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    • 专利摘要:
    The present invention relates to a method of searching for a glucan hydrolase gene and plant diseases using the same of pepper hanbyeol varieties resistant, the pepper plant bacterial etiology pepper bacterial jeommunuibyeong (Xanthomonas campestris pv. Vesicatoria) peppers hanbyeol cultivars showing resistance to (Capsicum An isolated glucanase gene derived from annuum L. cv.Hanbyul) was constructed and a recombinant vector inserted therein was constructed to prepare a transformant, inoculated with phytopathogens, or infected with plant diseases. When treated with chemicals known as inducers, the mechanism of expression of glucan hydrolase genes can be investigated to provide a method for labeling the plant's defense against pathogens and searching for plant disease resistance.
    公开号:KR20020029467A
    申请号:KR1020000060207
    申请日:2000-10-13
    公开日:2002-04-19
    发明作者:황병국；정호원
    申请人:채문식；학교법인고려중앙학원；
    IPC主号:
    专利说明:

    Glucan Hydrolase Genes of Korean Red Pepper Varieties and Methods for Investigating Plant Disease Resistance Using the Same hanbyul and probing method of resistance for plant disease}
    [9] The present invention relates to a glucan hydrolase gene of red pepper varieties and a plant disease resistance screening method using the same. More specifically, the present invention isolates the glucanase gene from the Capsicum annuum L. cv. Hanbyul, which is known to be resistant to the bacterium bacterium bacterial bacilli. Analyze and investigate plant disease resistance by investigating glucan hydrolase gene expression mechanisms when plants are infected with other phytopathogens or treated with chemicals known as plant disease resistance inducers. The present invention relates to a method for detecting a defensive response due to disease occurrence in a plant.
    [10] Bacterial spot disease of red pepper, also called spot germ disease and beetle disease, forms spots on the leaves, stems, and fruits of red peppers. Especially, the spots on leaves are known to cause early deciduous leaves and reduce yield. to be.
    [11] Invasion of certain crops by harmful pests and pathogens is a serious problem that threatens survival even for humans, who rely on plants for the most part. Various control methods have been used to solve these problems and protect plants from emerging pathogens. Among them, the easiest and simplest method of controlling plant diseases is spraying pesticides, which are highly toxic and thus kill harmful organisms such as pathogens and other surrounding ecosystems, destroying ecosystems and polluting soil and water quality. Therefore, it is important to properly apply physical and biological control methods in consideration of the environment and conditions where plants are located, as well as chemical control means such as spraying pesticides, and in particular, to improve plant varieties that have their own resistance to plant diseases and Control by breeding and cultivation is necessary.
    [12] In crop production, the decline in production due to the occurrence of pests has been recorded earlier. In an effort to reduce the damage caused by pests, programs have been undertaken to develop disease-resistant crops for various crop-pathogen interactions, and resistance to disease-resistant plants for understanding and practical application of disease resistance. Mechanism studies have been conducted on various crops and model plants. Tobacco, tomato and rice are representative examples of the crops that are being studied intensively. Agiaceae, a type of weed, is used worldwide as a model plant for the study of plant disease resistance. Pepper, which is a representative eggplant with tobacco and tomatoes, has been used as a spice and seasoning for a long time in Korea, and is one of the main crops that make up the Korean diet with rice and cabbage. The incidence of disease in Korea reported during the cultivation of red pepper is indicated for viral diseases, bacterial diseases and fungal diseases. The main diseases in pepper plants are plague caused by fungi, anthrax caused by fungi and bacterial spots caused by bacteria. Has been. Even in this reality, studies on defensive reactions and reactions to environmental stresses, including pepper-resistant mechanisms, are insufficient.
    [13] Host plants trigger various cellular metabolism against plant diseases, triggering defense mechanisms. By this defense mechanism, the reaction between host plants and phytopathogens can be determined by whether the pathogenesis can be limited in the early stage of infection by the pathogen, and the reaction is divided into friendly and incompatible reactions. Recognizing the invasion of pathogens by plant recognition in incompatible reactions, in response to this, hypersensitive reactions such as local cell death in infected cells, accumulation of callose, and ligninization Has been reported to cause thickening of plant cell walls, generation of various types of antimicrobial substances such as phytoalexin, and pathogenesis related (PR) protein production. In addition, this defense mechanism is reported to be caused not only by the invasion of pathogens, but also by salicylic acid and aminobutyric acid. This response is commonly referred to as induction resistance, which is evaluated as a signaling pathway that plays an important role in plants' ability to defend against pathogens. This inductive resistance is of interest as a model for signal transmission and is of practical value. By understanding the biochemical changes that cause this resistance, it is possible to develop genetically engineered plants that increase disease resistance or to develop plant protection chemicals that work by promoting the plant's genetic resistance.
    [14] Over the years, disease-producing proteins-1 (PR-1), chitin hydrolase, and glucan hydrolase have antimicrobial effects and these proteins play an important role in plant disease defense. This has been known. In particular, in the case of glucan hydrolase, it has the ability to degrade beta-1,3-glucan, which is a cell wall component of lower microorganisms such as fungi including late blight bacteria and mozzarlock bacteria, and fungi along with chitin hydrolase Has been reported to increase the inhibitory effect. It has been reported that glucan degrading fragments, which are degraded from fungal or plant cell walls by the action of glucan hydrolase, serve as signaling agents that promote various disease defense responses in plants. The distribution of glucan hydrolase in the flora is very diverse, such as in tobacco, tomatoes, potatoes and Arabidopsis.
    [15] In view of the above facts, the present inventors focus on the glucan hydrolase gene cloned from pepper varieties of Korean varieties, which are resistant to red pepper bacterial spot pattern disease, and a method for searching for other defense reactions in disease occurrence in red pepper plants using the same. It was. That is, the present invention isolates the glucan hydrolase gene that can represent the disease defense reaction of plants, and understands the disease defense reaction of pepper plants at the molecular level, and uses them in the molecular breeding program of pepper plants in the future. It suggests a method of searching for a disease resistance inducer of pepper plants, including the step of confirming the expression of the glucan hydrolase gene and suggesting the possibility of.
    [16] Accordingly, it is an object of the present invention to provide a nucleotide sequence of the glucan hydrolase gene CABGLU isolated from pepper varieties showing resistance to red pepper bacterial spot disease and amino acid sequence inferred therefrom in view of the above facts. Another object of the present invention is to provide a recombinant vector constructed by inserting the glucan hydrolase gene and a transformant prepared by introducing the recombinant vector into Escherichia coli. Still another object of the present invention is to provide a method for detecting plant disease resistance of plants by identifying differential expression of glucan hydrolase genes by pathogen infection and abiotic resistant derivatives using the glucan hydrolase genes. have.
    [17] The above object of the present invention is to inoculate cDNA by using reverse transcriptase after separating mRNA from pepper leaves inoculated by capsicum annuum L. cv. Hanbyul, inoculated with pepper bactericidal bacillus (Xanthomonas campestris pv.vesicatoria). Prepared and PCR amplified using a known primer to prepare a cDNA library, and screened it to select the glucan hydrolase gene and determine its nucleotide sequence, the bacterial pathogens, fungal pathogens and abiotic derivatives in pepper ethephon on the salicylic acid, methyl jasmonate, β- amino acid, such as a Bt H. inoculated and one was extracted by gel electrophoresis and transferred to a night is just the RNA of the plant and then by the present invention labeled with 32 P spray This was accomplished by reacting with a glucan hydrolase gene probe and looking for the degree of its induced expression.
    [18] Hereinafter, the configuration of the present invention will be described in detail.
    [1] Figure 1 shows the nucleotide sequence of the glucan hydrolase gene cloned from red pepper bacterial spot disease resistant varieties Capsicum annuum L. cv. Hanbyul.
    [2] Figure 2 shows the amino acid sequence inferred from the glucan hydrolase gene cloned from the pepper bacterial spot disease resistant varieties Capsicum annuum L. cv. Hanbyul.
    [3] FIG. 3 shows a cleavage map of the recombinant vector pCABGLU constructed by introducing a glucanase gene cloned from Capsicum annuum L. cv. Hanbyul.
    [4] Figure 4 shows the results of inducing glucan hydrolase gene expression in infected leaves when inoculated with the pathogenic or non-pathogenic strains of Xanthomonas campestris pv.vesicatoria causing pepper bacterial spotting It is shown.
    [5] Figure 5 shows the results of induction of glucan hydrolase gene expression when the pathogenic or non-pathogenic strain of Phytophthora capsici causing pepper blight was inoculated into the stem of a red pepper.
    [6] Figure 6 shows the results of induction of glucan hydrolase gene expression after treatment of the leaves of red pepper starch treated with etepon 10mM, salicylic acid 5mM, methyl jasmonate 100μM, beta-aminobutyric acid 19.4mM, BHT 95.2μM, respectively.
    [7] Figure 7 shows the expression induction of the glucan hydrolase gene according to the elapsed time after treatment with etepon.
    [8] Figure 8 shows the expression induction of glucan hydrolase gene with elapsed time after treatment with methyl jasmonate.
    [19] The present invention is inoculated with pepper pathogenic bacteria ( Xanthomonas campestris pv. Vesicatoria ) with mild pathogenic bacteria ( Capsicum annuum L. cv. Hanbyul), and the leaves are not infected with mRMA and pathogens of red pepper leaves. Isolating mRNA; Reverse transcription of the mRNA to prepare cDNA and PCR amplification to prepare cDNA library; Selecting individual cDNA clones from the cDNA library and constantly adsorbing them to the nitran membrane; Performing reverse northern hybridization using a single stranded cDNA probe; Selecting a glucan hydrolase gene CABGLU from the individual cDNA gene clones and determining a nucleotide sequence thereof; Inserting the glucan hydrolase gene CABGLU into a phagemid vector pBluescript SK (−) to construct a recombinant vector pCABGLU; Introducing the recombinant vector pCABGLU into E. coli to prepare a transformant Escherichia coli CABGLU (CABGLU); Among red pepper bacterial spot bacteria, non-pathogenic strain Bv5-4a, which reacts with plants and incompatible with plants, was inoculated into pepper leaves, treated with moisture, and RNA was isolated and electrophoresed. Confirming the induction of the expression of the glucan hydrolase gene by bacterial bacterial streptococcal infection in pepper leaves by transferring the glucan hydrolase gene with a 32 P-labeled probe; The inoculated strains of pathogenic strain S197, which reacts kindly with plants, and non-pathogenic strain CBS178.26, which reacts unfavorably with pepper, were inoculated into pepper stems, RNA was isolated and electrophoresed, and then transferred to the nylan membrane. Confirming the induction of glucan hydrolase gene by red pepper bacterium infection in red pepper stem by reacting the hydrolase gene with a probe labeled with 32 P; Ethephon, salicylic acid, methyl jasmonate, β-aminobutyric acid, and BH, which are known to induce plant disease resistance to plants, were treated with foliar spraying, and after 24 hours, RNA was isolated and electrophoresed. Transforming to the membrane and reacting the glucan hydrolase gene with a 32 P-labeled probe to confirm the induction of expression of the glucan hydrolase gene by the plant disease resistance inducer.
    [20] Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.
    [21] Example 1 pepper varieties ( Capsicum annuum cv. Isolation and Determination of Glucanase Gene from Hanbyul)
    [22] Isolation of glucan hydrolase genes obtained by inoculation of non-pathogenic pepper bacterial spot bacterium ( Xanthomonas campestris pv.vesicatoria ) was isolated from the red pepper varieties of resistance-induced red pepper cultivars, and prepared by cDNA library. The nucleotide sequence was analyzed.
    [23] Capsicum annuum L. cv. Hanbyul was inoculated with non-pathogenic pepper bacterial spot bacteria ( Xanthomonas campestris pv. Vesicatoria ) and incubated for 18 hours and then infected with bacterial spot bacteria and showed hypersensitivity reactions. Purified mRNA of pepper leaves was isolated and cDNA libraries were prepared from them. Individual cDNA clones were selected from the prepared cDNA library and adsorbed to the nitrile membrane uniformly, and then reverse hybridization was performed using single-stranded cDNA probes obtained from healthy red pepper leaves and red pepper leaves with hypersensitivity reactions. ) Was performed. Single stranded AMV- RT enzyme (reverse transcriptase) here by separating total RNA from the pepper leaves are infected with the bacterial strain showed a resistance jeommunuibyeong lesion of hypersensitivity reactions in order to manufacture those cDNA probe used this time and the oligo d (t) 18 A single-stranded cDNA probe was prepared by reacting the primers (obtained from Roche) and the dNTP mixture containing dUTP -DIG (obtained from Roche) for 10 minutes at 25 degrees, 60 minutes at 42 degrees, and 5 minutes at 99 degrees. The jar was prepared. The 5'-terminal sequence of the individual cDNA clones showing specific accumulation in the hypersensitivity reactions thus obtained was used to determine the total nucleotide sequence of the clone identified as the glucan hydrolase gene, and the base sequence and the analogy thereof. One amino acid sequence is shown in FIGS. 1 and 2.
    [24] Pepper glucanase gene CABGLU of the present invention is composed of all of the base sequence of 1332 bp, consisting of a total of 359 amino acids. Comparing these amino acid sequences with those of other plants, the glucan hydrolase genes derived from Korean varieties are relatively 77.4% to tobacco glucan hydrolase genes, 64.6% to potato genes and 58.7% to tomato genes. It showed high homology and was found to be basic protein with isoelectric point 9.46. On the other hand, the glucan hydrolase gene of the present invention isolated from the red pepper varieties showed a low homology of 42.6% with the glucan hydrolase gene of the acidic tomato can be expected to be a basic protein. Based on this, it can be seen that the glucan hydrolase gene of the red pepper starch cloned as described above is a new glucan hydrolase gene which is distinguished from the protein gene present in other plants.
    [25] Example 2 The glucan hydrolase gene of the phagemid state obtained through screening of E. coli ( E. coli Process for preparing and transforming transformants by transformation
    [26] The glucan hydrolase gene CABGLU of the present invention was introduced into a pBluescript SK (-) phagemid vector provided by stratagene to construct a recombinant vector pCABGLU and a cleavage map thereof is shown in FIG. 3.
    [27] Strain XL1-Blue MRF 'and SOLR strains, E. coli strains provided by Stratagene, were shaken overnight at 30 ° C in an LB liquid medium, and 0.5 ml was added to 50 ml LB liquid medium again for 2-3 hours at 37 ° C. While shaken (OD 600 = 0.2-0.5). XL1-Blue MRF 'was centrifuged (1,500 xg) and diluted with 10 mM MgSO 4 to an OD 600 value of 1.0. 200 µl of this strain, 250 µl of the phage stock obtained through screening, and 1 µl of ExAssist helper phage were placed in a 50 ml conical tube and shaken at 37 ° C for 15 minutes. This was further treated with 3 ml of LB liquid medium, shaken at 37 ° C. for 2 hours, and treated at 70 ° C. for 15 minutes, followed by centrifugation (4000 × g). In a new tube, add 100 µl of the supernatant and SOLR strain with an OD 600 value of 1.0, incubate for 15 minutes at 37 ° C, and add 10-50 µl to LB agar medium containing 50 µg / ml of ampicillin. Spread and incubate overnight at 37 ° C to form a flora. The bacterium was inserted with the glucan hydrolase gene, which was incubated for 15 hours in a 5 ml LB liquid medium, and contained 812.5 µl and 187.5 µl of 80% glycerol and stored at -70 ° C.
    [28] The strain of the present invention was named Escherichia coli GABGLU ( Ecsherichia coli CABGLU), and deposited on October 2, 2000 with the Korean spawn association under accession number KFCC 11216.
    [29] Example 3 Induced Expression of Glucan Hydrolase Gene of Pepper Plants by Phytopathogens
    [30] First step. Induced Expression of Glucan Hydrolase Gene by Red Pepper Bacterial Spot Bacteria Ds1 and Bv5-4a
    [31] Jeommunuibyeong pepper bacterial strains of the peppers were cultured for a non-pathogenic strain Bv5-4a showing a pathogenic strain Ds1 and the incompatible reaction indicating the reaction of the plant-friendly hanbyeol varieties and 5X10 8 cfu / ㎖ sprayers associated with the vacuum pump to 6 leaf stage of the plants at a concentration RNA was isolated by infiltrating (infiltration) using the inoculation and wetting for 18 hours and taking pepper leaves after 6, 12, 18, 24 and 30 hours, respectively. The isolated RNA was electrophoresed on a gel containing formaldehyde and then transferred to a nitlan membrane (Hybond N +). Recombinant plasmid vector pCABGLU containing the glucan hydrolase gene obtained in Example 1 was digested with restriction enzymes Eco RI and Xho I, and only the inserted DNA was recovered and labeled with 32 P. The reaction solution [0.25 M phosphoric acid] Buffer, 7% SDS, 1 mM EDTA, 5% dextran sulfate] and hybridization was performed by incubating at 65 ° C. for 16-24 hours. With a 32 P labeling of the DNA probe is nitro is and stick to the mRNA attached to the membrane is nitro is to photosensitive the DNA is X-ray films labeled with 32 P when lay the X-ray film over a film the expression levels in which I could recognize it. As a result, it was found that the glucan hydrolase gene was induced in all friendly incompatible interactions (FIG. 4).
    [32] Glucan hydrolase genes were induced in infected leaf tissues when different strains of red pepper bacterial spot pathogen were inoculated on the leaves of red pepper plants. In addition, it was found that the expression of glucan hydrolase genes was differentially expressed in pathogenic and non-pathogenic bacteria. In leaves infected with pathogenic bacteria, expression began at 18 hours after inoculation, increased rapidly at 24 hours and maintained for up to 30 hours, whereas expression in leaves infected with non-pathogenic bacteria was inoculated with pathogenic bacteria. Although the expression level of glucan hydrolase was 24 hours after infection, the expression of glucan hydrolase was significantly different from that of pathogenic bacteria invaded.
    [33] Second step. Induced Expression of Glucan Hydrolase Gene by Red Pepper Bacteria S197 and CBS178.26
    [34] Among the causative bacteria, pathogenic strain S197, which shows a friendly response to plants, and non-pathogenic strain CBS178.26, which shows an incompatible reaction, were inoculated into the pepper stem and sampled by date to isolate RNA. The isolated RNA was electrophoresed on a formaldehyde-containing gel and transferred to a nitrile membrane (Hybond N +), and then the recombinant plasmid vector pCABGLU containing the glucan hydrolase gene obtained in Example 1 was inserted into a restriction enzyme Eco RI. And Xho I, respectively, and the inserted DNA were recovered and labeled with 32 P, followed by 65 ° C., 16 − in a reaction solution (0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% dextran sulfate). Incubated for 24 hours. As a result, it was found that the amount of glucan hydrolysis genes expressed at low levels in healthy red pepper stem increased after 2 days in friendly reaction and then decreased after 2 days, and gradually decreased after 3 days in incompatible reaction. (Figure 5).
    [35] When the pathogenic and non-pathogenic bacterium of pepper germ pathogens were inoculated into the pepper stem, the pepper glucan hydrolase gene was specifically induced in the pepper stem. The expression level of glucan hydrolase gene was increased up to 2 days after the infection of the pathogenic bacteria, and decreased rapidly from the 3rd day of the death of the plant, and increased until 3 days after the inoculation of the non-pathogenic bacteria. It showed a tendency to decrease gradually.
    [36] As such, plants exhibiting resistance to disease show various protective reactions during invasion and infection of pathogens, and it is found that glucan hydrolase genes are induced in the leaves and stems of pepper plants during invasion of various bacterial and fungal pathogens. Can be. Induction and expression of this glucan hydrolase gene can be seen as molecular markers that indicate local and systemic acquisition resistance in infected areas. In addition, by using the glucan hydrolase of the present invention, plant disease resistance can be induced and plant diseases can be controlled by applying to plant disease control.
    [37] Example 4: Induction of glucan hydrolase gene expression by abiotic derivatives of plants
    [38] First step. Induction of glucan hydrolase gene expression by disease resistance inducer
    [39] In order to determine which signaling pathways induce the expression of glucan hydrolase genes in pepper plants, substances known as plant disease resistance inducing substances in other plants were treated externally in pepper plants.
    [40] 10 mM Ethephon, 5 mM salicylic acid, 100 µM methyl jasmonate, 19.4 mM DL-β-amino-n-butyric acid, known to induce disease resistance in plants , 95.2 μM of benzothiadia-zole (BTH) was treated by foliar spraying, and total RNA was isolated from the plants after 24 hours. After the isolated RNA was electrophoresed on a formaldehyde-containing gel and corrected with a nitran membrane (Hybond N +), the recombinant plasmid vector pCABGLU containing the glucan hydrolase gene obtained in Example 1 was inserted into the restriction enzyme Eco. After digestion with RI and Xho I, only the inserted DNA was recovered and labeled with 32 P, followed by 65 [deg.] C., 16 in a reaction solution [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% dextran sulfate]. Incubated for -24 hours.
    [41] As a result, it was found that the glucan hydrolase gene was strongly expressed in etepon and methyl jasmonate and was not induced and expressed by salicylic acid and BT and beta-aminobutyric acid (FIG. 6). In addition, the experiments to determine whether the byproducts hydrochloric acid and phosphorous acid produced in the process of the decomposition of etepon to ethylene affect the induction of the glucan hydrolase gene was confirmed that these by-products have no effect at all. As a result, the expression of the glucan hydrolase gene is related to the signaling process by ethylene or methyl jasmonate rather than the known salicylic acid signaling process.
    [42] Second step. Induction of Glucan Hydrolase Genes by Ethephon Induces Disease Resistance
    [43] After the treatment of ethylene-producing ethene, the glucan hydrolase gene accumulates over time, 10mM of etepon was added to red pepper leaves and 2, 6, 12, 18 and 24 hours later. Was taken to isolate total RNA. After the isolated RNA was electrophoresed on a formaldehyde-containing gel and transferred to a nitran membrane (Hybond N +), the recombinant plasmid vector pCABGLU containing the glucan hydrolase gene obtained in Example 1 was inserted into a restriction enzyme Eco. After digestion with RI and Xho I, only insert DNA was recovered and labeled with 32 P, followed by 65 ° C., 16 in reaction solution [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% dextran sulfate]. Incubated for -24 hours.
    [44] As a result, the glucan hydrolase gene began to accumulate 6 hours after treatment, increased to 18 hours, and maintained its expression level up to 24 hours. The expression results of the glucan hydrolase genes examined by time after treatment with etepon are shown in FIG. 7.
    [45] Third step. Induced Expression of Glucan Hydrolase Gene by Methyl Jasmonate Induces Disease Resistance
    [46] In order to know the effect of methyl jasmonate concentration and time on the expression of glucan hydrolase gene, 100 μM of methyl jasmonate was treated in pepper and total RNA was isolated after 2, 6, 12, 18 and 24 hours. After the isolated RNA was electrophoresed on a formaldehyde-containing gel and corrected with a nitran membrane (Hybond N +), the recombinant plasmid vector pCABGLU containing the glucan hydrolase gene obtained in Example 1 was inserted into the restriction enzyme Eco. After digestion with RI and Xho I, only the inserted DNA was recovered and labeled with 32 P, followed by 65 [deg.] C., 16 in a reaction solution [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% dextran sulfate]. Incubated for -24 hours.
    [47] As a result, it was found that the expression of the glucan hydrolase gene began 12 hours after methyl jasmonate treatment and the expression amount was maintained up to 24 hours (Fig. 8).
    [48] As described above, the glucan hydrolase gene product of the present invention, which is known to induce expression in the hypersensitivity reaction, which is a resistance reaction in red pepper, and is known to have an antimicrobial effect, can induce plant disease resistance and apply plant disease control to plant disease control. I can control it.
    [49] As is apparent from the above examples, the present invention isolates the gene of glucan hydrolase that exhibits plant disease defense reaction from Capsicum annuum L. cv. Hanbyul, which is resistant to red pepper bacterial spot disease. By determining the sequence, it can be used as a genetic material for labeling plant defense reactions and molecular breeding of plant disease resistant crops, and also inducing the glucan hydrolase gene by red pepper bacterial spot bacteria, red pepper bacterium and abiotic derivatives. By providing a method for measuring the expression level and understanding the biochemical changes of the plant causing the resistance at the molecular level, it is possible to develop pepper molecule sarcoma with enhanced disease resistance or to confirm the expression of the glucan hydrolase gene by using it. Exploration method of plant disease resistance of pepper plants, including process It is a very useful invention for the agricultural industry and the plant seed industry because it has an excellent effect of developing a plant protection chemical that promotes the resistance genetic mechanism of plants.
    权利要求:
    Claims (7)
    [1" claim-type="Currently amended] Nucleotide sequence of the following gene CABGLU encoding a plant disease resistant glucan hydrolase from Capsicum annuum L. cv. Hanbyul.

    [2" claim-type="Currently amended] The following amino acid sequence encoded by the plant disease resistant glucan hydrolase gene CABGLU derived from Capsicum annuum L. cv. Hanbyul described in claim 1 above.

    [3" claim-type="Currently amended] The recombinant vector pCABGLU constructed by inserting the plant disease resistant glucan hydrolase gene CABGLU derived from Capsicum annuum L. cv. Hanbyul as described in claim 1 into pBluescript SK (-).
    [4" claim-type="Currently amended] Transformant Escherichia coli characterized by introducing the recombinant vector pCABGLU according to claim 3 comprising the plant disease resistant glucan hydrolase gene CABGLU from Capsicum annuum L. cv. Hanbyul CABGLU ( Eherichia coli CABGLU) (KFCC 11216).
    [5" claim-type="Currently amended] RNA was isolated from pepper plants inoculated or treated with pathogens or abiotic chemicals, followed by electrophoresis and transfer to nitrile membranes, followed by reaction with the glucan hydrolase gene probe of claim 1 labeled with 32 P. A plant disease resistance screening method for plants characterized by identifying differential expression of glucan hydrolase genes against infection.
    [6" claim-type="Currently amended] The method of claim 5, wherein the pathogen is pepper bacterial spot pattern bacterium or pepper bacteriophage bacterium.
    [7" claim-type="Currently amended] The method of claim 5, wherein the abiotic chemical is ethephon or methyl jasmonate.
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    同族专利:
    公开号 | 公开日
    KR100396210B1|2003-09-17|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2000-10-13|Application filed by 채문식, 학교법인고려중앙학원
    2000-10-13|Priority to KR10-2000-0060207A
    2002-04-19|Publication of KR20020029467A
    2003-09-17|Application granted
    2003-09-17|Publication of KR100396210B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR10-2000-0060207A|KR100396210B1|2000-10-13|2000-10-13|Glucanase gene of Capsicum annuum L.cv. hanbyul and probing method of resistance for plant disease|
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