KR100900928B1 - CaRma1H1 gene increasing plant stress resistance and transgenic plants transformed by CaRma1H1 gene - Google Patents

Abstract

The present invention provides CaRma1H1 protein derived from pepper which increases resistance to biological or abiotic stress, gene encoding the protein, enzymatic activity of the protein, recombinant vector comprising the gene, plant transformed with the recombinant vector, The present invention relates to a method of increasing resistance of a plant to stress using the gene, a transformed plant having increased stress resistance by the method, and a seed thereof. The CaRma1H1 gene isolated from the present invention and the CaRma1H1 protein expressed from the gene may increase resistance to stress of plants.

Chili, stress resistant, CaRma1H1, transformation, ubiquitation

Description

CaRma1H1 gene increasing plant stress resistance and transgenic plants transformed by CaRma1H1 gene

1 shows sequence analysis of the CaRma1H1 gene. The CaRma1H1 gene consists of 1369 nucleotides, and the region to be encoded is gray.

Figure 2 shows the amino acid sequence of the CaRma1H1 gene. It is a protein composed of 252 amino acids and shows a comparative analysis with a protein with high similarity to Arabidopsis. Consistent amino acids are shown in gray and ring finger domains are shown in red.

Figure 3 shows the Northern blot analysis of the CaRma1H1 gene in abiotic stress conditions.

Figure 4 shows the enzyme activity analysis of CaRma1H1 protein. MBP (maltose binding protein) was attached to CaRma1H1 protein, followed by auto ubiquitination, and UBA1, UBC8, ubiquitin, CaRma1H1 were added, and MBP-specific antibodies were used to confirm protein changes over time. Blot analysis was performed.

(a) 40 mM Tris-HCl, pH 7.5, 5 mM MgCl ₂ , 2 mM ATP, 2 mM dithiothreitol (DTT), 300 ng / μl ubiquitin, 25 μM MG132, 500 ng UBA1, 500 ng UBC8, MBP -CaRma1H1 500 ng into each tube and incubated in a 30 ℃ incubator, after 5, 10, 20, 40, 60 minutes each 2X sample buffer 30 μl and boiled at 100 ℃ for 5 minutes and anti-MBP Western blot is performed using the antibody.

The results of Western blot using anti-MBP antibody after the removal of UBA1 (E1), UBC8 (E2), and ubiquitin (E1) and ubiquitin in the same composition as (b) and (a).

(C) Anti-MBP Western blot analysis showed that the enzyme activity was not shown when one of cystine and histidine was mutated in CaRma1H1 protein having a C3HC4 type ring finger domain.

(d) The domain of CaRma1H1 protein is shown schematically. The 58th, 61st, 89th, and 115th amino acids were respectively converted into corresponding amino acids.

Figure 5 shows 35S: CaRma1H1 This is the result of analyzing the phenotype of Arabidopsis transformed with recombinant vector. Wild-type Baby Pole (WT) and 35S grown for 4 weeks: CaRma1H1 The Arabidopsis transformed with the recombinant vector was not watered for 14 days, and then water was supplied again and the plants were observed after 5 days.

6 is 35S: CaRma1H1 Statistical processing for dry stress of Arabidopsis and wild species transformed with recombinant vector is shown. (a) After growing for 3 weeks, the plant was left without water for 11 days, and water was supplied again to observe the phenotype of the plant after 3 days. (b) CaRma1H1 gene overexpression of each transformant was confirmed by RT-PCR. (c) Relatively compared survival rate of transgenic plants and wild species.

The present invention uses CaRma1H1 protein derived from red pepper, which increases resistance to stress, the gene encoding the protein, the enzymatic activity of the protein, a recombinant vector comprising the gene, a plant transformed with the recombinant vector, and the gene. The present invention relates to a method for increasing the resistance of plants to stress, transgenic plants having increased stress resistance by the method and seeds thereof.

All living organisms are constantly exposed to biological and abiotic stresses throughout their lives. Animals can be avoided when exposed to danger, but plants are sticking organisms that are more affected than animals for various stresses. Therefore, plants can quickly detect abiotic stresses (eg, moisture, temperature, light, salinity, metals and soils) and biological stresses (eg, viruses, bacteria, fungi, predators) to protect themselves. And respond. Plants have been developed with sophisticated networks for environmental stress and defense / stress response (Yang Y. et al., 1997, 11: 1621-1639). Therefore, since plants are immobile, they have a mechanism for quickly recognizing and responding to environmental stimuli.

Ubiquitin, meanwhile, is a protein expressed in all eukaryotes, consisting of 76 amino acids and covalently linked to other proteins. Substrate proteins that are ubiquitous are so diverse that almost all of the physiological activities in the cell are involved in ubiquitination, and many diseases are caused by poor binding of these diseases. Ubiquitin has ubiquitin itself as its substrate, which can form covalently linked ubiquitin chains. Therefore, there are forms of ubiquitin monomer and one ubiquitin covalently bound protein, ubiquitin chain-bound protein, and ubiquitin chain. Ubiquitination is achieved by the E1-E2-E3 chain enzyme reaction, which is the reaction of ubiquitin activation, ubiquitin bonds to ubiquitin linkages, respectively. Ubiquitination is known to be in charge of various functions as a mechanism not only of plants but of all higher life forms. In particular, little is known about genes involved in abiotic stress.

Patents and commercialization of useful genes isolated from important plant resources such as domestic native plants occupy a large part of the biotechnology field, and the development of overexpressed plants and the suppression of gene expression involved in stress control of plants have led to the development of various new functional plants. Expectations for the possibility of producing and industrializing (eg, delayed aging plants, changing the color and size of red pepper, plants that resist various external stresses, and changing root lengths) are increasing. Furthermore, it can be applied to various agriculturally important crops and contribute to productivity improvement. Research on environmental stress of such plants is actively underway, and Korean Patent Laid-Open No. 2006-80235 discloses a method for improving stress resistance in plants and methods thereof.

Accordingly, the present inventors have completed the present invention by studying CaRma1H1 gene derived from red pepper, and finding that CaRma1H1 gene increases resistance to plant stress while studying biological or abiotic stress resistance of plants.

Accordingly, it is an object of the present invention to provide CaRma1H1 protein derived from red pepper which increases resistance to stress.

Another object of the present invention is to provide a gene encoding the CaRma1H1 protein.

Another object of the present invention is to provide enzymatic activity of the CaRma1H1 protein.

Another object of the present invention is to provide a recombinant vector comprising the gene.

Another object of the present invention is to provide a plant transformed with the vector.

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

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

In order to achieve the above object, the present invention provides a CaRma1H1 protein derived from red pepper made of an amino acid sequence represented by SEQ ID NO: 2 to increase the resistance to stress.

In the present invention, a new gene CaRma1H1 derived from red pepper is identified, and in the present invention, CaRma1H1 means that the ring finger domain is well preserved in the protein. (Capsicum annuum Ring finger protein with Membrane Anchor1 Homolog1).

The range of CaRma1H1 protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from red pepper and a functional equivalent 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. "Substantially homogeneous physiological activity" means activity that increases the stress resistance of a plant upon overexpression in the plant.

In the present invention, the stress may be dry, low temperature, wound, jasmonic acid (JA), salicylic acid (SA), hydrogen peroxide, chitosan, NaCl, cadmium, ultraviolet irradiation, protein kinase inhibitor or ozone, but is not limited thereto. .

The present invention also provides a gene encoding the CaRma1H1 protein. Genes of the invention include both genomic DNA and cDNA encoding the CaRma1H1 protein. Preferably, the gene of the present invention may include the nucleotide sequence represented by SEQ ID NO: 1.

In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene comprises a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1. 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 gene according to the present invention. The recombinant vector is preferably a recombinant plant expression vector.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. 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 plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.

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 (phosphinothricin), kanamycin, G418, bleomycin, hygromycin, chloramphenicol There are antibiotic resistance genes such as, but not limited to.

In the plant expression vector according to an embodiment 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 a region of DNA upstream from a 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 various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

The terminator may be a conventional terminator, and examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to 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 terminators is highly desirable in the context of the present invention.

The present invention also provides a plant transformed with the recombinant vector according to the present invention.

Plant transformation refers to 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 dicotyledonous plants as well as monocotyledonous 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. Preferred methods according to the invention include 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 "plant cells" used for plant transformation may be any plant cells. The plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells.

"Plant tissue" refers to the tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.

In addition, the present invention is CaRma1H1 according to the present invention It provides a method of increasing resistance to stress by overexpressing genes in plants. The stress may be dry, low temperature, salt, jasmonic acid (JA), salicylic acid (SA), hydrogen peroxide, chitosan, NaCl, cadmium, ultraviolet irradiation, protein kinase inhibitors or ozone, but is not limited thereto.

As a method for over-expressing a gene in a plant CaRma1H1 it may be performed by introducing a gene into a plant CaRma1H1 that does not contain the plant or CaRma1H1 gene containing CaRma1H1 gene. "Overexpression of the gene" in the above means that the expression of the CaRma1H1 gene above the level expressed in wild-type plants. As a method of introducing a CaRma1H1 gene into a plant, there is a method of transforming a plant using an expression vector containing a CaRma1H1 gene controlled by a promoter. The promoter is not particularly limited as long as it can overexpress the insertion gene in the plant. Examples of such promoters include, but are not limited to, 35S RNA and 19S RNA promoters of CaMV; Full length transcriptional promoters derived from Peak Water Mosaic Virus (FMV) and Coat protein promoters of TMV. In addition, the ubiquitin promoter can be used to overexpress the CaRma1H1 gene in monocotyledonous or woody plants.

Plants that can be increased stress resistance by the method of the present invention include food crops, including pepper, wheat, barley, corn, soybeans, potatoes, red beans, oats, millet; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops including lygras, redclover, orchardgrass, alphaalpha, tolskew, perennial licegrass and the like.

The present invention also provides a transgenic plant with increased stress resistance produced by the above method.

Plants with increased stress resistance according to the present invention can be obtained through the sexual propagation method or asexual propagation method which is a conventional method in the art. More specifically, the plant of the present invention can be obtained through the oily breeding process of producing seeds through the pollination process of flowers and breeding from the seeds. In addition, after transforming a plant with a recombinant vector comprising the CaRma1H1 gene according to the present invention can be obtained through the asexual propagation method which is a process of induction, rooting and soil purification of the callus according to a conventional method. That is, the sections of plants transformed with the recombinant vector containing the CaRma1H1 gene are incubated in a suitable medium known in the art and then cultured under appropriate conditions to induce the formation of callus, and when shoots are formed, they are transferred to a hormone-free medium. Incubate. After about 2 weeks, the shoots are transferred to rooting medium to induce roots. Plants with increased stress resistance can be obtained by inducing roots and then transplanting them into the soil to purify them. In the present invention, the transformed plant may include not only the whole plant, but also tissues, cells, or seeds obtainable therefrom.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example

Materials and methods

One. cDNA  Clone manufacturing

In the present invention, Lamda ZapII pepper leaf cDNA library was screened using a fragment clone of CaRma1H1 gene to obtain the full-length cDNA of the water stress induced CaRma1H1 fragment clone obtained through DD-PCR.

That is to say, E. coli XL1-Blue MRF 'host cells infected with recombinant phage infected 150 mm LB plates and plaques were formed, and the LB plates were left at 4 ° C. for at least 2 hours. After transferring the plaque to a Hybond-N + membrane (Amersham), the membrane was denatured in a solution containing 1.5 M NaCl and 0.5 M NaOH, then neutralized in a solution consisting of 1.5 M NaCl, 0.5 M Tris-HCl (pH 8.0) and 0.2 Washed with M Tris-HCl (pH 7.5) and 2 X SSC solution and dried. The membrane was then left at 80 ° C. for 2 hours to cross-link DNA to the membrane and hybridize with pCaRma1H1 probe labeled with ³² P (5 × Dendardt's, 6 × SSPE, 0.5% SDS, 55 ° C., 14 h). After hybridization, the nylon membrane was washed three times for 25 minutes at 60 ° C. with a washing solution (1 × SSPE, 0.1% SDS). The washed membrane was photosensitive at -70 ° C for 2-7 days using X-ray film and intensifying screen and then developed.

In order to separate pBluescript phagemid with recombinant DNA from UniZAP XR vector, a system using ExAssist helper phage / SOLR cells was used. Single phage stock (> 1X10 ⁵ phage particle), XL1-Blue MRF '(OD ₆₀₀ = 1) and ExAssist helper phage (> 1X10 ⁵ phage particle), completed until the second screening, were incubated at 37 ° C for 15 minutes and then LB medium. Incubated with shaking for more than 4 hours to cause monoclonal leaching, and the supernatant with phagemid was taken by centrifugation after treatment for 15 minutes at 70 ℃. A certain amount was mixed with 200 μl of SOLR cells (OD ₆₀₀ = 1) and incubated at 37 ° C. for 15 minutes, and 50 ml of LB / Amp solid medium was incubated at 37 ° C. for 16 hours or more. Plasmids were separated from the generated colonies to confirm the size of the inserted DNA and transformed into E. coli (DH5α strain).

Sequenase Ver. Sequencing was performed using dideoxy chain termination using 2.0 (USB). Plasmid DNA was denatured with alkali, followed by DNA sequencing primers bound and labeled with (35S) -dATP followed by addition of ddNTP to terminate at 37 ° C. The reaction product was subjected to electrophoresis on 6% modified polyacrylamide gel and then sensitized on an X-ray film to read the base sequence.

2. The genome derived from pepper DNA  extraction

In the present invention, genomic DNA was isolated from the pepper leaves of about 4 weeks. 3 ml of extract buffer (8.0 M urea, 50 mM Tris-Cl, pH 7.5), 20 mM EDTA, 250 mM NaCl, 2% (w / v) sarcosyl, 5% (v / v) phenol and 20 mM 2-mercaptoethanol) were mixed. DNA was concentrated by ethanol precipitation after a series of phenol / chloroform / isoamyl alcohol (25: 24: 1, v / v) processes. The pellet was dissolved in 10 mM Tris-Cl (pH 7.5) and 1 mM EDTA, 1.5 g mL ^-1 of cesium chloride was added and centrifuged at 200,000 g for 24 hours. After DNA collection, ethyl bromide was removed using water-saturated 1-butanol and dissolved in 10 mM Tris-Cl (pH 7.5) and 1 mM EDTA after ethanol precipitation.

3. Plant Growth Conditions and Sampling

Capsicum annuum L. cv. Pukang seeds stored at 4 ° C. were sterilized in 30% Lax and 0.025% Triton X-100 for 20 minutes and washed with water at least 10 times. The treated seeds were planted vertically in MS medium consisting of 3% sucrose, B5 vitamin (12 mg / L) and 0.8% agar, and then grown in a growth chamber (16 hours light / 8 hours dark) for about 2 weeks. Was carried out with the material. If the whole whole plant is used as a material, the seeds are soaked in running water for one day, and then planted in a flowerpot mixed with burmaculite and soil in a ratio of 1: 1 for 16 weeks. The plant grown under the conditions) was used as a material. For ethylene treatment, each treatment was maintained for a specific time in a 3 L bottle containing air or 20 ppm of ethylene.

4. Stress (salt, low temperature, dry, wound) treatment

In the present invention, in order to confirm the stress resistance of CaRma1H1 gene, the dry stress was sampled leaves and root tissues when the water content is reduced by 5% and 10% after exposing pepper seedlings grown for 2 weeks in the air. Salt stress was only sampled for root tissue after 2 hours and 5 hours after treatment with 300 mM sodium chloride in the roots of pepper plants grown for 4 weeks. Cold stress was incubated for 3 hours and 6 hours in the incubator maintained at 4 degrees in the incubator maintained for 2 weeks in the medium and sampled the leaves and root tissues, and in the case of wound treatment with a knife on the leaf tissue of the pepper plants grown for 2 weeks Samples were taken 30 minutes, 1 hour, and 2 hours later.

Various stressed pepper tissues were ground in a mortar with liquid nitrogen and then ground to powder, then 2 ml of extraction buffer per gram (4 M guanidine-HCl 20 mM, 10 mM EDTA, 10 mM EGTA, 0.5% sarcosyl). , pH 9) and β-mercaptoethanol, extracted and transferred to conical tubes, mixed with the same amount of PCI (phenol: chloroform: isoamyl alcohol = 25: 24: 1), shaken for 5 minutes, and then 3,500 rpm Centrifuge for 25 minutes at (Hanil Centrifuge, HA-1000-3). After centrifugation, the upper organic solvent layer was removed, the same amount of PCI was mixed and shaken, and then the centrifugation process was repeated twice. The aqueous solution layer formed at this time was subjected to twice ethanol precipitation and one time LiCl precipitation. RNA was isolated using. The isolated RNA was quantified using a Beckman DU 650 spectrophotometer, and each 30 μg of RNA was electrophoresed on a 1% agarose gel (1.85% formaldehyde). Electrophoretic gels were transferred to N + nylon membranes (Amersham, USA) using Trans-vac (Hoferfer Scientific Instruments, CA, USA), which hybridized ³² P labeled CaRma1H1 gene with probe (5 × Dendardt, 6 × SSPE, 0.5% SDS, 62 ° C., 14 hours). After hybridization, the nylon membrane was washed three times for 25 minutes at 65 ° C. with a washing solution (1 × SSPE, 0.1% SDS). The washed membrane was photosensitive and developed at -70 ° C for 2-7 days using an X-ray film and an amplification screen.

5. Quantitative Reverse transcriptase ( RT )- PCR

Whole RNA was extracted from the transgenic plants and wild leaves to synthesize single-stranded cDNA using oligo dT primers and MMLV reverse transcriptase. 20 ng of cDNA was used as a template for PCR. Two primers were used at 10 pmole, 5 ml of 10 x Taq polymerase buffer, 8 ml of dNTP mixture (1.25 mM each), and 1 unit of Taq DNA polymer. Laze was added to make a total volume of 50 ml.

PCR was performed with a Perkin Elmer DNA thermal cycler. First denatured at 94 ° C. for 2 minutes, followed by repeating 25 times at 94 ° C. for 30 minutes, 52 ° C. for 30 minutes, and 72 ° C. for 25 minutes. It was then stored frozen at -20 ℃. Primers of CaRma1H1 and ubiquitin genes used in PCR are as follows.

CaRma1H1 forward primer: 5'-ATGGAAGTCAATGAACGATG-3 '(SEQ ID NO: 3)

CaRma1H1 reverse primer: 5'-CTGCTGTTGATAACCTCGTC -3 '(SEQ ID NO: 4)

Ubiquitin forward primer: 5'-ATGCAGATCTTTGTTAAGACTCTC-3 '(SEQ ID NO: 5)

Ubiquitin reverse primer: 5'-TCAACCACCACGGAGCCTGA-3 '(SEQ ID NO: 6)

6. CaRma1H1  Design of vector constructs

In the present invention, a primer set (5'-gaattcatgaatcaagatatggcc) having a sequence corresponding to EcoRI restriction enzymes on the 5 'and 3' sides of the coding portion of CaRma1H1 for recombination of the plasmid to be expressed by fusing the protein bound to maltose with CaRma1H1 PCR was performed using -3 '(SEQ ID NO: 7), 5'-gaattctcaaaacaagattagac-3' (SEQ ID NO: 8)). PCR results and pMAL-X vector (New England Biolabs, Beverly, MA) were cut with EcoRI restriction enzyme and then ligated using T4 DNA ligase (New England Bio Lab) enzyme. Recombinant MBP-CaRma1H1 was expressed in Escherichia coli BL21-CodonPlus (DE3) RIL (stratizine) and then purified using amylose column chromatography. Protein was quantified using BSA as standard protein.

In addition, in the present invention, in order to confirm the specific activity of CaRma1H1 protein, an important amino acid near the ring domain was performed using a mutation system derived from QuikChange (Strateidine, USA). Point mutations were overlapped with the forward and reverse oligonucleotides at the desired site, cut for 12 hours with DpnI restriction enzyme after 12 cycles of amplification, and then transformed into DH5a Escherichia coli and sequencing analysis confirmed that the mutations were correct. It was. The mutated CaRma1H1 thus named CaRma1H1 H58A, CaRma1H1 C61S, CaRma1H1 C89S, CaRma1H1 K115R, respectively. The primer sequence of the vector used in the present invention is as follows.

CaRma1H1 H58A Forward Primer

5'-TAACTTTATGCGGTGCTCTTTACTGCTGGC-3 '(SEQ ID NO: 9)

CaRma1H1 H58A Reverse Primer

5'-GCCAGCAGTAAAGAGCACCGCATAAAGTTA-3 '(SEQ ID NO: 10)

CaRma1H1 C61S Forward Primer

5'-GCGGTCATCTTTACAGCTGGCCTTGCATT-3 '(SEQ ID NO: 11)

CaRma1H1 C61S Reverse Primer

5'-AATGCAAGGCCAGCTGTAAAGATGACCGC-3 '(SEQ ID NO: 12)

CaRma1H1 C89S Forward Primer

5'-CGCAATGCCCTGTTAGCAAGGCTGAAGTC-3 '(SEQ ID NO: 13)

CaRma1H1 C89S Reverse Primer

5'-GACTTCAGCCTTGCTAACAGGGCATTGCG-3 '(SEQ ID NO: 14)

CaRma1H1 K115R Forward Primer

5'-CCATCCGAAGAAAGGGCTCCGAATCTTGG-3 '(SEQ ID NO: 15)

CaRma1H1 K115R Reverse Primer

5'-CCAAGATTCGGAGCCCTTCCTTCGGATGG-3 '(SEQ ID NO: 16)

In addition, in the present invention, pBI121, which is a binary vector containing a CaMV 35S promoter and a NOS terminator, was used for transformation of Arabidopsis for the production of transformants. For transformation, PCR was performed using high fidelity EX-tag polymerase (Takara, Kyoto, Japan) using pCaRma1H1 cDNA as a template. As a result, a cDNA fragment completely containing the translational region of the protein was obtained, which was cut with EcoRI and BamH1 restriction enzymes and introduced into the pBI121 vector.

7. To Agrobacterium CaRma1H1  Transformation

The constructed construct was transformed into Agrobacterium (GV3101) using electroporation, and the presence or absence of the gene was confirmed by PCR. The ground part of about 4 weeks old Arabidopsis (columbia [Col-0]) was transformed by immersion in MS medium containing 0.05% silwet for 1 minute and 30 seconds (clough and Bent, 1998, Plant J 16; 735). -743). After culturing in a growth chamber at 23 ° C. for about 3 weeks and receiving seeds (T1), individuals who survived in a medium containing 25 μg / ml kanamycin and 250 μg / ml carbenicillin were selected for the presence of the transgene. Validation was by RT-PCR and Northern blot.

8. CaRma1H1  Enzyme Activity Analysis of Proteins

In the present invention, for the enzymatic activity analysis of CaRma1H1 protein, subcloning of the ORF of CaRma1H1 gene to the maltose binding protein frame was performed on the pMAL C2 vector. 40 mM Tris-HCl, pH 7.5, 5 mM MgCl ₂ , 2 mM ATP, 2 mM Dithiothreitol (DTT), 300 ng / μl Ubiquitin, 25 μM MG132, 500 ng UBA1, 500 ng UBC8, MBP-CaRma1H1 500 ng was put in each tube and incubated in a 30 ℃ incubator. After 5, 10, 20, 40 and 60 minutes, 30 μl of 2X sample buffer was added thereto, and then boiled at 100 ° C. for 5 minutes, and Western blot was performed using anti-MBP (New England Bio Labs) antibody.

Example  One: CaRma1H1 Genetic Characterization and Functional Analysis

Pepper protein encoded by a separate gene from (Capsicum annuum L.CV Bukang) is CaRma1H1 which means that the ring finger domains been well preserved (Capsicum annuum Ring finger protein with Membrane Anchor1 Homolog1).

CaRma1H1 isolated from the present invention The gene cDNA (SEQ ID NO: 1) is composed of 252 amino acid polypeptides of 1369 bp in length.The amino acid sequence of the CaRma1H1 gene (SEQ ID NO: 2) and the Arabidopsis At4g28270 are 34%, AtRma1 is 34%, At4g27470 is 29% similarity. It confirmed that it was seen.

Example  2: for various stress CaRma1H1 Analysis of gene expression patterns

In the present invention, CaRma1H1 according to abiotic stress treatment To check gene expression, various environmental stress conditions such as drying, salt, low temperature, wound, etc. (NaCl 300 mM, drying condition: when the water content of red pepper seedlings reached 5%, 10%, low temperature: 4 ℃) Northern blot analysis of CaRma1H1 gene expression in the leaves and roots of red pepper was performed. CaRCI gene used to compare with the gene in the present invention is a gene with high similarity to RCI2a of Arabidopsis is well known as a gene induced expression by salt stress, CaPINII gene is similar to the protease inhibitor II As a high pepper gene, it is known as a marker gene whose expression is very well induced by ethylene and wound stress. As a result of confirming the expression pattern of CaRma1H1 gene, as shown in FIG. 3, it was confirmed that the expression of CaRma1H1 gene was significantly increased after 3 hours in the leaves treated with cold stress (4 ° C.). In addition, it was observed that the expression of CaRma1H1 gene increased in the roots over time. When the salt was treated, it was confirmed that the expression was significantly increased in the roots after 2 hours and then the expression amount was decreased as time passed. On the other hand, when the dry stress was treated, the expression of the gene did not show a clear change as the water content was reduced by 5% and 10%, but the expression of the CaRma1H1 gene was increased when the water content was decreased. When the wound was treated, the amount of expression increased slightly after 30 minutes, but it was confirmed to decrease with time.

Example  3: CaRma1H1  Enzyme Activity Analysis of Proteins

In the present invention, after the addition of UBA1, UBC8, ubiquitin and CaRma1H1 to analyze the enzymatic activity of CaRma1H1 protein, the change of CaRma1H1 protein was measured by Western blot after 5, 10, 20, 40 and 60 minutes. As can be seen in Figure 4a, as time passes, more and more bands were observed as compared to the initial stage, which indicates that the MBP-CaRma1H1 protein was autoubiquitated. Therefore, CaRma1H1 protein was found to have E3 ligase enzyme activity. In addition, in the present invention, after removing the UBA1 (E1), the UBC8 (E2), and ubiquitin one by one, changes in CaRma1H1 protein were confirmed (see FIG. 4B). In FIG. 4C, it was confirmed that when one of these amino acids was mutated to confirm the function of cystine and histidine, which are important effects on the enzymatic activity of CaRma1H1 protein having a ring finger domain of C3HC4 type, the enzymatic activity did not appear. .

Example  4: 35S: CaRma1H1  Phenotypic analysis of Arabidopsis transformed with vector

In the present invention, the phenotype of the Arabidopsis transformed with wild-type Arabidopsis and 35S: CaRma1H1 vector was observed for functional analysis of the CaRma1H1 gene. As can be seen in Figure 5, wild species Arabidopsis cultivated for 4 weeks and the Arabidopsis transformed with CaRma1H1 gene without watering 14 days without dry stress after supplying water again after 5 days of wild species It was confirmed that the drying resistance of transformed Arabidopsis increased compared to.

Example  5: 35 S: CaRma1H1  Phenotypic statistical analysis of Arabidopsis transformed with vector and CaRma1H1  Gene Expression Analysis

Four independent transgenic plant groups were used to perform statistical analysis on dry stress with wild species. After eleven days without watering to the Arabidopsis grown for three weeks, the water was supplied again and the plants were observed after three days. Compared with wild species, transgenic Arabidopsis was found to have resistance to dry stress at a rate of 33% to 91% (see FIGS. 6a and c). In addition, as a result of analyzing the degree of overexpression of each transformant through Northern blot, it was confirmed that the CaRma1H1 gene is significantly expressed in all lines (see FIG. 6B).

CaRma1H1 gene derived from red pepper isolated from the present invention and CaRma1H1 protein expressed from the gene may increase resistance to plant stress.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> CaRma1H1 gene increasing plant stress resistance and transgenic          plants transformed by CaRma1H1 gene <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 1369 <212> DNA <213> Capsicum annuum <400> 1 ggcacgaggc tttgcttttc tccttctaaa gactcaatat cttacttgtg tgttagttca 60 caaacgttct tgatacaatt atactacttg ggcattagca gataatttta ttacatcatg 120 aatcaagata tggccttaga acagcttgac actaccttta acaaacacga tactccatta 180 gggaaatgga agtcaatgaa cgatgaagtt gaagagaata tttctggtgg cttcgactgt 240 aacatatgcc tggattgtgt gcacgaacct gtgataactt tatgcggtca tctttactgc 300 tggccttgca tttacaaatg gatttatttc cagagtgttt cttcagaaaa ttcggatcag 360 caacaaccgc aatgccctgt ttgcaaggct gaagtctcag aaaaaacctt gattccactc 420 tatggacgcg gtggtcaatc tacaaaacca tccgaaggaa aggctccgaa tcttggcata 480 gtgatcccac aaaggccccc tagtccaagg tgtggtggtc acttcttgtt accaactact 540 gattcaaatc catcccagct acttcaacga cgaggttatc aacagcagtc tcaaacacgt 600 caaccggctt atcagggtag ctacatgtct tcgcccatgc tcagccctgg tggtgcgact 660 gcgaatatgt tacaacactc catgattgga gaagtagcct atgcaagaat ttttggcaac 720 tcatcaacaa ctatgtatac atatccaaac tcttataatc tagcaatcag cagtagccca 780 agaatgagaa ggcaattatc acaggctgat agatcacttg gcagaatatg ttttttccta 840 ttttgttgct ttgtcacatg tctaatcttg ttttgaacac ttttttcctc ctgtcttttg 900 cttgtacttt gtacaaaaag atttggtgtt gtagtctcag taaaaaaaaa gggcagcctg 960 gtgcatctaa actcctgcta tctatgtgca gggtttcgga aagggctgga caacaacggt 1020 acgttatagt cccttatagt agataagaaa atgtacaaca gcctcggcta aatgtgtaac 1080 aaatcataga agcttttgcc tctttgattg ttgctaaaca ctcttggaat tagagtgctt 1140 tcattgaacc cttttttgat attataatct aagaaatgag gtttgaagtt ctatccatta 1200 tggtgcatag agactcatac tatgttatgt aacaatcaat ttgatatggt cgctaacaat 1260 caatttgata tggtcgcacc gtttcatttt ttctctttca tttggccttt gcttatatct 1320 aattatccct ttttatcttt tttttataaa aaaaaaaaaa aaaaaaaaa 1369 <210> 2 <211> 252 <212> PRT <213> Capsicum annuum <400> 2 Met Asn Gln Asp Met Ala Leu Glu Gln Leu Asp Thr Thr Phe Asn Lys   1 5 10 15 His Asp Thr Pro Leu Gly Lys Trp Lys Ser Met Asn Asp Glu Val Glu              20 25 30 Glu Asn Ile Ser Gly Gly Phe Asp Cys Asn Ile Cys Leu Asp Cys Val          35 40 45 His Glu Pro Val Ile Thr Leu Cys Gly His Leu Tyr Cys Trp Pro Cys      50 55 60 Ile Tyr Lys Trp Ile Tyr Phe Gln Ser Val Ser Ser Glu Asn Ser Asp  65 70 75 80 Gln Gln Gln Pro Gln Cys Pro Val Cys Lys Ala Glu Val Ser Glu Lys                  85 90 95 Thr Leu Ile Pro Leu Tyr Gly Arg Gly Gly Gln Ser Thr Lys Pro Ser             100 105 110 Glu Gly Lys Ala Pro Asn Leu Gly Ile Val Ile Pro Gln Arg Pro Pro         115 120 125 Ser Pro Arg Cys Gly Gly His Phe Leu Leu Pro Thr Thr Asp Ser Asn     130 135 140 Pro Ser Gln Leu Leu Gln Arg Arg Gly Tyr Gln Gln Gln Ser Gln Thr 145 150 155 160 Arg Gln Pro Ala Tyr Gln Gly Ser Tyr Met Ser Ser Pro Met Leu Ser                 165 170 175 Pro Gly Gly Ala Thr Ala Asn Met Leu Gln His Ser Met Ile Gly Glu             180 185 190 Val Ala Tyr Ala Arg Ile Phe Gly Asn Ser Ser Thr Thr Met Tyr Thr         195 200 205 Tyr Pro Asn Ser Tyr Asn Leu Ala Ile Ser Ser Ser Pro Arg Met Arg     210 215 220 Arg Gln Leu Ser Gln Ala Asp Arg Ser Leu Gly Arg Ile Cys Phe Phe 225 230 235 240 Leu Phe Cys Cys Phe Val Thr Cys Leu Ile Leu Phe                 245 250 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 forward primer <400> 3 atggaagtca atgaacgatg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 reverse primer <400> 4 ctgctgttga taacctcgtc 20 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Ubiquitin forward primer <400> 5 atgcagatct ttgttaagac tctc 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ubiquitin reverse primer <400> 6 tcaaccacca cggagcctga 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gaattcatga atcaagatat ggcc 24 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gaattctcaa aacaagatta gac 23 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 H58A forward primer <400> 9 taactttatg cggtgctctt tactgctggc 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 H58A reverse primer <400> 10 gccagcagta aagagcaccg cataaagtta 30 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 C61S forward primer <400> 11 gcggtcatct ttacagctgg ccttgcatt 29 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 C61S reverse primer <400> 12 aatgcaaggc cagctgtaaa gatgaccgc 29 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 C89S forward primer <400> 13 cgcaatgccc tgttagcaag gctgaagtc 29 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 C89S reverse primer <400> 14 gacttcagcc ttgctaacag ggcattgcg 29 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 K115R forward primer <400> 15 ccatccgaag aaagggctcc gaatcttgg 29 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CaRma1H1 K115R reverse primer <400> 16 ccaagattcg gagcccttcc ttcggatgg 29  

Claims (12)

delete delete delete delete delete delete delete A method of increasing resistance to dry stress by overexpressing in a plant a gene encoding CaRma1H1 protein derived from red pepper consisting of the amino acid sequence represented by SEQ ID NO: 2. delete According to claim 8, The plant is rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, sorghum crops; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops, including lygras, red clover, orchardgrass, alpha alpha, tolsque cue, perennial lygras, and the like. A transgenic plant with increased dry stress resistance produced by the method of claim 8 or 10. delete

Images