcagacatg tggtcatgtg gtgttatact ctatgtcttg 840 cttgcaggtt acttgccttt cgatgattct aatctaatga atctttataa gaaaatttca 900 tctggtgaat tcaactgtcc tccatggcta tcacttggcg ccatgaaact catcactcga 960 atcttagatc ctaaccccat gactcgtgta acaccacaag aggtgtttga agatgaatgg 1020 ttcaagaaag attacaagcc tccagtgttt gaggagaagg atgattcaaa catggacgat 1080 gttgatgctg ttttcaagga ctccgaggaa catcatgtta ctgaaaaaaa ggaagagcag 1140 ccagctgcaa tcaatgcctt tgagatcatc tcaatgtcaa gggggcttaa cttggaaaac 1200 ctgtttgatc ctgaacagga atttaagaga gaaacaagga taactctgag aggaggcgca 1260 aacgaaatca tcgacaagat agaagaagct gcaaagcctc tcggttttga tgtgcaaaag 1320 aagaactaca agatgaggct tgagaatgtg aaggctggga gaaaagggaa tctcaatgtg 1380 gcaacagaga tattccaagt agcaccaagt cttcatatgg ttcaagtttc gaaatcaaaa 1440 ggggacactc tcgaattcca caagttttat aagaagctgt ctaattctct ggagaatgta 1500 gtctggacga acaacgaagt gaagaaagcg aaagtgaagg gtgggggaaa agggaatttc 1560 aaaggggcca ccgagatatt tcaagtagcc cccagttttc atatggttca agttttgaaa 1620 tccaaagggg gcccttttga atttccccag ttttttaaga aactgtttaa ttttttgggg 1680 aaagttgttt gggcgaacaa cgaaatgaag aaaactggaa aatgaagttc cgtatgaaag 1740 gttgggtttt ttggggccag cttacttttt ttgtttttga gctttttttt tggtgatccg 1800 gctttacttt tgtaaggatt gatgagtgag tggggttggt ttgtgtaagg ggtatatatg 1860 aactgccaaa tcccttataa aatcaaatcg ttggttgaac aaaaaaaaaa aaaaaactga 1920 ggccataagg gcctctagag gatccccggg gcggccgcga gctcgaattc cccgatcgtc 1980 aa 1982 <210> 2 <211> 502 <212> PRT <213> Brassica rapa <400> 2 Met Asn Arg Arg Gln Gln Val Lys Arg Arg Val Gly Lys Tyr Glu Val   1 5 10 15 Gly Arg Thr Ile Gly Glu Gly Thr Phe Ala Lys Val Lys Phe Ala Arg              20 25 30 Asn Ser Glu Thr Gly Glu Pro Val Ala Leu Lys Ile Leu Asp Lys Glu          35 40 45 Lys Val Leu Lys His Lys Met Ser Glu Gln Ile Arg Arg Glu Ile Ala      50 55 60 Thr Met Lys Leu Ile Lys His Pro Asn Val Val Gln Leu Tyr Glu Val  65 70 75 80 Met Ala Ser Lys Thr Lys Ile Phe Ile Ile Leu Glu Tyr Val Thr Gly                  85 90 95 Gly Glu Leu Phe Asp Lys Ile Val Asn Asp Gly Arg Met Lys Glu Asp             100 105 110 Glu Ala Arg Arg Tyr Phe Gln Gln Leu Val His Ala Val Asp Tyr Cys         115 120 125 His Ser Arg Gly Val Tyr His Arg Asp Leu Lys Pro Glu Asn Leu Leu     130 135 140 Leu Asp Ala Tyr Gly Asn Leu Lys Ile Ser Asp Phe Gly Leu Ser Ala 145 150 155 160 Leu Ser Gln Gln Val Arg Asp Asp Gly Leu Leu His Thr Ser Cys Gly                 165 170 175 Thr Pro Asn Tyr Val Ala Pro Glu Val Leu Asn Asp Gly Gly Tyr Asp             180 185 190 Gly Ala Thr Ala Asp Met Trp Ser Cys Gly Val Ile Leu Tyr Val Leu         195 200 205 Leu Ala Gly Tyr Leu Pro Phe Asp Asp Ser Asn Leu Met Asn Leu Tyr     210 215 220 Lys Lys Ile Ser Ser Gly Glu Phe Asn Cys Pro Pro Trp Leu Ser Leu 225 230 235 240 Gly Ala Met Lys Leu Ile Thr Arg Ile Leu Asp Pro Asn Pro Met Thr                 245 250 255 Arg Val Thr Pro Gln Glu Val Phe Glu Asp Glu Trp Phe Lys Lys Asp             260 265 270 Tyr Lys Pro Pro Val Phe Glu Glu Lys Asp Asp Ser Asn Met Asp Asp         275 280 285 Val Asp Ala Val Phe Lys Asp Ser Glu Glu His His Val Thr Glu Lys     290 295 300 Lys Glu Glu Gln Pro Ala Ala Ile Asn Ala Phe Glu Ile Ile Ser Met 305 310 315 320 Ser Arg Gly Leu Asn Leu Glu Asn Leu Phe Asp Pro Glu Gln Glu Phe                 325 330 335 Lys Arg Glu Thr Arg Ile Thr Leu Arg Gly Gly Ala Asn Glu Ile Ile             340 345 350 Asp Lys Ile Glu Glu Ala Ala Lys Pro Leu Gly Phe Asp Val Gln Lys         355 360 365 Lys Asn Tyr Lys Met Arg Leu Glu Asn Val Lys Ala Gly Arg Lys Gly     370 375 380 Asn Leu Asn Val Ala Thr Glu Ile Phe Gln Val Ala Pro Ser Leu His 385 390 395 400 Met Val Gln Val Ser Lys Ser Lys Gly Asp Thr Leu Glu Phe His Lys                 405 410 415 Phe Tyr Lys Lys Leu Ser Asn Ser Leu Glu Asn Val Val Trp Thr Asn             420 425 430 Asn Glu Val Lys Lys Ala Lys Val Lys Gly Gly Gly Lys Gly Asn Phe         435 440 445 Lys Gly Ala Thr Glu Ile Phe Gln Val Ala Pro Ser Phe His Met Val     450 455 460 Gln Val Leu Lys Ser Lys Gly Gly Pro Phe Glu Phe Pro Gln Phe Phe 465 470 475 480 Lys Lys Leu Phe Asn Phe Leu Gly Lys Val Val Trp Ala Asn Asn Glu                 485 490 495 Met Lys Lys Thr Gly Lys             500 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HPT-Fw primer <400> 3 ggatttcggc tccaatgtcc tgacgga 27 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HPT-Rv primer <400> 4 cttctacaca gccatcggtc caga 24 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pBigs_SfiI-Fw primer <400> 5 tattcggaga gggtacgtat ttttac 26 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pBigs_sfiI-Rv primer <400> 6 gcaacaggat tcaatcttaa gaaact 26 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CIPK3-Fw primer <400> 7 atcggaagca agtcaagcg 19 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CIPK3-Rv primer <400> 8 ctccatcgta gcctccgtca t 21

Claims (12)

Consisting of the amino acid sequence of SEQ ID NO: 2, Chinese cabbage (Brassica rapa) non-biological (abiotic) stress tolerance related BrCIPK3 (Brassica rapa calcium CBL-interacting protein kinase 3) of the resulting protein. A gene encoding the protein of claim 1. 3. The gene according to claim 2, wherein the gene consists of the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 4. A method of increasing the abiotic stress resistance of a plant comprising the step of transforming the recombinant vector of claim 4 into a plant cell, overexpressing the BrCIPK3 gene. Transforming a plant cell with the recombinant vector of claim 4; And
A method for producing a transgenic plant having increased abiotic stress resistance, comprising: regenerating a plant from the transformed plant cell. A transgenic plant with increased abiotic stress tolerance produced by the method of claim 7. The transgenic plant of claim 8, wherein the abiotic stress is salt, aluminum, low temperature or oxidative stress. The transgenic plant of claim 8, wherein the plant is a monocotyledonous plant. Seeds of plants according to claim 8. A composition for increasing abiotic stress resistance of plants comprising the BrCIPK3 gene consisting of the nucleotide sequence of SEQ ID NO: 1.

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