KR101460743B1 - Disease resistance-related gene CaChitIV, and transgenic plants using the same - Google Patents

Abstract

The present invention relates to a disease resistance-related gene CaChitIV (Capsicum annuum Chitinase class IV), a transgenic plant using the same, and a method for searching plant disease resistance. According to the present invention, the gene is a novel resistance-related gene interacting with pepper kinase CaPIK1 related to plant disease resistance, which has an advantage of being useful as a gene stock for efficient and effective molecular breeding for researches of defensive reaction of plants against attacks of pathogen and manufacture of disease resistant plant.

Description

Disease resistance-related gene CaChitIV, and transgenic plants using the same.

The present invention relates to a disease resistance-related class forchitis gene CaChitIV ( Capsicum annuum Chitinase class IV ) And a transgenic plant using the same. More specifically, the present invention relates to a bacterium of the pepper plant, which comprises a bacterium belonging to the genus Xanthomonas campestris pv. specific to the new resistance-related gene expression is induced encoding a plant pathogen resistance related pepper kinase gene, a protein interacting with a protein that is encoded pepper receptor-like cytoplasmic protein kinase (CaPIK1) which when infected by pathogenic microorganisms in vesicatoria) the CaChitIV provides the nucleotide sequence and amino acid sequence of the gene accordingly relates to disease resistant transgenic plant and pathogen resistance of the plant search method using the same.

As economic development and living standards are improved, interest in crop production by environmentally friendly organic farming methods is increasing. On the other hand, the environment is increasingly dependent on chemical pesticides which are harmful to humans and the natural environment due to natural disasters such as earthquakes, floods and heavy snow, more frequent occurrence of warming, and increased incidence of pests. Due to the excessive use of these pesticides, environment-friendly green growth is becoming a hot topic for the general public because of environmental pollution and harmfulness to constructions. In line with this trend of public opinion, international cooperation for green growth is being sought, and it is becoming increasingly compulsory for all industries and society to change their overall lifestyles in an environmentally friendly way.

In order to achieve environmentally friendly green growth in agriculture, it is urgent to find ways to improve and breed disease-resistant cultivars that do not require chemical control such as pesticides. Studies on the defense mechanism of plants with resistance to plant disease resistance or environmental stress have technological value to identify in vivo mechanisms not yet known. In addition, information on plant resistance genes can be used not only as a direct material for molecular genetic breeding of crops, but also for traditional genetic breeding. In order to increase environmentally friendly food production, crop breeding using various genes related to disease defenses can reduce food production costs by minimizing losses caused by economically important pathogens, weeds, and environmental stresses, and by providing high quality food, And contributes to the revitalization of various industries associated with the development of new varieties.

Plant disease control of pepper ( Capsicum annuum L.), which is intensively cultivated in Korea and as a major economic crop, is of great importance and provides a good experimental system for studying the signaling pathway of defense against plant diseases. The isolation and function analysis of the genes involved in the defense reaction in the interaction of pepper with the bacterial spotty pathogen of pepper resulted in a scientific understanding of the plant defense response through overexpression in the model plant, Arabidopsis, And is the basis for genetic resources for the development of disease resistant transgenic plants expressing resistance-related genes in pepper plants.

When a plant is infected with a pathogen, the host plant activates a defense mechanism by activating various intracellular metabolic systems to counteract it. These defenses may be caused by the recognition of host cells for small pathogens, by the addition of intracellular Ca ^{2 +} , increased reactive oxygen species, callose deposition, generation of secondary metabolites, phenolic antimicrobials, Reactions, and a variety of defense-related proteins. However, there are very few researches on the factors controlling the hypersensitivity reaction and its mechanism.

As a background of the present invention, Korean Patent No. 10-0757300 (Sep. 11, 2007) describes a kittinase having a plant disease control effect isolated from strain C-61 of the genus Chromobacterium strain , Korean Patent No. 10-0757298 (Sep. 11, 2007) discloses a kittinase having a plant disease control effect isolated from Lysobacter enjymogenes C-3 strain. However, there has been no disclosure of a disease-resistant class forchitinase gene that directly interacts with plant disease resistance-related red pepper kanease to regulate the hypersensitive reactive response.

Korean Patent No. 10-0757300 (September 11, 2007) Korean Patent No. 10-0757298 (September 11, 2007)

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems occurring in the prior art, and the inventors of the present invention confirmed that the protein encoded by the novel resistance-related gene CaChitIV directly interacted with the CaPIK1 protein to regulate the hypersensitive reaction, , Which is expected to contribute substantially to the breeding of disease resistant crop varieties.

Therefore, an object of the present invention is to provide a method for screening a class forkitinase gene CaChitIV ( Capsicum annuum Chitinase class IV ) is separated and decoded to provide the nucleotide sequence information and the amino acid sequence information therefrom.

It is also an object of the present invention to provide a transgenic plant which is resistant to plant diseases using the CaChit IV gene.

Furthermore, the present invention aims to provide a method for searching for resistance to plant diseases using the CaChit IV gene.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for isolating a class forchitis gene ( Capsicum ) derived from the isolated pepper of the following (a) or (b), which codes for class forchitis (Chitinase class IV) annuum Chitinase class IV , CaChit IV ): (a) isolated genes encoding proteins having the amino acid sequence of SEQ ID NO: 2; Or (b) an isolated gene having the nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides a method for producing a class of protein from a separated pepper having a amino acid sequence of SEQ ID NO: 2 ( Capsicum annuum Chitinase class IV , CaChit IV ).

According to still another aspect of the present invention, there is provided a recombinant vector comprising the gene ( CaChit IV ).

According to still another aspect of the present invention, there is provided a transformed plant produced using the gene or the recombinant vector.

According to still another aspect of the invention there is provided CaChitIV by separating mRNA from plants after pathogen inoculation The present invention provides a method for searching for a disease resistance of a plant including a step of identifying differential expression of a gene.

Hereinafter, the configuration of the present invention will be described in detail with reference to the accompanying drawings.

The above object of the present invention is achieved by a process for producing annuum cv. Nockwang) is induced to express specifically when infected with non-pathogenic capsular bacterium ( Xanthomonas campestris pv. Vesicatoria strain Bv5-4a) accompanied by hypersensitive cell death, and a novel resistance-related protein encoding a protein interacting with the CaPIK1 protein The gene CaChitIV1 was identified and its nucleotide sequence was determined and the interaction of CaPIK1 protein and CaChitIV protein was examined using Yeast Two Hybrid technique, BiFc (Bimolecular fluorescence complementation) technique and Co-IP (co-immunoprecipitation) Check, and explore the expression of each of the pepper plant tissue CaChitIV, and to investigate whether the expression of the pathogen through CaChitIV tobacco processing the location of the cell CaChitIV protein (Nicotiana benthamiana ) plants, and CaPIK1 , CaChit IV The effect of transient expression of the gene on the apoptotic effect was confirmed, and CaChitIV gene was suppressed through VIGS, and transgenic Arabidopsis plants overexpressing this gene ( Arabidopsis thaliana ) was confirmed by confirming the plant resistance resistance induction function of CaChitIV gene.

The present invention relates to a method of isolating mRNA from a green pepper cultivar which exhibits an irritant-resistant reaction to a non-pathogenic strain of pepper-bloody pathogenic fungus, preparing a cDNA library therefrom, and using yeast two-hybrid screening ) Was performed to identify a cDNA clone that interacted with CaPIK1. This clone was obtained by separation and purification from yeast cells. Then, the present inventors confirmed the nucleotide sequence of the class fork nutrition coding sequence (SEQ ID NO: 1) and the corresponding amino acid sequence (SEQ ID NO: 2) (see FIG. 1).

(A) an isolated gene ( CaChit IV ) encoding a protein having the amino acid sequence of SEQ ID NO: 2, or (b) a gene having the nucleotide sequence of SEQ ID NO: 1, which encodes a class forchitinase The isolated gene ( CaChit IV ) is provided. In the CaChitIV gene of the present invention, the nucleotide sequence may be any nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, but is preferably SEQ ID NO: 1.

Further, the present invention provides a separated protein (CaChit IV) having the amino acid sequence of SEQ ID NO: 2.

The coding region (ORF) of the CaChitIV1 cDNA according to the present invention is composed of 834 nucleotide sequences encoding 277 amino acids. The sequence of the CaChitIV protein sequence from the N-termini to the signal peptide (1-28 ), A chitin binding domain (ChtBD; 30-61), and a glycohydrolase domain (Glyco_hydro_19; 77-277) (see Fig. 2). The amino acid sequence of the identified CaChit IV and the amino acid sequence coded by the above sequence are shown in Fig. 1, and the amino acid sequence homology between the chitinase amino acid sequence of other crops (tobacco, grape, Arabidopsis and rice) and CaChit IV is shown in Fig. 2 Respectively.

Next, the present invention relates to the interaction between the CaPIK1 protein and the CaChitIV protein in yeast by using the Yeast Two Hybrid technique, a BiFc (Bimolecular Fluorescence Complementation) technique, a Co-IP (co-immunoprecipitation) technique Tobacco ( Nicotiana benthamiana ) was confirmed and shown in Fig.

Next, the present invention separates the RNA from each organ (leaf, stem, root, flower, blueberry , red fruit) of the plant and isolates the mRNA after the pathogen treatment on the leaf surface of the plant to discriminate the CaChitIV gene The method comprising the steps of: More specifically, the biological treatment may be exemplified by pathogenic bacteria such as pathogenic or non-pathogenic bacteria of the bacterium of the red pepper. In order to confirm the expression of CaChitIV gene, various methods for measuring mRNA expression in addition to northern blotting and real-time RT-PCR can be used. The CaChitIV gene according to the present invention was differentially induced and expressed by plant pathogens through experiments (see FIG. 4). Therefore, the CaChit IV gene according to the present invention can be usefully used for searching resistance of plants.

The present invention also provides a recombinant vector comprising the gene ( CaChit IV ). The recombinant vector is preferably a recombinant plant expression vector. Such recombinant vectors include, but are not limited to, for example, pGADT7: CaChitIV , pGBKT7: CaChitIV , pSPYNE: CaChitIV , pSPYCE: CaChitIV , pBIN35S: CaChitIV : HA , or TRV2: CaChitIV .

Furthermore, the present invention provides a transformed plant produced using the gene or the recombinant vector. Transformation of a plant means any method of transferring DNA to a plant. Any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable plant cells. The method is based on the 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) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), the infiltration of plants or the transformation of Agrobacterium tumefaciens Infection by viruses (non-integrative) in virus-mediated gene transfer (EP 0 301 316), and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery.

In addition, the invention of which temporarily expressing CaChitIV in pepper plant with a recombinant vector containing the gene of CaChitIV plant production, specifically to production of the plant inhibition CaChitIV gene, CaChitIV The present invention provides a method for producing a new disease resistant transgenic plant and a material for resistant molecular breeding by confirming whether the transgenic Arabidopsis plants overexpressing the gene are resistant to plant disease. More specifically, the present invention observed a cell apoptosis phenomenon, a reactive oxygen species (ROS) and a nitric oxide (NO), both of which are expressed by coexpression of a CaPIK1 gene and a CaChitIV gene in pepper plants (see FIGS. 5 to 6) VIGS plants produced for inhibiting the expression of CaChitIV gene (see Fig. 7 to Fig. 9), a recombinant vector containing the gene CaChitIV1 (pBIN35S: CaChitIV) and Agrobacterium tumefaciens (Agrobacterium using tumefaciens strain GV3101) strain, Arabidopsis (Arabidopsis thaliana plants (see Figs. 10 to 11), and provides disease-resistant transgenic plants through the expression of the CaChitIV gene.

As described above, CaChitIV , a novel disease resistance-related gene according to the present invention, is a factor that directly controls apigenetic apoptosis by interacting directly with a protein encoded by CaPIK1 , a gene related to plant disease resistance. Thus, the present invention can contribute substantially to studying the hypersensitive cellular killing mechanism and breeding crop varieties resistant to major plant diseases. In particular, by confirming that the expression of CaChitIV gene is suppressed or overexpressed, it contributes to the resistance of the plant disease. Thus, CaChit IV according to the present invention can promote the development of resistance to plant disease and development of resistant cultivars as a material for breeding of disease resistant plants The present invention is a very useful invention in the plant breeding industry.

1 is nokgwang pepper cultivars by the present invention (Capsicum annnuum L. cv. A view showing the nucleotide sequence and amino acid sequence encoded by the nucleotide sequence of the gene cloned from pepper CaChitIV Nockwang),
FIG. 2 is a graph showing the homology between the CaChit IV amino acid sequence according to the present invention and the chitinase amino acid sequence of another crop.
FIG. 3 is a view showing the results of checking the interaction between the CaPIK1 protein and the CaChitIV protein using the Yeast Two Hybrid technique, BiFc (Bimolecular fluorescence complementation) technique and Co-IP (co-immunoprecipitation)
FIG. 4 is a graph showing the results of inducing the expression of the CaChitIV gene according to the present invention by pathogen treatment,
FIG. 5 and FIG. 6 are graphs showing changes in cell death upon simultaneous expression of CaChit IV and CaPIK 1,
FIGS. 7 to 9 show the results of immunohistochemical staining of CaChit IV according to the present invention using virus-induced gene silencing (VIGS) The pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a on the plants of the green pepper cultivars inhibited the expression of the gene. The bacterium of Xanthomonas campestris pv. vesicatoria ), showing the change in disease resistance, and Fig.
10 to 11 are diagrams showing changes in the disease resistance of plants caused by overexpression of CaChitIV gene according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

[ Example  One] CaChit IV Determination of base / amino acid sequence of gene

The following test was carried out to provide a base sequence of the pepper class (CaChitIV) -coding gene, which is one of the disease resistance-related proteins for the red pepper bacterial spotty disease, and the amino acid sequence deduced therefrom.

Capsicum annuum cv. Nockwang) were sprayed with red pepper bacillus fungi ( Xanthomonas campestris pv. vesicatoria ) and treated for 15 hours in a humidified condition. Then, the plants exhibiting the susceptible cell death response, which is a resistant disease, were taken out of the wet and the leaves were collected.

Next, mRNA was extracted from the leaf sample and reverse transcribed, followed by PCR (Polymerase Chain Reaction) amplification to prepare a cDNA library. Then, yeast two-hybrid screening was performed using the cDNA library thus constructed to identify cDNA clones that interact with CaPIK1, a disease resistance-related protein of pepper plants. This clone was isolated and purified from yeast cells.

After sequencing the obtained clones, the chitinase protein coding gene was confirmed by comparing with a gene database registered in GenBank using a blast program, and CaChit IV Gene. The nucleotide sequence (SEQ ID NO: 1) of CaChitIV identified and the amino acid sequence (SEQ ID NO: 2) encoded by the nucleotide sequence are shown in FIG.

[ Example  2] CaChit IV  Protein and CaPIK1  Identification of protein interactions

In order to confirm the interaction between CaChitIV protein and CaPIK1 protein obtained in Example 1, the following test was conducted.

[Step 1]

CaChitIV cDNA was inserted into a yeast two-hybrid vector to determine whether it interacted with CaPIK1. pGADT7: CaChitIV , PGBKT7 : CaPIK1, and pGBKT7: CaChitIV and pGADT7: CaPIK1 were grown in a selective medium (SD-AHLT) free of alanine, histidine, leucine and tryptophan, and cultured in a medium containing x-alpha-galactosidase (Fig. 3), confirming the yeast growth. The negative controls Lam and SV40-T failed to grow in the starter medium and did not decompose galactose, resulting in no color development. The positive controls, Lam and SV40-T, grew vigorously in the starter medium and decomposed galactose to give a green color reaction. CaChitIV and CaPIK1 grew vigorously in the starter medium and showed blue coloration.

[Step 2]

BiFc (Bimolecular fluorescence complementation) and Co-IP (co-immunoprecipitation) were performed with CaPIK1 and CaChitIV, respectively, to determine whether CaChitIV interacts with CaPIK1 in the plant. BiFc has N-termini and C-termini of YFP, which are fluorescent proteins, in pSPYNE and pSPYCE vectors, so that when the proteins encoded by the inserted genes in the vector interact, a complete YFP is generated and the fluorescent protein is observed through the confocal microcope can do. A recombinant vector was constructed by inserting CaChitIV and CaPIK1 cDNA into pSPYNE and pSPYCE. The Co-IP technique is based on the fusion of different epitope tags to the gene, followed by precipitation with one of the tagged proteins, followed by the detection of the other protein in the precipitated product, . p35S: CaChitIV : cMyc and p35S: CaPIK1 : HA were prepared, respectively. The prepared recombinant vector was transformed into Agrobacterium tumefaciense strain GV 3101) by electroporation, and the GV3101 strain containing the recombinant vector was introduced into Nicotiana It was inoculated into leaf used, mesophyll scanning (infiltration) of the method the syringe benthamiana) temporarily induces the expression of genes was performed BiFc and Co-IP method. In the case of Co-IP, proteins were extracted from plant leaf tissues, and proteins were precipitated through cMyc agarose and immunoblotting was performed using HA antibody to confirm protein interactions (see FIG. 3).

[ Example  3] in pepper plants CaChit IV  Exploration of gene expression

In order to confirm the use of the CaChitIV gene obtained in Example 1 for the search for disease resistance, the following test was conducted.

[Step 1]

First, in order to present the resistance search method concretely, the pathogenic bacteria such as red pepper bacterial spotty pathogen ( Xanthomonas campestris pv. vesicatoria ) and non - pathogenic strain Bv5-4a were inoculated to the back of 5th and 6th leaf of 6th green pepper cultivar at 10 ⁸ cfu / ㎖ concentration by syringe. After the inoculation, the cells were treated with wet treatment at 28 ° C and 100% relative humidity for 15 hours. The leaves were sampled at each time point after each inoculation of the pepper plants and inoculation, RNA was isolated, electrophoresed on formaldehyde gel, and transferred to Hybond N membrane.

Next, the CaChitIV gene obtained in Example 1 was labeled with α- ³² P [dCTP] with an isotope using a Klenow enzyme, and the resultant was added to the reaction solution [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% dextran sulfate) at 65 ° C for 24 hours to perform hybridization. When the isotope-labeled DNA probe is attached to the mRNA attached to the nylon membrane (Hybond N +) and the X-ray film is placed on the nylon membrane (Hybond N +), So that the presence and the degree of the expression can be recognized.

The results are shown in FIG. 4. As a result, the CaChitIV gene was expressed in the flower tissue of healthy plant and strongly expressed after 5 hours of inoculation with the non-pathogenic capsicum.

[ Example  4] CaChit IV and CaPIK1  Cell death at the time of simultaneous expression

To investigate the effect of CaChitIV interacting with CaPIK1 on CaPIK1 - induced apoptosis in pepper plants, expression of apoptosis was examined by simultaneous expression. The CaChitIV gene obtained in Example 1 was introduced into the pBIN35S vector for plant overexpression to prepare pBIN35S: CaChitIV and then Agrobacterium ( Agrobacterium tumefaciens) tumefaciense strain GV 3101) was introduced by electroporation and the GV3101 strain containing the recombinant vector was introduced into pBIN35S: CaPIK1 And CaCl2 and CaPIK1 genes were induced by inoculation on leaves of pepper plants at 6 weeks .

[Step 1]

A recombinant vector overexpressing CaChitIV (35S: CaChitIV), recombinant vectors overexpressing CaPIK1 (35S: CaPIK1) and any genes control vector (35S: 00) that does not over-expressing transformants were Agrobacterium tyume Pacific conversion using Enschede ( Agrobacterium tumefaciense strain GV 3101) was shake cultured in a yeast-peptone (YEP: 10 g yeast extract, 10 g peptone, 5 g / L sodium chloride) medium and the bacterial suspension concentration was measured by using a spectrophotometer with an optical density of 600 , OD ₆₀₀ ) with CaPIKl 1.0, CaChit IV 1.0, respectively. The cells were inoculated on leaves of pepper plants, and the increase of conductivity due to electrolyte leakage by cell death was measured at 0, 24, 48, 72 and 96 hours. The results are shown in FIG. On the third day after inoculation, apoptosis was observed in leaves inoculated with Agrobacterium expressing CaPIK1 . No apoptosis was observed in leaves inoculated with Agrobacterium expressing CaChit IV . In the leaves inoculated with CaPIK1 and CaChitIV , strong cell apoptosis was observed as compared with the leaves inoculated with CaPIK1 alone, and apoptosis was quantified and visualized (Fig. 5).

[Step 2]

CaPIK1 was fixed at 0.2, and CaChit IV 0.05, 0.1, and 0.5, respectively, and the mixture was mixed tobacco ( Nicotiana benthamiana ) leaves and 0, 24, 48, 72 and 96 hours later, the increase in conductivity due to electrolyte leakage due to cell death was measured. The results are shown in Fig. The higher the concentration of CaChit IV, the higher the level of apoptosis. It was confirmed by immunoblotting that CaChitIV protein-dependent increase was also observed (Fig. 5).

[Step 3]

CaPIK1 was fixed at 0.2, and CaChit IV 0.05, 0.1, and 0.5, respectively, and the mixture was mixed tobacco ( Nicotiana benthamiana) after each inoculation the leaves 24, 48 and observed radicals through the DAB staining after 72 hours, the cells were between an increase in the amount of free radicals produced at higher levels of the results of quantifying the hydrogen peroxide CaChitIV. In addition, a result of using a DAF-2DA (4,5-diaminofluorescein diacetate ) 24 and 48 hours of nitric oxide (Nitric Oxide, NO) after inoculation after dye sensitive to NO, staining was observed in leaf tissue through the confocal microscope also of CaChitIV 6 shows that NO production is increased as the concentration is increased.

[ Example  5] CaChit IV  Changes in disease resistance due to inhibition of gene expression

CaChitIV from pepper plants In order to investigate whether expression of other genes related to the development of the disease was decreased when the expression of the gene was specifically suppressed and whether the susceptibility to the disease was increased, the CaChit IV The recombinant vector TRV2: CaChitIV was prepared by introducing CaChitIV gene into TRV2 vector using TRV (Tobacco Rattle Virus) used in virus-induced gene silencing (VIGS) (Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing, Curr. Opin. Plant Biol., 2: 109-113).

The thus-prepared recombinant vector TRV2: CaChitIV was transformed into Agrobacterium tumefaciense strain GV 3101) by electrophoresis, and then the GV3101 strain containing the recombinant vector was inoculated to the cotyledon of the pepper seedlings by a syringe and infiltration method to introduce into the pepper plant Induced the inhibition of CaChitIV gene expression. In addition, existing TRV2: was produced in a plant having suppressed expression of both were suppressed CaPIK1 using CaPIK1 plant and CaChitIV and CaPIK1.

[Step 1]

In order to investigate the changes in the resistance to bacterial diseases in the plants in which the expression of CaChitIV was inhibited, the pepper plants inhibited the expression of CaChitIV gene and the pepper plants were inoculated with the TRV2: 00 vector as a control. Xanthomonas campestris pv. Vesicatoria ) and non-pathogenic strain Bv5-4a were inoculated at 5 x 10 ⁴ cfu ml ^-1 and bacterial growth was measured on infected plant tissues at 0 and 3 days after inoculation. As shown in FIG. 7, bacterial growth of pathogenic bacteria and non-pathogenic bacteria in the infected tissues after the inoculation of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a in the plants in which the expression of CaChit IV was inhibited by VIGS was observed in CaChit IV Growth of non-pathogenic bacteria was increased in plants with inhibited expression. Bacterial growth was significantly increased in plants inhibited with both CaChitIV and CaPIK1 compared with plants inhibited by one gene.

[Step 2]

Was inoculated CaChitIV, pathogenic strains Ds1 and non-pathogenic strains of Bv5-4a expression of CaPIK1 pepper bacterial jeommunuibyeong bacterium (Xanthomonas campestris pv. Vesicatoria) in the plant and the two genes both inhibit plant each inhibitory to 10 ⁷ cfu ml ^-1 inoculation Ion conductivity was measured at 12, 24, and 48 hours. As shown in FIG. 8, the ionic conductivity was decreased in plants inhibiting CaChitIV and CaPIK1 , respectively , compared to the control plant (TRV: 00), and decreased most in plants suppressed with both genes.

[Step 3]

CaChitIV, inhibit the expression of CaPIK1 each plant and pepper on both the two genes inhibit bacterial flora jeommunuibyeong bacteria (Xanthomonas campestris pv. vesicatoria ) and non ^- pathogenic strain Bv5-4a were inoculated at 10 ⁷ cfu ml ^{- 1} , and hydrogen peroxide, a kind of active oxygen, was measured at 6, 12 and 24 hours after inoculation. As shown in FIG. 9 , the production of reactive oxygen species was decreased in the plants which inhibited CaChitIV and CaPIK1 , respectively , compared with the control plant (TRV: 00)

[ Example  6] CaChit IV  Production of transgenic plants using genes CaALDH1  Variation of disease resistance of Arabidopsis plants by overexpression of genes

In order to investigate whether the resistance to the disease upon overexpression of the CaChitIV gene in Arabidopsis thaliana plants used as a model plant was increased, the pBIN35S: CaChitIV transformed Agrobacterium tumefaciens ( Agrobacterium tumefaciens) strain GV 3101) was introduced into Arabidopsis thaliana through floral dipping method to produce a transgenic Arabidopsis thaliana inducing overexpression of CaChitIV gene and the following test was carried out.

In the CaChitIV gene is overexpressed plants to investigate whether or not the resistance to bacterial disease increase, was observed with the expression of CaChitIV gene in wild-type Arabidopsis thaliana plant as the CaChitIV gene is overexpressed Arabidopsis thaliana with the control and the results 10. # 1, # 2, and # 3 represent the phylogenetic number of the transgenic plants used. As a result, the CaChitIV gene was overexpressed.

As mentioned above, inoculation of Haialoparonospora 2 nodule strain to CaChit IV overexpressed plants and observation of nociceptive growth in the infected tissues revealed that nociceptive growth in the plants overexpressing CaChitIV gene resulted in infection of wild type plant infected tissue (Fig. 11).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Disease resistance-related gene CaChit IV, and transgenic plants          using the same <130> P11-130910-03 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 990 <212> DNA <213> Capsicum annuum cv. Nockwang <400> 1 aagatgggca ttaattctag tcatttaaaa aattcactgt tggcctactg gacacttaac 60 taatcaagat gaacttctct tcatcaacaa aatatttctt tctctttgta gcattagcta 120 taatagctga tgtaccaaga ctaatcttgg cacaaaactg tgggtgtgca gcaaatttat 180 gttgtagcaa atggggttat tgtggagagg gaaaggatta ttgtggtgaa gggtgtcaag 240 gggggccatg ttttagtact acaccatcag gcaataatgg cggttcagtt tctgatattg 300 tatctgatgc attctttaat gggatagttg atcaagccgc ttctaattgt gaaggaaaag 360 ggttttattc aagggataaa ttctttgaag ctcttaaatc ttatcctaac tttggaactg 420 tgggttctaa tgatgactct aaacgtgaaa ttgctgcttt ctttgctcat gtcacccacg 480 aaactggcca catgtgctac ataaatgaga taaatggtcc atcaggcgac tattgtgatg 540 aggacaacaa agagtaccct tgtgtatcag gcaagaacta ctatggtcga ggaccaattc 600 aactatcatg gaacttcaac tacggacctg ctggaaaatc cattggattt gatggcctaa 660 atgaccctga catagttgca agagatgctg ttatttcctt caagacagca ttgtggtatt 720 ggatgaacaa ttgtcattca ctaattactt ctggacaagg ttttggtcca actattagag 780 ctattaatgg tagacttgaa tgtgatggtg gtaatcctca aactgttgct agaagggttg 840 agtattacac tcagtattgt caacaacttg gtgttgatgc tggggataat ctcacgtgtt 900 agtttgcaag ttagaataaa cactatgatt acactaagtc gctcacagac tatacttatg 960 agattataca ttcaaaaaaa aaaaaaaaaa 990 <210> 2 <211> 277 <212> PRT <213> Capsicum annuum cv. Nockwang <400> 2 Met Asn Phe Ser Ser Ser Thr Lys Tyr Phe Phe Leu Phe Val Ala Leu   1 5 10 15 Ala Ile Ile Ala Asp Val Pro Arg Leu Ile Leu Ala Gln Asn Cys Gly              20 25 30 Cys Ala Ala Asn Leu Cys Cys Ser Lys Trp Gly Tyr Cys Gly Glu Gly          35 40 45 Lys Asp Tyr Cys Gly Glu Gly Cys Gln Gly Gly Pro Cys Phe Ser Thr      50 55 60 Thr Pro Ser Gly Asn Asn Gly Gly Ser Val Ser Asp Ile Val Ser Asp  65 70 75 80 Ala Phe Phe Asn Gly Ile Val Asp Gln Ala Ala Ser Asn Cys Glu Gly                  85 90 95 Lys Gly Phe Tyr Ser Arg Asp Lys Phe Phe Glu Ala Leu Lys Ser Tyr             100 105 110 Pro Asn Phe Gly Thr Val Gly Ser Asn Asp Asp Ser Lys Arg Glu Ile         115 120 125 Ala Ala Phe Phe Ala His Val Thr His Glu Thr Gly His Met Cys Tyr     130 135 140 Ile Asn Glu Ile Asn Gly Pro Ser Gly Asp Tyr Cys Asp Glu Asp Asn 145 150 155 160 Lys Glu Tyr Pro Cys Val Ser Gly Lys Asn Tyr Tyr Gly Arg Gly Pro                 165 170 175 Ile Gln Leu Ser Trp Asn Phe Asn Tyr Gly Pro Ala Gly Lys Ser Ile             180 185 190 Gly Phe Asp Gly Leu Asn Asp Pro Asp Ile Val Ala Arg Asp Ala Val         195 200 205 Ile Ser Phe Lys Thr Ala Leu Trp Tyr Trp Met Asn Asn Cys His Ser     210 215 220 Leu Ile Thr Ser Gly Gln Gly Phe Gly Pro Thr Ile Arg Ala Ile Asn 225 230 235 240 Gly Arg Leu Glu Cys Asp Gly Gly Asn Pro Gln Thr Val Ala Arg Arg                 245 250 255 Val Glu Tyr Tyr Thr Gln Tyr Cys Gln Gln Leu Gly Val Asp Ala Gly             260 265 270 Asp Asn Leu Thr Cys         275

Claims (5)

A classpoxinase gene ( Capsicum (R)) derived from the isolated pepper of the following (a) or (b) that codes for class plant species (Chitinase class IV) annuum Chitinase class IV , CaChit IV ):
(a) an isolated gene encoding a protein having the amino acid sequence of SEQ ID NO: 2;
(b) an isolated gene having the nucleotide sequence of SEQ ID NO: 1. The isolated capsicum-derived class &lt; RTI ID = 0.0 &gt; forchitis &lt; / RTI &gt; protein ( Capsicum annuum Chitinase class IV , CaChit IV ). A recombinant vector comprising the gene ( CaChit IV ) of claim 1. A plant transformed with the recombinant vector of claim 3. Isolating mRNA from the plant after inoculation of the pathogen, and identifying the differential expression of the gene ( CaChit IV ) according to claim 1.

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