KR20110051578A - Brcpi gene enhancing plant tolerance to abiotic stress and uses thereof - Google Patents

Abstract

PURPOSE: A Brassica rapa-derived BrCPI(Brassica rapa cysteine protease inhibitor) gene is provided to reduce abiotic stress and to enhance crop yield. CONSTITUTION: A Brassica rapa-derived BrCPI protein has an amino acid sequence of sequence number 2. A gene encoding the BrCPI protein has a base sequence of sequence number 1. Resistance against environmental stress is enhanced by overexpression of BrCPI gene through transformation. The environmental stress is an abiotic stress such as salt, low temperature, or ABA(abscisic acid).

Description

{BrCPI gene enhancing plant tolerance to abiotic stress and uses}} to increase plant resistance to abiotic stress

The present invention relates to a BrCPI gene and its use to increase plant resistance to abiotic stress, more specifically BrCPI ( Brassica rapa cysteine protease inhibitor) protein derived from turnips, genes encoding the protein, including the gene Recombinant vector, a host cell transformed with the recombinant vector, a method for producing a transgenic plant using the vector, a method for increasing resistance to environmental stress of a plant using the vector, and an environmental stress produced by the method. It relates to a transgenic plant and its seed with increased resistance to.

Turnip ( Brassica rapa L.) is a temperate plant belonging to the Cruciferaceae, which was introduced and cultivated in Korea around the 13th century. Cruciferous plants in the world have been divided into 3,000 species of 350 genera including cabbage, cabbage, broccoli, mustard, and rapeseed, and have shown great results in genetic and sarcoma studies through the establishment of the theory of genomic evolution.

The genome of the main cruciferous plants is turnip ( B. rapa 2n = 20 AA), radish ( Raphanus sativus , 2n = 18), cabbage ( B. oleracea , n = 9, CC), cabbage and cabbage rapeseed ( B. napus , 2n = 4 × = 38, AACC), cabbage and black mustard hybridized ( B. juncea , 2n = 4 × = 36, AABB), etc. . Chinese cabbage is one of the four major vegetable crops, accounting for about a quarter of the total vegetable growing area, producing about 2.4 million tons per year at about 40,000 ha.

Many Korean cabbage researchers have been cultivating under high temperature and high humidity conditions for a long time and have been trying to improve the yield, cabbage type, maturity and disease resistance. . Nevertheless, the economic damage caused by various diseases of cabbage year after year is enormous.

The main diseases that are in question in domestic cabbage cultivation are Downy mildew disease, Witch disease, Wart disease, Rotting disease, White rust disease, Mosaic disease, etc.A large amount of chemical spray is required for the control of these diseases. In particular, in case of insomnia, the damage is so severe that it is the biggest obstacle in the production of Chinese cabbage in summer, not only in Korea but also in Japan and China.

Recently, efforts to develop crops resistant to pests due to the development of molecular biology have emerged as an important research topic in many countries, but many studies have been carried out, but the exact mechanism of defense has not been elucidated. Research on cabbage genes in Korea focuses on several universities and research centers, including gene bank construction, isolation and molecular characterization of functional genes, control of gene expression by RNAi, enhanced gene expression, comparative analysis of EST, and microarray techniques. The gene expression analysis used is being actively conducted.

Various physiological reactions related to the metabolism in the plant, especially biodefense, proceed in such a way that the specific protein is activated through proteolysis. Cysteine protease inhibitors protect cells from unfavorable proteolytic activity by cysteine proteases from inside or outside the cell and play an important role as a biological defense against invaders.

Until now, cysteine protease inhibitors are cathepsin B, H, L, S, and papain, which inhibit the action of cysteine-type protease with thiol groups, and are widely distributed in plants and animals and have various types and characteristics. About 30 plant-derived cysteine protease inhibitors are known, are present in an unsulfated bond and active site of QxVxG in a size of 12 to 16 kDa and have very high homology with animal cysteine protease inhibitors derived from eggs. According to the nucleotide sequence, plant-derived cysteine protease inhibitors are classified as a new subfamily in the term of phytocystatin. These proteins show different expression patterns in plant growth and development, and are known to be sensitive to biological and abiotic stresses. The role of these proteins in plant cells protects cells by inhibiting cysteine protease action, which has a reserve of biological activity for use in embryonic development, seed germination and programmed cell death. In addition, the defense mechanism for protecting plants from invasive beetles, nematodes, fungi and viruses is an important immune factor because inhibitors of cysteine protease play an important role as digestive enzymes in cell replication and gut. do. In particular, cysteine protease inhibitors in abiotic stress response showed high mRNA accumulation in cold tissues of barley grown in cold shock and dark conditions, and high expression levels in the roots and leaves of chestnut trees treated with cold, saline and high temperature stress. . In addition, phytocystatin is used as a supplement in the food industry and applied to biotechnology. The plants of the genus Brassica, including broccoli, cabbage and kale, have been studied as a model plant for the study of self-incompatibility. But one of the most important small crops in Asia is Turnip ( Brasssica There is no research on the defense mechanism in rapa L.).

The present invention is derived from the above-mentioned demands, and in the present invention, a phytocystatin-related gene (BrCPI1) is isolated from a bud of the turnip ( Brassica rapa ), and the structural analysis and expression characteristics of the gene are determined. The present invention has been completed by revealing that the introduced plants have increased resistance to abiotic stress.

In order to solve the above problems, the present invention is BrCPI ( Brassica) derived from turnip rapa cysteine protease inhibitor) 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 for producing a transgenic plant using the vector.

The present invention also provides a method of increasing the resistance to environmental stress of plants using the vector.

The present invention also provides a transgenic plant and its seed having increased resistance to environmental stress produced by the method.

According to the present invention, since the turnip BrCPI gene of the present invention is thought to act to reduce abiotic stress, a plant highly resistant to various stresses such as salt and low temperature can be effectively obtained. Therefore, it is expected to contribute greatly to the increase in yield of economic crops and the like.

According to an aspect of the invention, the invention provides a turnip derived BrCPI (Brassica rapa cysteine protease inhibitor) protein.

The range of BrCPI protein according to the present invention is turnip (Brassica rapaProtein having an amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of the protein. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2.

The present invention also provides a gene encoding the BrCPI protein. Genes of the invention are involved in abiotic stress resistance and include both genomic DNA and cDNA encoding BrCPI proteins. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene is a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% sequence homology with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

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

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of recombinant vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer a portion of themselves, the so-called T-region, to plant cells. 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 a chemical method, which corresponds to all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin (kanamycin), G418, bleomycin, hygromycin (hygromycin), chloramphenicol and There is the same antibiotic resistance gene, but it is not limited thereto.

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

In the recombinant vectors of the present invention, conventional terminators can be used, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens) Terminator of the octopine gene, but 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. The host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains.

In addition, when transforming a vector of the present invention to eukaryotic cells, yeast ( Saccharomyce) as a host cell cerevisiae ), insect cells, human cells (eg, CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), and plant cells. The host cell is preferably a plant cell.

The method of 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 .

The present invention also provides a method of increasing resistance to environmental stress of a plant, comprising the step of transforming plant cells with the recombinant vector of the present invention, overexpressing the BrCPI gene. The environmental stress is preferably abiotic stress, the abiotic stress may preferably be, but not limited to, salt, low temperature or abscisic acid (ABA).

Transformation of a plant means any method of transferring DNA to a plant. 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 terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the 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. 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.

The present invention also provides a transgenic plant having increased resistance to environmental stress produced by the method. The transgenic plant is Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrots, buttercups, parsley, cabbage, cabbage, gall, watermelon, melon, Dicotyledonous plants such as cucumbers, pumpkins, gourds, strawberries, soybeans, green beans, kidney beans, peas, or monocotyledonous plants such as rice, barley, wheat, rye, corn, sugarcane, oats, and onions, preferably dicotyledonous It is a plant, More preferably, it is a baby pole.

The present invention also provides a seed obtained from a transgenic plant with increased resistance to environmental stress.

The invention also comprises the steps of transforming a plant cell with a recombinant vector according to the invention; And

It provides a method for producing a transformed plant comprising the step of regenerating a transformed plant from the transformed plant cells.

The method of the present invention comprises the step of transforming plant cells with the recombinant vector according to the present invention, wherein the transformation can be mediated by Agrobacterium tumefaciens . The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

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

1. Plant Materials

Turnip ( Brassica rapa L.) Seeds were induced in the glass greenhouse of the Department of Horticulture, Hankyong National University by maintaining the low temperature below 12 ℃ for a certain period of time. Before flowering, roots, stems, leaves, ovules, petals, stamens, calyxes, flowers, etc. were separated, frozen in liquid nitrogen, and stored in a -80 ° C freezer to be used as RNA extraction material. Also, in order to obtain the plant material at the beginning of germination, turnip seeds were germinated on a filter paper in the dark at 25 ° C., and then seedlings of 1, 2, 4, 8, and 10 days were collected and placed in liquid nitrogen and frozen. Total RNA was isolated by storage in a freezer at 80 ℃.

2. Total RNA  detach

0.1 g of plant tissue was measured and ground with liquid nitrogen using a mortar and stick prepared beforehand. The ground tissue was mixed in a 1.5 ml centrifuge tube containing 1 ml of TRIzol-reagent (1 ml TRIzol-reagent / 50-100 mg tissue) using a measuring spoon, and placed at room temperature for 15 minutes to completely separate the nucleoprotein. 0.2 ml chloroform was added to 1 ml TRIzol-reagent and vortex for 15 seconds. The mixture was left at room temperature for 5 minutes and then centrifuged at 11,000 rpm for 15 minutes at 4 ° C., so that only the upper layer was transferred to a new tube. The upper layer was mixed well with 0.5 ml of isopropanol (per 1ml TRIzol-reagent) and 0.25ml of high salt precipitation solution (0.8M sodium citrate / 1.2M NaCl) and left for 10 minutes at room temperature to precipitate RNA. The extracted product was centrifuged at 11,000 rpm for 10 minutes at 4 ° C to discard the supernatant, and 1 ml of 75% ethanol was added to the RNA precipitate, followed by centrifugation at 11,000 rpm for 5 minutes at 4 ° C. The supernatant was then removed and the RNA precipitate was dried for 5-10 minutes in vacuo. Finally, 50 μl-100 μl of DEPC-treated sterile water was dissolved, and the amount of RNA was quantified using a spectrophotometer.

3. cDNA  Synthetic and RT - PCR  analysis

Total RNA was performed according to the RNase H-Reverse Transcriptase (Gibco SuperscriptII) protocol and the first strand cDNA was synthesized from 5 μg total RNA. To 5 μg of RNA template, 1 μl of 50 mM oligo-dT, 1 μl of 10 mM dNTP, and 10 μl of DEPC water were added to 17 μl, denatured in a 65 ° C. incubator for 5 minutes, cooled on ice, and cooled to 5 × 1 strand buffer 4 1 μl and 0.1 μl DTT, 1 μl of RNase inhibitor (40 units / μl), and 1 μl of SuperscriptII RTase (200 units / μl) were added to prepare a final volume of 20 μl, followed by 5 minutes at 25 ° C. and 1 hour at 42 ° C. The enzyme was inactivated by heating at 72 ° C. for 15 minutes. 2 μl of synthesized cDNA as a template, 5 μl of 10 × PCR buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1.5 μl of 50 mM MgCl ₂ , 1 μl of 10 mM dNTP mix, 0.4 μl of Taq DNA polymerase (5 units / μl), 1 μl each of the forward primer (10 μM) and the reverse primer (10 μM) was added, and the final volume was 50 μl with 38.1 μl of sterile water, followed by PCR. The primer used was synthesized from the information obtained from the NCBI database (5'-GAACTTGGAAGGTACTG-3 '; SEQ ID NO: 3), (5'-AGTTTTGCGCTTCAGGCA-3'; SEQ ID NO: 4). The PCR reaction was premodified at 94 ° C. for 4 minutes, denatured at 94 ° C. for 1 minute, annealed at 55 ° C. for 1 minute, and extended at 72 ° C. for 2 minutes to 35 cycles, and finally extended at 72 ° C. for 10 minutes. . PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide to identify bands. The amplification product was cloned into pGEM T-Easy vector (Promega) to examine the nucleotide sequence, and the homology comparison between genes was performed by BLAST analysis.

4. Cloning

The cDNA was cloned into the pBig vector using A-tailing at the 3′-end.

vector DNA Preparation

In the restriction enzyme treatment of the pBig vector containing the GUS gene, 0.5 μl of SfII (10 units / μl), 1.5 μl of 10 × buffer, 5 μl of vector DNA (200 ng / μl) were added and 7.5 μl of sterile water was added to a final volume of 15 μl. It was. After reacting at 37 ° C. for 2 hours, it was confirmed on a 0.8% agarose gel. The vector DNA was reextracted from the gel for dephosphorylation. For BAP treatment, 34 μl of vector DNA, 5 μl of 10 × buffer, 1.5 μl of BAP, and 9.5 μl of sterile water were collected in a tube and reacted at 55 ° C. for 1 hour.

Competent cell  Ready

E. coli DH5α strains stored in -80 ° C. cryogenic freezer in 50% glycerol stock in LB agar medium were streaked and incubated at 37 ° C. for 16 hours. Single colonies were incubated for 16 hours at 37 ° C. and 200 rpm in a 50 ml tube containing 10 ml of sterile LB broth [tryptone 10 g / L, yeast extraction 5 g / L, sodium chloride (NaCl) 5 g / L]. 2.5 ml of the culture was added to 250 ml of LB broth and incubated at 37 ° C. at 200 rpm for 4 hours until OD 600 = 0.6. After cooling for 10 minutes on ice, the culture solution was divided into 200 ml centrifuge tubes and placed at 5,000 rpm. Centrifuged at 4 ° C. for 10 minutes. The supernatant was discarded, and the same amount of sterile water was added to the precipitated cells, followed by centrifugation three times for 10 minutes at 5,000 rpm at 4 ° C. Finally, 400 μl of 10% glycerol was added to the precipitated cells, and 50 μl was aliquoted into a 1.5 ml tube, and immediately placed in liquid nitrogen and frozen, and stored in an ultra-cold freezer at -80 ° C.

With plasmid vector insert DNA of Ligation

For ligation, prepare a ratio of vector (pBig) to insert DNA (CPII gene) of 1: 3, 1 μl of T4 DNA ligase 10 × buffer, 1 μl of T4 DNA ligase (3 Weiss units / μl), vector DNA (50ng) 1μl, Insert DNA (20ng) was added 1μl and the final volume was adjusted to 10 with sterile water and reacted at 4 ℃ for more than 16 hours.

Transformation Using Electroporation

The competent cells stored in the cryogenic freezer at -80 ℃ were taken out, dissolved in ice, mixed with 1-3μl of ligation-completed DNA, injected into the electroporation cuvette, and transformed by applying an electric shock at 1440V. Then, 1 ml of SOC medium was added thereto, mixed, put into a sterile test tube, and incubated at 37 ° C. for 1 hour. Subsequently, 10 μl of 839 mM IPTG (Isopropyl-Thio-β-D-Galactopyranoside) and 50 mg / ml X-gal (5-Bromo-4-Chloro-3-Indolyl-β-) were added to LB agar medium containing 50 mg / L of kanamycin. 40 μl of D-Galactopyranoside) was dropped onto the medium and spread evenly with a stirrer, and then placed at 37 ° C. for 10 minutes, and then 200 μl of the culture solution was dropped by pipetting. Colonies were observed by incubating the curry in a 37 ° C. thermostat for 16 hours. All white colonies were subjected to colony PCR analysis to observe the size of the plasmid and to confirm the ligation. PCR was used to confirm the introduction using primers of the CPI gene obtained from the NCBI database.

Plasmid DNA  extraction

A total of 12 samples were selected by colony PCR and plasmid DNA was extracted. Single bacterial colonies were inoculated in 5 ml of LB medium containing 50 mg / L of kanamycin and shaken at 200 rpm in a 37 ° C. incubator for 16 hours. 1 ml of the culture was dispensed into a 1.5 ml tube and centrifuged for 1 minute at 12,000 rpm at 4 ° C. To the precipitated cells, 200 μl of cold GTE solution I [glucose 2.25g, 1M Tris-Hcl (pH 8.0) 6.25ml, 0.5M EDTA 5ml per 250ml] was added and mixed and solution II (0.2N NaOH, 1% SDS). ) 200 μl was added followed by tapping. Immediately, 200 μl of solution III (3M potassium acetate pH 7.5) was added thereto, and then tapped and stored at −20 ° C. for 5 minutes, followed by centrifugation at 4 ° C. at 12,000 rpm for 5 minutes. Transfer the supernatant to another tube, add 3µl of RNase (10mg / ml), leave for 30 minutes at 37 ℃, and add 500µl of PCI (phenol / chloroform / pediatric wheat alcohol, 25: 24: 1). Centrifuged at 12,000 rpm for 5 minutes at room temperature. 1 ml of 100% ethanol was added to the supernatant, stored in a -20 ° C freezer for 30 minutes, and centrifuged at 4 ° C and 12,000 rpm for 15 minutes to precipitate plasmid DNA. Thereafter, 500 µl of 70% ethanol was added thereto, followed by tapping, centrifugation at 12,000 rpm for 5 minutes, and the final precipitate was dried and used in 50 µl of sterile water.

5. Gene expression analysis according to abiotic stress treatment

In order to determine the response to the abiotic stress of the BrCPI1 gene, the expression patterns of the genes in the tissues obtained after the stress treatment were examined by RT-PCR analysis. The types of stress were 100 mM ABA, dry, osmotic (15% PEG6000), 250 mM NaCl, 4 degreeC cold, etc. were used. After seed germination, 14-day-old seedlings were used in Yosida nutrient solution (Yosida et al. 1976, Philippines: IRRI. 83) to grow at 25 ° C and 70-75% of relative humidity to grow leaves of plant tissues at different times. And roots were harvested. Total RNA extraction and RT-PCR analysis were performed using Intron Bio's commercial plant RNA extraction kit and one-step RT-PCR kit, respectively. Primers that can be specifically amplified for each gene were selected in consideration of the GC content ratio of the 3'-UTR region of the gene. Primer synthesis was made by Takara. RT-PCR reaction conditions were carried out in reverse transcription reaction for 30 minutes at 45 ℃, 5 minutes at 94 ℃, repeated 40 times in steps of 20 seconds at 94 ℃, 40 seconds at 58 ℃, 1 minute at 72 ℃, Final amplification at 72 ° C. for 5 minutes. The amplified PCR product was electrophoresed on 1.5% agarose gel to confirm the expression level.

6. Agrobacterium for transformation Tumer faciens ( Agrobacterium tumefaciens ) Strain creation

Plant Expression Vector Construction

The pBig vector containing the GUS gene was subjected to SfII treatment, and an 881 bp CPI1 gene was inserted at that position. The recombinant vector was transformed into E. coli and plasmid was extracted from a single colony selected from LB medium containing 50 mg / L of kanamycin to confirm the presence of insert DNA.

Agrobacterium In Tumerfaciens  Transformation

Plasmids identified for vector construction of plant expression were transformed into Agrobacterium tumerfaciens LBA4404. Agrobacterium tumerfaciens competent cell LBA4404 prepared in the same manner at 28 ℃ was dissolved in ice and mixed with 1 μl of plasmid DNA and injected into the electroporation cuvette. Then, transformed by applying an electric shock at 1440V, 1ml SOC medium was injected and mixed, and then put into a sterile test tube and incubated at 28 ℃ 200rpm for 1 hour. The culture medium was AB agar medium containing 50 mg / L of kanamycin (stock I: ₃ g of K ₂ HPO ₄ , stock II: 1 g of NaH ₂ PO ₄ , NH ₄ Cl 1 g, MgSO ₄ 7H ₂ O 0.3 g, KCl 0.15 g, CaCl ₂ 0.01 g, stock III: 2.5 mg FeSO ₄ · 7H ₂ O, 5 g / L glucose) was pipetted and smeared, and the colonies were observed by incubating the sare for 2-3 days in a 28 ° C. thermostat. All white colonies were transformed by colony PCR, and the cultures of the identified strains were inoculated in AB liquid medium to which 50 mg / L of kanamycin was added, followed by incubation at 200 rpm in a 28 ° C. incubator for 2-3 days. The culture solution was added to the same amount of 50% glycerol and stored in the cryogenic freezer (-80 ℃).

7. Genetic analysis of transgene

Total DNA  detach

Total DNA separation was performed using CTAB (Cetyltrimethyl ammonium bromide) method. 0.5 g of leaves from transformants and control plants were finely ground with liquid nitrogen and 1.5 ml centrifuged with 400 μl of DNA extraction buffer [100 mM Tris-HCl (pH 8.0), 50 mM EDTA, 500 mM NaCl]. Put the ground tissue in the separation tube and mixed 20 times up and down. Then 200 μl of 2 × CTAB buffer [2% (w / v) CTAB, 100 mM Tris-HCl (pH8.0), 20 mM EDTA, 1.4 M NaCl, 1% pvp-40 (polyvinylpyrrolidine)] was added to the same method. After mixing with 10% SDS and mixed up and down about 10 times, it was allowed to stand for 20 minutes in a 65 ℃ constant temperature water bath. Add 200 μl of 5M potassium acetate (pH 7.5) and mix up and down about 50 times. Add 700 μl of PCI [Phenol / Chloroform / Isoamyl Alcohol (25: 24: 1)] and mix up and down 30 times. The protein was separated by centrifugation at 12,000 rpm for 10 minutes. About 400 μl of the upper layer was transferred to a new 1.5 ml tube and the same amount of isopropanol was added and stored in a -20 ° C. freezer for 20 minutes, followed by centrifugation at 4 ° C. at 12,000 rpm for 15 minutes to precipitate DNA. Precipitated DNA was washed with 1 ml of 70% ethanol, dried thoroughly at room temperature, and sufficiently dissolved in 50 µl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH7.4) to which 2 µl of RNase (1 mg / ml) was added. It used for 1 hour at 37 degreeC.

For transgene testing PCR  analysis

The PCR reaction to confirm the introduction of the CPI1 gene was used 20ng template DNA, and synthesized three primers as follows. Pre-denaturation with hygromycin resistance gene (HPT) specific amplification primer set (forward 5'-GCGTGACCTATTGCATCTCC-3 '; SEQ ID NO: 5, reverse 5'-TTCTACACAGCCATCGGTCC-3'; SEQ ID NO: 6) After 30 seconds of denaturation at 94 ° C, annealing at 58 ° C for 30 seconds, and extension for 2 minutes at 72 ° C was performed 30 cycles, and finally extension at 72 ° C for 5 minutes. Premodified at 94 ° C for 4 minutes with a primer set capable of specifically amplifying BrCPI1 gene (forward 5'-ATGGCTTCCAAAGTTCAACTCGTG-3 '; SEQ ID NO: 7, reverse 5'-TCAAGTTTCAGAAACCATCTT-3'; SEQ ID NO: 8) At 1 minute denaturation, annealing at 55 ° C. for 1 minute, extension process at 2 ° C. at 72 ° C. for 35 cycles, and extension at 72 ° C. for 10 minutes were performed. The resulting amplified product was run on a 1.5% agarose gel and then stained with ethidium bromide for 10 minutes to identify bands.

8. RT - PCR On by BrCPI1  Gene expression analysis

In order to analyze the expression level of the BrCPI1 gene of the transformant, the mRNA was isolated from the leaf tissue (0.2 g) and subjected to RT-PCR analysis. The isolated mRNA was converted to cDNA using reverse transcriptase (Promega, USA) and used as a template a uniformly adjusted cDNA by spectrophotometer. The primer set of BrCPI1 gene used for RT-PCR was synthesized with forward 5'-ATGGCTTCCAAAGTTCAACTCGTG-3 '(SEQ ID NO: 9) and reverse 5'-TCAAGTTTCAGAAACCATCTT-3' (SEQ ID NO: 10). PCR conditions were premodified at 94 ° C. for 4 minutes, followed by denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, and extension at 72 ° C. for 2 minutes to 35 cycles, and finally extension at 72 ° C. for 10 minutes. Actin gene was used as a quantitative reference for RT-PCR. Primer set of β-actin gene was used by synthesizing forward 5'-ATGGTTGGGATGGGTCAAAAA-3 '(SEQ ID NO: 11), reverse 5'-TCTTTAATG TCACGGACGATT-3' (SEQ ID NO: 12). The PCR reaction was premodified at 94 ° C. for 4 minutes, then denatured at 94 ° C. for 1 minute, annealed at 58 ° C. for 1 minute, and extended to 1 minute at 72 ° C. for 30 cycles, and extended at 72 ° C. for 5 minutes. PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide for 10 minutes to identify each band. Quantitative analysis of RNA quantified the expression of Actin gene with BrCPI1 gene and reference within the standard curve. 50 μl of cDNA stock (5 ng / μl) of each post-transgenic lineage was diluted and 5 μl per tube was added. For quantitative PCR, SmartCycler II (TaKaRa, Japan) and SYBR RT-PCR kit (Perfect Real Time) were used, and a method of detecting SYBR Green I bound to amplified DNA was used. Relative expression was calculated from the detected values using a standard curve, measured in two replicates for each sample and the average value was used for analysis. In addition, the expression level of the actin gene was corrected to 1 for each strain, and the expression level of the BrCPI1 gene was calculated and displayed as a graph.

Example  One. BrCPI1  Gene Isolation and Structural Analysis

1-1. Gene structure analysis

Primers Br1 (5'-GAACTTGGAAGGTACTG-3 '; SEQ ID NO: 13) and Br2 (5'-AGTTTTGCGCTTCAGGCA-3'; SEQ ID NO: 14) were synthesized by NCBI data analysis to isolate a turnip derived cysteine protease inhibitor related gene. RT-PCR analysis using these two primers yielded a 171 bp fragment, indicating that 88% homology with Arabidopsis-derived cysteine protease inhibitors was considered to be a cysteine protease inhibitor. Therefore, using the nucleotide sequence of the fragment, the full length was obtained by the RACE method. As a result, a 575 bp cDNA fragment was obtained at 3 'RACE and a 531 bp fragment at 5' RACE. The fragment obtained from the 3 'RACE contains 17 A tails, 209 bases exist as untranslated regions (UTRs), and the 125 fragments overlapped with the first fragment. In the fragment obtained from the 5 'RACE, the fragment separated from the first fragment was overlapped with 62bp, and the start codon ATG was present. Therefore, the gene related to cysteine protease inhibitor derived from turnip was 881bp and named BrCPI1. Structural analysis of the BrCPI1 gene was performed by the DNA star program. The start codon of the ATG was located at the 63rd position, and the end codon TGA was located at the 673th, after which the full length cDNA containing poly A was present (FIG. 1). . Therefore, the BrCPI1 gene was composed of 202 amino acids in an ORF region composed of 609 bases, and predicted pI = 4.6 with a size of 15.5kDa.

As a result of hydrophobicity analysis and von Heijne method using the amino acid sequence of BrCPI1 gene, the sequence having a signal was A ₂₃ . In addition, L ₁₀₃ amino acids BrCPI1 gene product of the judging that the amino acids of the L ₁₀₃ and P ₁₀₄ present in the first to the N-terminus of BrCPI1 genes in the comparison of the number of cysteine protease showed high homology of the protein with the known It is thought to be the end region of the protein structure that constitutes it. Cysteine proteases usually require the removal of amino acid terminal regions to produce active enzymes.

The amino acid terminal region is called a proregion and plays a role not only in controlling enzyme activity but also in correctly chaining newly produced proteins.

And it protects against denaturing effect when pH condition changes suddenly. The N-terminal amino acid sequence of the cysteine protease known to date and phytocystatin, the BrCPI1 gene product isolated in this study, are very similar in structure and are 15.5 kDa. Therefore, the papain cysteine protease according to the proregion region can be divided into cathepsin B consisting of about 60 amino acids in the N-terminal region and the subfamily of cathepsin L consisting of about 100 amino acids (FIG. 2).

Moreover, there exists the sequence QxVxG motif which preserves only a cysteine protease among amino acid sequences. Therefore, cystatin, the product of BrCPI1 gene, was cathepsin L in the papain subfamily.

1-2. Gene Strain  analysis

A strain was created between the amino acid sequence of the BrCPI1 gene isolated from this experiment and the previously reported gene. Lineages were replaced with phytocistatin-related gene names obtained from NCBI search. As a result of the generation of strains, it was found that the currently reported cysteine protease inhibitor-related gene group is largely divided into four groups.

Since there is little information on the function of the gene, its function is unpredictable, suggesting the possibility of functional separation between the four groups of genes. BrCPI1 isolated in the present invention was confirmed to form clusters such as cystatin gene derived from Arabidopsis and cabbage (Fig. 3). From these results, the BrCPI1 gene is expected to have functions similar to Arabidopsis and cabbage derived genes.

Example  2. BrCPI1  Gene copy  Number and Expression Analysis

To determine the number of copies of the BrCPI1 gene on the turnip genome, the turnip leaves were extracted and Southern blot analysis was performed using a 3 'terminal region specific nucleotide sequence with low homology with other cysteine protease inhibitors of turnips. All four restriction enzymes used were seen as single bands (FIG. 4).

Also, BrCPI1 by institution of turnips RT-PCR was performed with known CPI genes (BrCC2, BrCC3, BrCC4, BrCC5) in Chinese cabbage to analyze the expression patterns of the genes. It can be seen that it is expressed (Fig. 5).

In addition, the results of examining the expression pattern of BrCPI1 gene according to the development stage of turnip is as shown in FIG. The expression patterns of cabbage-derived CPI genes BrCC2, BrCC3, BrCC4, BrCC5 and BrCPI1 showed almost similar expression patterns except for BrCC4. The BrCPI1 gene increased expression after 15 days of germination, similar to BrCC2, BrCC3 and BrCC5. However, unlike the BrCC3 gene, which is strongly maintained until 20 days after germination, and the BrCC5 gene, which is maintained until 30 days after germination, BrCPI1 and BrCC2 were strongly expressed around 15 days after germination and then decreased in expression. Therefore, the turnip-derived BrCPI1 gene is expected to have the same function, considering that the cabbage-derived BrCC2 gene has a similar expression pattern.

Therefore, BrCPI1 gene is a gene that exists as a single copy in the turnip genome and has a defense mechanism in terms of tissue and organ expression. It is a gene mainly expressed in organ differentiation and growth point, and is considered to be a gene involved in seed development and plant growth.

Example 3. BrCPI1  Transgenic transformation and future generation

3-1. Plant expression binary  Vector structure analysis

To construct a plant-expressed binary vector, a gene fragment made by synthesizing a SfiI restriction enzyme site was inserted at both ends of a BrCPI1 gene isolated from turnips by SfiI treatment of the pBIG vector (FIG. 7). Therefore, BrCPI1 gene was expressed under the control of the 35S promoter. The selection marker gene, which contains the hygromycin resistance gene (HPT), is considered to be a strong candidate for regeneration.

3-2. Selection and Purification of Transgenic Baby Pole Pole, Generation Generation

Transformation of the BrCPI1 gene was performed by the spray method. Agrobacterium was moistly infected with the flower organs by spraying the Arabidopsis before flowering, and seeds were collected.

The obtained T1 seed was selected from 10 plants normally grown among 30 transformants in MS medium containing hygromycin to grow T2 generation (FIG. 8).

3-3. Identification and expression analysis of transgene

In order to know the presence or absence of the gene introduced from the transgenic plant, PCR analysis was performed using a primer set capable of specifically amplifying the HPTII and BrCPI1 genes using antibiotic-resistant T1 plants and control plants. As a result, amplification products could not be obtained from control plants without introducing the genes, but HPT gene products were identified from the transgenic plants. In addition, PCR analysis of the BrCPI1 gene confirmed the amplification product. Therefore, it can be said that the gene used as a selection marker in gene water and the BrCPI1 gene of interest are stably introduced into the cabbage genome. In order to confirm the expression of the BrCPI1 gene in the transgenic plant, RT-PCR analysis was performed by extracting total RNA from each strain to confirm that the BrCPI1 gene was normally transcribed into mRNA. From the above results, it can be said that BrCPI1 gene isolated from turnip was introduced into Arabidopsis genome and stably expressed.

3-4. Abiological  Due to changes in stress BrCPI1  Gene expression analysis

The BrCPI1 gene influences defense mechanisms and is thought to be highly involved in seed development and plant growth. Therefore, as a result of examining the expression level of BrCPI1 gene after ABA treatment among salts, cold and growth regulators, which are abiotic stresses affecting plant growth, ABA treatment showed almost the same expression level as control (FIG. 9). In addition, as a result of analyzing the expression level of the ground part by treating 250 mM NaCl, 4 ° C cold for the expression of BrCPI1 gene according to the environmental stress change, the expression level of the gene was increased when the plant was subjected to environmental stress. (FIG. 9). Salt stress is caused by Na + and Cl- ion concentrations derived from NaCl, leading to an increase in Ca2 + ions in the cell, and the Ca2 + ion concentration in the cytoplasm that is changed instantly acts as a second messager, signaling for cell stress. It activates the system and regulates the influx of K + ions and K + / Na + relationships that are involved in adapting to salt stress in cells. ABA is a plant hormone that plays an important role in plant abiotic stress and is known to mediate the regulation of genes involved in the stress response. Therefore, considering that BrCPI1 gene is increased in saline and cold stressed Arabidopsis leaves, plants under environmental stress have growth-related molecular mechanisms that allow the plant to maintain its normal expression for some time. I think it is not.

Therefore, it is thought that the expression of BrCPI1 gene in the cell results in a change in other enzyme activity in the cell, thereby showing a difference in activity by interaction between proteases in the cell. Proteases are known to be physiologically changed in the process of plant growth and development, such as metabolic signaling and amino acid synthesis in cells, and it is known that there are many changes in the aging process. Therefore, the BrCPI gene derived from turnips is involved in the defense mechanisms of plants, suggesting that they play a huge role in plant growth and development.

1 shows the nucleotide and deduced amino acid sequences of the BrCPI1 gene encoding cysteine protease inhibitors in turnip plants.

Figure 2 shows the deduced amino acid sequence of the coding region of the cystatin gene (AtCPI, BoCPI, GmCPI, OsCPI, RcCPI, ZmCPI; NCBI data, BrCPI1) and its alignment. Virtual signal peptides and active site domains are dimmed.

3 shows the lineage of plant cystatin gene by Clustal W. FIG. The number next to the bar represents bootstrap values from 100 iterations.

4 shows Southern blot analysis using BrCPI1 in turnip plants. A 3 'region specific for the BrCPI1 gene was used as a probe. Restriction enzymes used are as follows; M: molecular weight marker , B : BamHI , E : EcoRI , H : HindIII , P : Pst I.

5 shows BrCPI1 expression analysis in turnip plant organs. A: root, B; Stem, C; Young leaves, D; Mature leaves, E; Ovule, F; Surgery, G; Petals, H; Calyx, I; Landscape. BrCC2-BrCC5; Cysteine Protease Inhibitor Coding Genes in Cabbage.

6 shows analysis of BrCPI1 expression according to the stage of development of turnip plants. A; 5 days after germination, B; 10 days after germination, C; 15 days after germination, D; 20 days after germination, E; 30 days after germination. BrCC2-BrCC5; Cysteine Protease Inhibitor Coding Genes in Cabbage.

7 shows a Ti-plasmid vector construct for BrCPI1 gene overexpression. Nos-p; nos gene promoter, Nos-t; nos gene terminator, HPT; Hygromycin phosphotransferase gene, 35S-p; Cauliflower mosaic virus 35S-promoter, LB; left border, RB; right border.

8 shows Arabidopsis plants transformed with a pBIG vector comprising the BrCPI1 gene. Left photo; Transgenic plants on selection medium, right picture; Phenotype of Transgenic Plants.

Figure 9 shows the growth pattern (A) under salt, cold and ABA stress of transgenic Arabidopsis T2 plants comprising BrCPI1 gene and the expression pattern (B) of BrCPI1 gene under salt, cold and ABA stress.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> BrCPI gene enhancing plant tolerance to abiotic stress and uses          the <130> PN09231 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 880 <212> DNA <213> Brassica rapa <220> <221> gene (222) (64) .. (672) <223> BrCPI coding sequence <400> 1 atgatgcaaa gccgtttctc aatcttcctc ttcatcttcg tctcctctgt gatcgctagt 60 gacatggcct tgctcggcgg cgttcgcgat gtacctgctt atcagaacag cgacgaggtc 120 gagagcctcg ctcgtttcgc cgtcgatgaa cataacaaga aagagaatgt actgctagag 180 tttgcgaggg ttgtgaaagc gaaggaacaa gttgtggcgg gaacgatgca tcatttaacg 240 ctggagatcg tcgaggctgg gaagaagaag ctttacgaag ccaaagtgtg ggtgaagccg 300 tggttgaact tcaaggagtt gcaggagttc aagcctgcct ctgatggtgc tccttccatc 360 actccctctg atcttggctg caagaaaggt gaacatgaat ctgggtggag ggaagttcca 420 ggggatgatc cagaggtaca gcacgttgct gatcatgctg ttaagactat tcagcagagg 480 tctaactctt tgttccctta tgagcttcag gaggttgttc atgctaacgc tgaggttact 540 ggtgaggctg caaagtacaa catggttctg aagttgaaga gaggagacaa ggaggagaag 600 ttcaaggtgg aggttcacaa gaaccatcaa ggtgttcttc atctcaacca catggagcaa 660 caccatgact agccttattt gactaacttc atttctttgt tcgtaaagtc ccataatata 720 atcggacaaa aaccaaaatg tgtatgatgt gaatctatta gcaatgggtt aatagtttgg 780 tctataaaga tgttttacgt tgtacataaa taaaggtaaa atatgttatg aagatgtgat 840 aaattactga aattttgcgt cgtcaaaaaa aaaaaaaaaa 880 <210> 2 <211> 202 <212> PRT <213> Brassica rapa <400> 2 Met Ala Leu Leu Gly Gly Val Arg Asp Val Pro Ala Tyr Gln Asn Ser   1 5 10 15 Asp Glu Val Glu Ser Leu Ala Arg Phe Ala Val Asp Glu His Asn Lys              20 25 30 Lys Glu Asn Val Leu Leu Glu Phe Ala Arg Val Val Lys Ala Lys Glu          35 40 45 Gln Val Val Ala Gly Thr Met His His Leu Thr Leu Glu Ile Val Glu      50 55 60 Ala Gly Lys Lys Lys Leu Tyr Glu Ala Lys Val Trp Val Lys Pro Trp  65 70 75 80 Leu Asn Phe Lys Glu Leu Gln Glu Phe Lys Pro Ala Ser Asp Gly Ala                  85 90 95 Pro Ser Ile Thr Pro Ser Asp Leu Gly Cys Lys Lys Gly Glu His Glu             100 105 110 Ser Gly Trp Arg Glu Val Pro Gly Asp Asp Pro Glu Val Gln His Val         115 120 125 Ala Asp His Ala Val Lys Thr Ile Gln Gln Arg Ser Asn Ser Leu Phe     130 135 140 Pro Tyr Glu Leu Gln Glu Val Val His Ala Asn Ala Glu Val Thr Gly 145 150 155 160 Glu Ala Ala Lys Tyr Asn Met Val Leu Lys Leu Lys Arg Gly Asp Lys                 165 170 175 Glu Glu Lys Phe Lys Val Glu Val His Lys Asn His Gln Gly Val Leu             180 185 190 His Leu Asn His Met Glu Gln His His Asp         195 200 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gaacttggaa ggtactg 17 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agttttgcgc ttcaggca 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcgtgaccta ttgcatctcc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttctacacag ccatcggtcc 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 atggcttcca aagttcaact cgtg 24 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tcaagtttca gaaaccatct t 21 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 atggcttcca aagttcaact cgtg 24 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tcaagtttca gaaaccatct t 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 atggttggga tgggtcaaaa a 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tctttaatgt cacggacgat t 21 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gaacttggaa ggtactg 17 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 agttttgcgc ttcaggca 18  

Claims (12)

BrCPI ( Brassica rapa cysteine protease inhibitor) protein derived from turnip, consisting of the amino acid sequence of SEQ ID NO: 2. The gene encoding the BrCPI protein of claim 1. According to claim 2, wherein the gene is a gene, characterized in that consisting 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 resistance to environmental stress in a plant, comprising the step of transforming the plant cell with the recombinant vector of claim 4, thereby overexpressing the BrCPI gene. 7. The method of claim 6, wherein said environmental stress is abiotic stress. 8. The method of claim 7, wherein said abiotic stress is salt, low temperature, or abscisic acid (ABA). Transgenic plant with increased resistance to environmental stress produced by the method of claim 6. 10. The transgenic plant of claim 9, wherein the plant is a dicotyledonous plant. Seeds of plants according to claim 9. Transforming the plant cell with the recombinant vector of claim 4; And Regenerating a transformed plant from the transformed plant cells.

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