KR100781075B1 - 1 OsMSRPK1 gene increasing plant stress resistance - Google Patents

Abstract

An OsMSRPK1(Oryza sativa multiple signaling response protein kinase 1) gene and an OsMSRPK1 expressed from the gene are provided to increase the resistance of a plant against stress. An OsMSRPK1 protein increasing the resistance against stress such as jasmonic acid, salicylic acid, hydrogen peroxide, chitosan, NaCl, Cd, ultraviolet radiation, protein phosphorylase inhibitor and ozone includes an amino acid sequence described as SEQ ID : NO. 2. A gene encodes the OsMSRPK1 protein and consists of a sequence described as SEQ ID : NO. 1. A rice plant is transformed by a recombinant vector including the gene. A method for increasing the resistance against stress comprises a step of over-expressing the gene in a rice plant.

Description

OsMSRPK1 gene increasing plant stress resistance

Figure 1 shows the OsMSRPK1 gene was identified from the up-regulation of mRNA levels using dot-blot hybridization. This analysis was performed using 72 putative protein kinase, total cDNA probe of japonica rice cv. Nipponebare (NPB), and transgenic rice overexpressed OsEDR1 . Ospr1a Gene, B: OsEDR1 - OsMSRPK1 in the overexpression lines.

Figure 2 shows rice CDC2 and OsMSRPK1 The amino acid sequence of the gene is shown. Matching amino acids are indicated by asterisks (*) and similar ones by colons (:). OsMSRPK1 Three Conserved Domains of Genes and Rice CDK1 ( CDC2 ) Proteins are represented by I: PSTAIRE motifs, II: catalytic domains, III: T-ring regions, respectively. Conserved phosphorylation is indicated by arrows.

3 shows the amino acid sequences of the OsMSRPK1 gene and other CDC2 proteins. Asterisks indicated by an asterisk (*) indicate precisely conserved amino acid residues, amino acid similarity (·, :) based on the CLUSTAL-W regulation.

Figure 4 shows a schematic of the OsMSRPK1 gene and other CDC2 (CDK1) protein, the schematic was designed using CLUSTALW 1.83 (CLUSTALW: http // www.ebi.ac.uk / clustalw).

5 is OsMSRPK1 Genome Southern blot analysis of the gene is shown. Genomic DNA (1 μg) extracted from the leaves was digested with HindIII , EcoRI, SacI , XbaI , EcoRI Electrophoresis was performed on 0.8% (w / v) agarose gel to separate and analyzed on nylon membrane (Hybond-N +, Amersham). Membrane is [α- ³² P] dCTP labeled OsMSRPK1 with high stringency conditions Hybridization with cDNA probe. Marked with M represents molecular weight marker ( HindIII Enzyme cleaved λDNA)

6 shows OsMSRPK1 over time for various environmental stresses. This is a result of analyzing gene expression. Leaf fragments show JA, SA, Cd at 100 μM, 10 mM CHX, 0.1% CT, 1 M OA, 10 mM H ₂ O ₂ And 150 mM NaCl. After each treatment the leaf fragments were dried in Petri dishes and irradiated with ultraviolet (UVC 254 nm) at a distance of 15 cm. Leaves and leaf fragments were sampled over time and total RNA was extracted to synthesize cDNA by RT-PCR. Band intensities were analyzed by NIH image program and compared with band intensities of rice actin.

7 is a diagram showing the organization and specific developmental stages of wild type rice (Nipponbare, japonica type), (I). Roots: A) Young roots: 10 days after sowing, B) Adult roots: 90 days after sowing (II). Vinegar: 90 days after sowing, A) innermost organ (young stage), E) outermost organ (fully grown stage), III). Ears of grain A) Just before earing, B) When it is extruded, C) When it starts to bloom, D) When it is fully bloomed, E) When it is growing, F) When it is fully grown.

Figure 8 illustrates the expression patterns of genes in OsMSRPK1 tissue and the developmental stage of rice plant, OsMSRPK1 to confirm tissue-specific expression pattern With gene-specific primers RT-PCR was performed. Actin is a tissue nonspecific gene expression marker. I). Roots: a) Young roots: 10 days after sowing, b) Adult roots: 90 days after sowing II). Vinegar: 90 days after sowing, a) the innermost organ (young phase), e) the outermost organ (fully grown phase), III). Ear of grain a) Immediately before extrusion b) When to extrude c) When to start flowering d) When to fully bloom e) When to grow early f) When to grow fully.

9 was able to observe fluorescence in the nucleus by OsMSRPK1 :: GFP analysis performed for intracellular positioning. (A) O sMSRPK1 :: GFP Schematic diagram of fusion proteins. (B) GFP- tagged OsMSRPK1 fusion constructs were introduced into onion cells by particle spray method. Onion cells Incubated for 24 hours at 25 ℃ and observed the expression using a fluorescence microscope.

10 is OsMSRPK1 Expression of other genes involved in defense / development in transgenic rice overexpressed is shown. (A) The overexpression construct was inserted with OsMSRPK1 full ORF. Created using the pCamLA vector. (B) Total RNA was extracted from the transformed plants. RT-PCR is a primer set for tissue nonspecific gene expression [ PBZ1 ( Probenazole inducible gene), JA myb (jasmonic acid inducible gene), OsBZ8 (ABA inducible gene) and actin ]. CON; Untransformed control plants, R1, R2, R3, R4, and R5; Each transgenic rice plant line expressing OsMSRPK1 .

Figure 11 shows the analysis of the OsMSRPK1 promoter and the sMSRPK1 promoter :: GUS construct, (A) OsMSRPK1 using the signal scan program. Promoter Analysis (http: // www.dna.affrc.go.jp/PLACE/signalscan), ① Myc transcription factor ② Myb transcription factor ③ Pollen specific cis regulatory element, ④ Rice WRKY71 (W-box) binding part ⑤ Important element of gibberellin (GA) reaction complex.

12 is OsMSRPK1 gene control using the GUS protein patterns and various illustrates a change in the GUS accumulation of the hormone treatment, OsMSRPK1 promoter (2.1kb) :: it has 10 days after sowing plants of Arabidopsis thaliana with the GUS construct a variety of hormone treatment (IAA; 10 uM, ETP: 200 uM, JA; 25 uM, SA: 25 uM, GA; 10 uM, ABA: 50 uM, PBZ; 125 uM). A; Cotyledon, B; First leaf C; Shoot-hypocotyl junction D: junction E; Side roots, F; Freckles.

The present invention provides a rice-derived OsMSRPK1 protein that increases resistance to stress, the gene encoding the protein, a recombinant vector comprising the gene, a plant transformed with the recombinant vector, and a plant against stress using the gene. The present invention relates to a method of increasing resistance, a transgenic plant and a seed thereof having increased stress resistance.

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.

Protein phosphorylation / dephosphorylation processes in plants play a very important role in this adaptation (Umezawa T. et al., (2004) Mol. Gen. Genet. 224: 331-340), and certain protein kinase (PKs) Is known to play a role as a signaling factor associated with stress response in plants. For example, MAPKs (MAP kinase) cascades play a central role in regulating signaling pathways associated with biological and abiotic stresses in plants (Munnik T. et al., (1999) Plant J. 20: 185-211).

Cyclin-dependent protein kinase (CDKs), one of protein kinase (PKs), regulates circulation in eukaryotic cells, and cyclin-CDK complexes have been reported to play important functions in regulating cell circulation. CDKs are conserved in all eukaryotic cells and regulate intracellular processes and have three distinct conserved regions, including the PSTAIRE motif, catalytic domain and T-ring region.

Rice ( Oryza sativa L.) is an important model for plant research as it is used as a staple in many countries including Asia, and many studies are being actively conducted to date. Korean Patent Laid-Open No. 2006-80235 discloses a method for improving stress resistance in plants and methods thereof.

Accordingly, the inventors of the present invention, while studying the OsMSRPK1 gene derived from rice, revealed that the OsMSRPK1 gene increases resistance to stress of plants, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a rice-derived OsMSRPK1 protein that increases resistance to stress.

Another object of the present invention is to provide a gene encoding the OsMSRPK1 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 an OsMSRPK1 protein derived from rice to increase the resistance to stress, consisting of the amino acid sequence represented by SEQ ID NO: 2.

For the development of rice varieties we performed reverse phase northern hybridization with 72 signal candidate clones obtained from the Rice Genome Support Center (RGRC). The OsEDR1 gene is the first MAPKKKs found in Nipponbare, which is known to respond quickly to a variety of environmental factors. In the present invention, we identified a new gene OsMSRPK1 with much increased expression in transgenic rice overexpressed OsEDR1 gene, and confirmed that cDNA clone (Genbank accession number AK111593) is upregulated for various signals. In the present invention, OsMSRPK1 means a multiple signal in response to the cDNA protein kinase 1 Oryza sativa multiple signaling response protein kinase 1.

The range of OsMSRPK1 protein according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 isolated from rice and functional equivalents of the proteins. "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 jasmonic acid (JA), salicylic acid (SA), hydrogen peroxide, chitosan, NaCl, cadmium, UV irradiation, protein kinase inhibitor or ozone, but is not limited thereto.

The present invention also provides a gene encoding the OsMSRPK1 protein. Genes of the invention include both genomic DNA and cDNA encoding the OsMSRPK1 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 can do. 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 the natural form of the cell 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 virus 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 OsMSRPK1 according to the present invention It provides a method of increasing resistance to stress by overexpressing genes in plants. The stress may be 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 OsMSRPK1 it may be performed by introducing a gene into a plant OsMSRPK1 that does not contain the plant or OsMSRPK1 gene containing OsMSRPK1 gene. "Overexpression of the gene" in the above means that the OsMSRPK1 gene is expressed above the level expressed in wild-type plants. As a method of introducing the OsMSRPK1 gene into a plant, there is a method of transforming a plant using an expression vector containing the OsMSRPK1 gene under the promoter's control. 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 OsMSRPK1 gene in monocotyledonous plants or woody plants.

Plants that can be increased stress resistance by the method of the present invention include food crops, including rice, wheat, barley, corn, beans, potatoes, red beans, oats, sorghum; 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 OsMSRPK1 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 explants of plants transformed with the recombinant vector containing the OsMSRPK1 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

1. cDNA clone manufacturing

72 major signaling candidate clones related to pathogen resistance were supported by the Rice Genome Support Center (RGRC, Tsukuba, Japan).

2. Dot-blot Hybridization

The putative rice MAPK cDNA clone was supported by the Rice Genome Support Center (RGRC). cDNA (2 μg each) was denatured with DNA denaturing solution (0.5N NaOH and 1.5M NaCl) and heated at 65 ° C. for 5 minutes. Denatured DNA was mixed with 2XSSC and transferred to nylon membrane (HybondN ⁺ Amersham Pharmacia Biotech, USA) using 96-well dot blot manifold blotter. The membranes hybridized with [α- ³² P] dCTP-labeled total cDNA of transgenic rice overexpressed OsEDR1. This cDNA was synthesized from total RNA extracted from the transformed leaves using Trizol reagent (Clontech, USA). Hybridized membranes were washed with 2XSSC and 0.1% SDS for 1 hour at 65 ° C., washed once again with 0.2X SSC and 0.1% SDS for 1 hour at 65 ° C. and then exposed to X-ray film at −80 ° C. for 2 days. I was.

3. Plant Growth Conditions and Sampling

Seedlings Nipponebare ( Oryza sativa L. japonica type) and seeds used in the present invention are sterilized with 100% ethanol for 30 seconds, shaken with 2% bleach (NaClO) for 20 minutes, and then washed 4-5 times using distilled water. It was. It was seeded in sterile 1/2 MS (2.15 g MS + 15 g sucrose + 3 g phyta gel / 1 L) medium and then grown in a growth chamber (28 ° C., 70% humidity, white fluorescence). Seeds were germinated in a Petri-dish with wet Whatman 3MM filter paper and then transferred to the soil. Plant material was harvested and stored at -80 ° C until use.

4. Rice Genomic DNA Extraction

The rice sample was frozen with liquid nitrogen and ground finely using a mortar and pestle. Add 100-200 mg of the sample to a 1.5 ml microtube, and extract buffer solution [2% w / v CTAB, 1.4M NaCl, 20mM]. EDTA, 100 mM Tris-HCl, pH8.0, 0.2% beta-mercaptoethanol] 0.7 ml was added and then homogenized. 0.7 ml of a mixture of phenol / chloroform / isoamyl alcohol (25: 24: 1) was added to the homogenized solution and mixed carefully. The solution was incubated at 65 ° C. for 30 minutes while mixing upside down at five intervals. The culture was centrifuged at 13,000 rpm for 15 minutes, the supernatant was transferred to 1.5 ml of clean microtubes, and the same volume of chloroform / isoamyl alcohol (24: 1) was added and mixed slowly for 5 minutes. The solution was centrifuged at 13,000 rpm for 15 minutes, transferred to clean microtubes (1.5 ml) and equal volume of 100% isopropanol was added. Shake for 5 times and then incubated for 30 minutes at -2 ℃. The cultured solution was centrifuged at 5,000 rpm for 15 minutes, and washed with 1 ml of 70% ethanol. Then ethanol was removed, dried in air and dissolved in 50ul TE.

5. Southern Blot  analysis

Genomic DNA of rice ( Oryza sativa L. japonica type cv. Nipponebare) was extracted from leaves 2 weeks after sowing. Genomic DNA (5 μg) was digested with restriction enzymes HindIII, EcoRV , SacI, XbaI , EcoRI and isolated by electrophoresis on 0.8% agarose gel. For DNA denaturation, the agarose gel containing genomic DNA was washed with denatured solution and neutral solution. Genomic DNA was transferred to nylon membrane (HybondN ⁺ Amersham Pharmacia Biotech, USA) and hybridized with [α- ³² P] dCTP-labeled (Mgaprime DNA labeling system, Amersham) OsMSRPK1 gene specific probe. The hybridized membranes were weakly washed for 1 hour at 65 ° C. with 2X SSC and 0.1% SDS, then more strongly for 1 hour at 65 ° C. with 0.2X SSC and 0.1% SDS, and X-ray film at −80 ° C. for 2 days. Exposed to.

6. Chemical treatment

Rice ( Oryza sativa L. japonicatype cv. Nipponebare) plants were grown at 25 ° C., 70% relative humidity, white fluorescence (wavelength 390-500 nm, 150 mol m ⁻² s ⁻¹ , ¹² hr photoperiod). All treatments with the middle part (2 cm) of the grown leaves of the 2 week seedlings were carried out by the previously reported method (Agrawal et al., (2002) Biochem. Biophys. Res. Commun. 294 (5): 1009 -1016). Leaf fragments were cultured in Petri dishes containing Milli Q water and the other fragments were JA (jasmonic acid), SA (salicylic acid), Cd (cadmium chloride), 10 mM CHX (cycloheximide), 0.1% CT (chitosan), 1 M 100 μM each of OA (Okada acid), 10 mM H ₂ O ₂ (hydrogen peroxide) 150 mM NaCl (sodium chloride) solution was treated. The fragments were transferred to empty Petri dishes for drying. Ultraviolet light (UV-C, 254 nm) was irradiated at a distance of 15 cm using a Hitachi (Japan) germicidal lamp, and then treated with continuous light. Leaves or leaf fragments were sampled at various intervals and immediately frozen at -80 ° C. In the case of ozone (0 ₃₎ process, the plant oil in the pot is moved to the fumigation chamber and treated with O ₃ 0.2ppm. Control plants were filtered and left in the air. Ozone was prepared and used, ML9810 O ₃ Concentration was monitored with an analyzer (Lear Siegler Measurement Controls, USA).

7. Quantitative Reverse transcriptase (RT)- PCR

Total RNA was isolated from rice tissue using the QIAGEN Rneasy Plant Mini Kit (QIAGEN, Maryland, USA). Total RNA samples were treated with DNase-free DNase (Stratagene, La Jolla, CA, USA) before RT-PCR to remove genomes contained in the samples. Single stranded cDNA was prepared with 50 μl reaction mixture using StartaScript ™ RT-PCR kit (Stratagene, USA) according to the manufacturer-provided test method with 10 μg of total RNA extracted from rice. 50 μl reaction mixture was prepared with 1.0 μl of the single-stranded cDNA, 200 Mm dNTPs, followed by the respective specific primer set [ OsMSRPK1 Specific primers (5'-AGCAAGGAGCACTACCACCACC-3 '(SEQ ID NO: 3) and 5'-TCCAGACTCAGGGCTTGTAACC-3' (SEQ ID NO: 4))] 10 pmol and 0.5U taq polymerase (TaKaRa Ex Taq Hot Start Version, TaKaRa Shuzo Co Ltd., Shiga, Japan). Amplification cycle parameters are first denatured by pretreatment at 97 ° C. for 5 minutes, followed by amplification of this cycle 30 times in 45 seconds at 95 ° C., 45 seconds at 55 ° C., and 1 minute at 72 ° C. At the end of the last cycle, an additional step was further amplified at 72 ° C. for 10 minutes (TaKaRa PCR Thermal Cycle Dice, Model TP600, Tokyo, Japan). After the PCR was completed, 2.0 µl of 10X loading buffer solution was added to the entire reaction mixture, and the mixture was mixed vigorously, and 10 µl of this solution was mixed with 1.6% agarose gel (Agarose ME, Cat.No. 50013 R, Iwai Chemicals, Tokyo, Japan). And electrophoresis was performed at 100V for 30 minutes using a Mupid-ex electrophoretic device (ADVANCE, Tokyo, Japan) containing 1X TAE buffer solution. The gel was stained with a solution containing 20 μl of ethidium bromide (50 mg / ml) in 100 ml of 1X TAE buffer solution for 10 minutes, followed by washing the excess ethidium bromide solution and UV-transluminator (ATTO, Tokyo, Japan ) To observe the colored bands. The intensity of each band (area) was quantified by ATTO lane and spot analyzer (ATTO, Japan). RT-PCR controls quantified band values from relative areas using a specific rice actin gene primer set (5'-TCCATCTTGGCATCTCTCAG-3 '(SEQ ID NO: 5) and 5'-GTACCCGCATCAGGCATCTG-3' (SEQ ID NO: 6)) It was.

8. Designing Vector Constructs

OsMSRPK1 To design the vector overexpressed, the gene was amplified using oligomers (5'-TCT AGACGACTTGGCTTTCTCCTCCTCTC-3 '(SEQ ID NO: 7) and 5'-GGATCCATAGGCCAGATCATGCAGGTGGG-3' (SEQ ID NO: 8)) and the pBluescript SK ^- vector Subclones. pBluescript SK ^- ORF of OsMSRPK1 cDNA (2079 bp) in vector Digested with XbaI and BamHI, and were inserted into the lower portion of the cauliflower mosaic virus (CaMV) 35S promoter in the multi-cloning pCambia1300 part of the double vector XbaI and BamHI. Once the ORF of OsMSRPK1 cDNA was completed, the pK7WGF2 vector (VIB, Belgium) was subcloned for instantaneous location analysis. cDNA was completed using the Gateway DNA recombination system (Invitrogen, USA). The whole ORF of the OsMSRPK1 gene was amplified by PCR primers with matching attB sequences during the two-step PCR amplification process. PCR primers used in the first stage amplification were 5'- AAAAAGCAGGCT CCCGAGTAAGAAGAGGAAGCAATC-3 '(SEQ ID NO: 9) and 5'- AGAAAGCTGGGT TGAGTTATACACCAGCACCTCTCC-3' (SEQ ID NO: 10) (attB sequences are shown in bold). The obtained PCR product was amplified by adding the attB sequence to the first PCR product DNAs as a template for the second PCR reaction (5'- GGGGACAAGTTTGTAC AAAAAAGCAGGCT- 3 '(SEQ ID NO: 11) and 5'-GGGGACCACTTTGTACA AGAAAGCTGGGT -3' (SEQ ID NO: 6). No. 12)) The newly added attB sequence is underlined and the previously included attB sequence is indicated in bold). PK7WGF2 vector containing OsMSRPK1 ORF was used for localization in onion cells.

9. To the Agrobacterium OsMSRPK1  Transformation

OsMSRPK1 gene was transformed into ABAbacterium tumefaciens strain LBA4404 by cryolysis. Qualified cells are prepared by incubating overnight single colonies in 30 ml, 5 ml LB liquid medium, adding 50 ml LB liquid medium to 2 ml of this culture and incubating overnight until the cell density is OD ₆₀₀ = 0.4-0.6. It was. The culture was centrifuged at 4 ° C., 3,000 rpm for 5 minutes, and 20 mM CaCl ₂ solution in ice. 1 ml was added and resuspended. 2 μl DNA and 100 μl LBA4404 eligible cells were combined, frozen in liquid nitrogen for 1 minute, and then incubated for 5 minutes in a 37 ° C. thermostat. 1 ml LB liquid medium was added to the solution and incubated for 2-4 hours at 30 ° C. in a shaker incubator. The culture solution was centrifuged at 5,000 rpm for 5 minutes to collect the precipitated cells, sprinkled with LB kanamycin and spectinomycin, and incubated at 30 ° C. for 2 days.

10. Using Agrobacterium Callus  Transformation

In Nipponebare (Japonica type) OsMSRPK1 In order to design the overexpressed double vector of the gene, the standard experimental method (Hiei Y et al., (1994) Plant J. 6 (2): 271-282) was slightly modified using the Agrobacterium tumerfaciens strain LBA4404. Switched. Transformed callus may be selected from selective media [(2N6 medium (1 L), hygromycin (10 mg / L) and / or phosphinothricin (PPT, 3), in which the level of hygromycin is gradually increased during culture in vitro . mg / L), Celltaxim (250 mg / L)).

The selected callus was regenerated medium [MS 4.3g, MS vitamin (10% inositol, 0.05% nicotinic acid, 0.05% pyridoxine, 0.1% thiamine hydrochloride) 1 ml, 3% sucrose, 3% sorbitol, 0.2% casamino acid, After autoclaving 1.1% MES, pH5.8, 0.4% gellan gum, NAA (2 mg / L), kinetin (1 mg / L), cytotaxime (250 mg / L), hygromycin (30 mg / L Added)]. After shooting and rooting the transformed callus, the seedlings were acclimated for 3-4 days in water, and then grown in soil in a plastic house.

Seeds from the T ₁ line were analyzed for hygromycin resistance and seedlings with hygromycin resistance were used for functional analysis and T ₂ homozygote selection.

11. Particle Impact Analysis

GFP fusion protein is full-length OsMSRK1 with GFP C, N-terminus in CaMV 35S promoter Constructed with genes. The N-terminal fusion portion was designed with primers (5'-AAAAAGCAGGCTGGGGAGCGATTCATG-3 '(SEQ ID NO: 13) and 5'-AGAAAGCTGGGTTCTTTTTGGCTAA-3' (SEQ ID NO: 14)) and amplified using this primer. The second PCR product was then amplified using attB1,2 primers (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3 '(SEQ ID NO: 15) and 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3' (SEQ ID NO: 16)). After amplification, the resulting PCR product was subcloned with a pDONR vector (invitrogen, gateway vector), and read back with a pK7WGF2 (invitrogen, gateway vector) vector. The C-terminal fusion portion was determined by OsMSRPK1 using the XbaI and BamHI moieties in the MCS of the Cambia :: GFP cassette bivector. The open reading frame (ORF) of the gene was cloned.

For transient localization analysis, Pro35S: GFP: OsMSRPK1 construct DNA was purified into plasmids using the Qiagen plasmid miniprep kit (Qiagen, USA). Plasmid DNA was mixed with tungsten particles and particles covered with the Pro35S: GFP: OsMSRPK1 construct plasmid were transformed into onion cells by particle bombardment. Onion samples were replaced with MS medium in Petri plates. GFP images expressed in epidermal cells were confirmed using a fluorescence microscope using a GFP-optimized ND filter set (Olympus, Japan). Analytical conditions were carried out under bright light, GFP filter (excitation; 450 500 nm, splitter; FT510 nm, excitation; BP515-565 nm), DAPI filter and FITC filter (excitation; 450 490 nm, splitter; FT510 nm, emission; LP515 nm). Digital images were measured using an Olympus IX 70 fluorescence microscope (Olympus) equipped with an I.CAMSCOPE digital camera (Sometech, Korea) and software (MicroFire, USA).

12. Gus staining analysis

OsMSRPK1 For promoter analysis, the present invention utilizes rice genomic sequencing to determine OsMSRPK1 from a pBI101 vector with a GUS report gene. The 2.1 kb promoter DNA sequence of the gene was designed (see FIG. 10A). After cloning, T ₃ Gus staining was performed using Arabidopsis transgenic homozygotes. After 10 days, cold acetone was added to the seedlings for 30 minutes to remove chloroplasts, and then left in distilled water for 5 minutes. Gus activity of transgenic plants was 100 mM NaPO ₄ , a GUS histological buffer solution. (pH 7.0), 0.5 mMK ₃ [Fe (CN ₆ )], 10 mM EDTA, 0.5 mM K ₄ [Fe (CN ₆ )], 0.5 mg / ml X-glu, 0.1% Triton X-100 and total volume Measurement was made by adding 1/5 methanol and incubating. Plant tissues were incubated at 37 ° C. for 24 hours. After Gus staining, wash buffer (20% methanol, 0.24N hydrochloric acid) was added to the sample and left at 60 ° C. for 40 minutes. The samples were incubated for 20 minutes in a room temperature, 60% ethanol (0.7% NaOH added) buffer solution and then 40%, 20% ethanol was added to wash the samples. Samples were left in 50% glycerol solution for at least 1 hour. Gus activity was measured using Ziess imaging microscope 2.0 (Japan).

Example  One: OsMSRPK1 Genetic Characterization and Functional Analysis

Nipponebare ( Oryza sative L. japonica -type) OsEDR1 , the first MAPKKKs family found Genes It has been found to react very quickly to various external biological and abiotic stresses initially. In the present invention, 72 important signaling candidate clones are provided by the Rice Genome Support Center (RGRC) to determine the genes involved in plant defense / stress signal transcription pathways. reverse northern hybridization) was performed.

MRNA was extracted from wild-type rice and transformed rice plants, and then new PKs were selected and identified using the synthesized probe total cDNA (see FIG. 1). Based on its expressed profile, the present invention uses OsMSRPK1 , which means Oryza sativa multiple stress-response protein kinases. Named gene.

OsMSRPK1 The cDNA consisted of 693 amino acid polypeptides containing an open reading frame (ORF) of 2097 nucleotides, 3116 bp in length. The amino acid sequence of the OsMSRPK1 gene was 49% and 47% identical to rice OsCDC2a and OsCDC2b, respectively. The amino acid sequence alignment of this gene was shown to be distinct from CDK1 (CDC2) with serine / threonine kinase domain. OsMSRPK1 though Although the gene is a MAPK-like gene group, OsMSRPK1 Domain analysis proved to be classified into CDK1 (CDC2) group and PK group. CDKs have three characteristic conserved regions, including the PSTAIRE motif, catalytic domain, and T-ring region, and are divided into two types (PSTAIRE group, non-PSTAIRE group). OsMSRPK1 Although the gene has three characteristic conserved regions of the CDK protein, it also contains a non-PSTAIRE group (see Figure 2).

CDK proteins of rice and other plants were analyzed using the NCBI database with the CLUSTALW program 1.83 (http://www.ebi.ac.uk/clustalw/).

OsMSRPK1 The gene has a longer C-terminal portion than other CDK proteins, and the amino acid polypeptides interpreted by this gene showed 45-55% amino acid identity with other CDKs groups (see Figure 3). OsMSRPK1 and other CDC2 (CDK1) proteins showed 100% identity with rice OsCDC2L2 and 68% homology with rice OdCDC2L1. This showed some difference between plants and animals (see FIG. 4).

Example  2: OsMSRPK1 Gene Genome Southern Analysis

In the present invention, Southern blot was performed to find a gene copy of OSMSRPK1 in the whole rice genome. Genomic DNA was digested with HindIII , EcoRV , SacI , XbaI , EcoRI cleavage enzyme. OSMSRPK1 , using DNA gel blotting It was found that this rice was a single copy of the entire genome.

Example  3: for various environmental factors OsMSRPK1 Time lapse analysis of genes

OsEDR1 OsMSRPK1 in the transgenic rice Since the expression levels of the genes have been significantly upregulated, the role of these genes in defensive signaling can be attributed to various environmental factors (JA, SA, H ₂ O ₂ , CT, NaCl, cadmium, UV-C irradiation, protein kinase inhibitors, ozone). Has been found to respond. Signaling molecules [JA, SA, H ₂ O ₂ , fungal attractants (CT)] include defense / stress signaling responses in monocotyledonous and dicotyledonous plants. RT-PCR used a specific rice actin gene primer set (5'-TCCATCTTGGCATCTCTCAG-3 '(SEQ ID NO: 17) and 5'-GTAC CCGCATCAGGCATCTG-3' (SEQ ID NO: 18)).

Transcriptional expression was observed 30 minutes after expression by kinase in response to internal and external stimuli (see FIG. 6). OsMSRPK1 The transcriptional behavior of the genes was tested in a 30-120 minute time frame to precisely analyze the patterns of expression according to various stress treatments. OsMSRPK1 markedly expressed at 30 min when wound and JA treated Genes rapidly decreased in transcription level over time, whereas after 90 minutes, they gradually decreased. In the case of treatment with SA and hydrogen peroxide, the transcription level was accumulated at 30 minutes and gradually decreased to almost the initial level after 120 minutes. Upon exposure to NaCl and drought stress, mRNA levels increased significantly at 30 minutes, with little change until 90 minutes. The level of transcription following UV-C treatment showed a slight increase over 60 minutes, but little change over time, and markedly increased at 30 minutes with CHX (inhibitor of eukaryotic protein synthesis). As time passed, it was confirmed that little change (see FIG. 6). The difference in transcription level according to these various stresses is OsMSRPK1 It indicates that the gene is involved in the defense system as a positive regulator.

Example  4: OsMSRPK1 Developmental stages and tissue specific expression patterns of

Nipponebare ( Oryza sativa L. japonica) OsMSRPK1 in Rice The action of genes to find out, Developmental control patterns of OsMSRPK1 were analyzed on leaves, vines, and roots as well as ear at different stages of maturity using RT-PCR. RT-PCR was performed with each of five lines in the same step to avoid organ specificity.

Expression was confirmed in all the growth period and all organs of the rice except the root, there was a difference in the amount of expression (see Fig. 8). After 90 days after sowing, RT-PCR was carried out with five different growth stages of leaves and stems. In the case of the ear, it was carried out by dividing into six stages immediately before the ear protrusion, immediately after the ear protrusion, the start of flowering, the flowering full bloom, the seed slightly ripened, and the seed fully ripened (see FIG. 7). As a result, a slight expression was observed just before the ear protrusion, and the degree of expression increased rapidly until the beginning of flowering, and then rapidly decreased from the beginning of flowering, and it was not observed that the seeds began to ripen. These results demonstrate that the OsMSRPK1 gene plays a more important role in defensive mechanisms and development during childhood than before the plant is fully grown.

Example  5: in cells OsMSRPK1  Gene location

To identify the position of OsMSRPK1 GFP fusion protein, two constructs ( OsMSRPK1 :: GFP, GFP :: OsMSRPK1 ) were used in the present invention (see FIG. 9A). OsMSRPK1 :: GFP was detected with stronger fluorescence than GFP :: OsMSRPK1 in onion epidermal cells. OsMSRPK1 :: GFP constructs were located primarily in the nucleus, and GFP vectors were located both in the nucleus and cytoplasm (see FIG. 9B).

OsMSRPK1 The results of the amino acid sequence analysis of the gene can be found in [Psort version 6.4 (http // psort.ims.u-tokyo.ac.jp /)]. OsMSRPK1 The gene had a signal located in the PAKKKA nucleus at amino acids 236-240. These results are OsMSRPK1 The gene indicates that it serves to express the gene against the next growth factor and external stress located in the nucleus.

Example  6: Functional Analysis Using Transgenic Rice

For the functional analysis of OsMSRPK1 gene, two transformation lines (overexpression and gene expression inhibitor constructs) were constructed in the present invention. We have ten overexpressing transformation lines (T ₂ seeds) and are making T ₃ generations. As a result of the functional analysis, no significant difference was observed in comparison with the wild type. So in the present invention OsMSRPK1 using RT-PCR Differences in mRNA levels were identified.

OsMSRPK1 using RT-PCR Analysis of the overexpressed transformation lines resulted in different mRNA levels for each individual. The same amount of disease resistance associated with increased mRNA expression An increase in PBZ1 mRNA levels was also observed (see FIG. 10B). These results indicate that OsMSRPK1 is involved not only in defense but also in developmental stages in plants.

Example  7: using heterogeneous systems OsMSRPK1  Promoter Analysis

In the present invention, after the promoter analysis of OsMSRPK1 (2.1kb DNA sequence) gene using a promoter analysis program (http://www.dna.affrc.go.jp/PLACE/signalscan.html), four constructs are designed. (See FIG. 11A, B). OsMSRPK1 using Arabidopsis pole In promoter analysis, GUS-activity was not found in the main root, lateral root, and dominant apex, but was somewhat active in the cotyledon and the first leaf. The myo- hypodermis showed a relatively stronger GUS-activity. On the other hand, in order to recognize OsMSRPK1 reactivity OsMSRPK1 by various hormones 10 day old Arabidopsis plants, including promoter (2.1 kb) :: GUS construct, were treated with various hormones (IAA; 10 uM, ETP: 200 uM, JA; 25 uM, SA: 25 uM, GA; 10 uM, ABA: 50 uM, PBZ; 125 uM) It was. After treatment with hormones, GUS-activity was observed under a Ziess Image 2.0 (Japan) microscope, and when treated with JA, SA, GA, ABA, and PBZ , IAS showed stronger Gus-activity than the control without hormones, but with IAA And ETP showed a similar pattern to the control (see FIG. 12).

OsMSRPK1 gene isolated from the present invention and OsMSRPK1 protein expressed from the gene can increase the resistance to stress of plants.

Attach sequence list electronic file  

Claims (11)

Oryza sativa multiple signaling response protein kinase 1 protein derived from rice to increase the resistance to stress, consisting of the amino acid sequence represented by SEQ ID NO: 2. The protein of claim 1, wherein the stress is jasmonic acid (JA), salicylic acid (SA), hydrogen peroxide, chitosan, NaCl, cadmium, ultraviolet radiation, protein kinase inhibitor, or ozone. The gene encoding the OsMSRPK1 protein of claim 1. The gene of claim 3, wherein the gene consists of a nucleotide sequence represented by SEQ ID NO: 1. Recombinant vector comprising the gene of claim 3. Rice plant transformed with the recombinant vector of claim 5. The method of increasing the resistance to stress by overexpressing the gene of claim 3 in rice plants. 8. The method of claim 7, wherein the stress is jasmonic acid (JA), salicylic acid (SA), hydrogen peroxide, chitosan, NaCl, cadmium, ultraviolet radiation, protein kinase inhibitors or ozone. delete A transformed rice plant having increased stress resistance by the method of claim 7 or 8. Seed of the transgenic rice plant of Claim 10.

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