KR100578746B1 - 1 Pathogen induced pepper geneCAPIP1 sequence and probing method of plant disease resistance and environmental stresses using the same - Google Patents

Abstract

The present invention relates to a base sequence of a pathogen-induced pepper gene (CAPIP1) and a method for searching for plant disease resistance and environmental stress using the same, and more specifically, Capsicum annuum L. cv.Hanbyul), which isolates and sequencing the CAPIP1 gene, a resistance-related new gene induced by pathogens, provides the nucleotide sequence and its amino acid sequence, and uses this gene to induce biological / chemical / physical resistance induction. The present invention relates to a method for searching for plant diseases and environmental stress resistance that detects whether or not expression of induction resistance occurs. According to the present invention described above, it is possible to provide a genetic material for labeling the protective reaction against plant pathogens and molecular breeding of plant disease resistant crops, and to facilitate the search for plant disease resistance.

Pepper, CAPIP1 Gene, Plant Disease, Resistance, Environmental Stress, Bacterial Spot Disease

Description

Pathogen induced pepper gene (CAPIP1) sequence and probing method of plant disease resistance and environmental stresses using the same}

Figure 1 shows the nucleotide sequence and amino acid sequence of the CAPIP1 gene cloned from Capsicum annuum L. cv. Hanbyul according to the present invention.

Figure 2 shows the expression of the CAPIP1 gene according to the present invention in healthy and uninoculated leaves when inoculated with the red pepper bacterial spot bacterium ( Xanthomonas campestris pv.vesicatoria ) of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a Induced results are shown.

Figure 3 shows the expression induction results of the CAPIP1 gene according to the present invention according to the elapsed time after treating ethylene starch varieties.

Figure 4 shows the expression induction results of the CAPIP1 gene according to the present invention according to the elapsed time after the treatment of methyl jasmonate in the red pepper varieties.

Figure 5 shows the expression induction results of the CAPIP1 gene according to the present invention according to the elapsed time after the treatment of salicylic acid in the red pepper varieties.

Figure 6 shows the expression induction results of the CAPIP1 gene according to the present invention according to the time elapsed after treatment of the BH varieties of pepper.

Figure 7 shows the expression induction results of the CAPIP1 gene according to the present invention according to the elapsed time after treating the β-aminobutyric acid in the red pepper varieties.

Figure 8 shows the expression induction results of the CAPIP1 gene according to the present invention according to the elapsed time after the treatment of hydrogen peroxide varieties per capita.

Figure 9 shows the expression induction of the CAPIP1 gene according to the present invention according to the elapsed time after treating the wound in the red pepper varieties.

Figure 10 shows the expression induction results of the CAPIP1 gene according to the present invention according to the elapsed time after treating the sodium chloride in the red pepper varieties.

Figure 11 shows the expression induction of the CAPIP1 gene according to the present invention according to the elapsed time after the treatment of low-temperature varieties of pepper.

12 is a cleavage map of the recombinant vector pCAPIP1 into which the CAPIP1 gene is inserted according to the present invention.

Figure 13 shows the results of pathogenicity test against the bacterial pathogen Pseudomonas siringe Pasova tomato DC3000 strain in the transgenic Arabidopsis plants overexpressed the CAPIP1 gene according to the present invention.

Figure 14 shows the results of the resistance assay for sodium chloride and mannitol of the transgenic Arabidopsis plant seeds overexpressing the CAPIP1 gene according to the present invention.

Figure 15 shows the results of the resistance assay for sodium chloride of the transgenic Arabidopsis plants overexpressed the CAPIP1 gene according to the present invention.

Figure 16 shows the results of the resistance test for drying of transgenic Arabidopsis plants overexpressed the CAPIP1 gene according to the present invention.

The present invention relates to a base sequence of pepper gene (CAPIP1) induced by a pathogen, and to a method for searching for plant disease resistance and environmental stress using the same, and more particularly, pepper varieties ( Capsicum) known to have resistance to pepper bacterial spot disease. annuum L. cv. Hanbyul) isolates and sequencing the CAPIP1 gene, a new gene associated with resistance induced by pathogens, to provide its sequencing and amino acid sequences, and to use this gene to provide biological, chemical and physical resistance. The present invention relates to a method for searching for plant disease resistance and environmental stress for searching for expression of induction resistance during induction treatment.

Pepper, which is a representative eggplant with tobacco and tomatoes, is a representative crop that has been used as a spice or spice for a long time in our country. Viruses, pepper disease, fungal disease, and the like have been reported in various ways, among the deadly ones, including deadly diseases caused by fungi, anthrax caused by fungi, and bacterial spot diseases caused by bacteria.

Among them, the pepper bacterial spot pattern causes spots on the leaves, stems, and fruits of peppers to induce deciduous leaves and reduce the yield. It is commonly called dermis disease, spot bacteria, etc. Considering that, the damage is serious.

Until now, chemical control methods using pesticides have been mainly used to control pepper bacterial spot diseases. Due to its toxicity, there has been a harmful effect that destroys ecosystems and contaminates soil and water quality by killing not only pathogens but also living organisms. .

Therefore, in the future, it may be desirable to use a method of reducing pesticide use as much as possible by appropriately mixing physical and biological control measures in consideration of the environment and conditions in which plants are located, and more preferably, resistance to disease. Finding a way to suppress the disease itself through breeding and breeding of varieties.

In fact, as part of efforts to reduce the damage caused by pests without using pesticides, programs to cultivate disease-resistant crops for various crops and pathogens including pepper have been steadily progressed. For practical applications, research on resistance mechanisms in disease resistant plants has also been carried out on various crops and model plants, including pepper.

Once a plant is infected, the host plant activates several intracellular metabolic systems and initiates a defense mechanism, which divides the reaction between host plants and phytopathogens into friendly and incompatible reactions at the beginning of pathogen infection. It is determined whether or not it can limit the progress of the disease.

In an incompatible reaction, the infected plant recognizes the invasion of the pathogen by its own cognitive action, against which hypersensitive reactions such as local cell death in the cells of the infected site, accumulation of callose, lignin It shows the reaction of thickening of the cell wall by the formation of a cell, and produces various kinds of antimicrobial substances such as phytoalexin and produces a protective protein called pathogenesis related (PR) protein. It is known.

These defense mechanisms are reported not only when invading pathogens, but also by artificial chemical stimuli such as ethylene and methyl jasmonate, and by environmental stress such as physical wounds, salinity and temperature.

This resistance induced by artificial biological / physical / environmental stress is generally referred to as induction resistance, which is evaluated as a signaling pathway that plays an important role in the plant's ability to defend against pathogens.

This induction resistance is of interest and practical value as a model for signal transduction, and by understanding the biochemical changes that cause resistance, it is possible to develop genetically engineered plants that enhance disease resistance or to develop plant resistance genetic mechanisms. It can be used to develop plant protection chemicals that work to promote it.

In recent years, the induction resistance research has been much more accelerated by the intervention of genetic engineering techniques. In the past, resistance induction treatment has been performed to determine whether resistance is shown after cultivating plants and inoculating bottles to check for expression of resistance. While it was necessary to confirm whether or not the gene coding for the resistance-related protein to be induced now can be used to confirm the expression of resistance at the laboratory level, crop cultivation, field inoculation, This is because procedures such as checking for the appearance of lesions can be omitted.

This means that by shortening the cycle of the overall resistance induction test, more tests can be performed in a shorter time than in the past, which means that better resistance varieties can be developed.

However, this is possible on the premise that the gene sequence to be induced is identified and thus the corresponding gene clone is secured. In this sense, it is an urgent challenge to accumulate genetic information of various disease-producing proteins (PR proteins) that are resistant to plant diseases, thereby facilitating the search for plant disease resistance using the same and further promoting the development of plant disease resistant varieties. can do.

The present invention has been made to solve the above-mentioned problems in the prior art, an object of the present invention is to participate in the protective response to pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ) CAPIP1 which is one of the plant disease resistance related genes By separating and deciphering the pepper gene (CAPIP1) induced by a pathogen encoding a protein, the base sequence and the amino acid sequence encoded thereby are provided.

Another object of the present invention is to provide a method for detecting plant disease resistance and environmental stress using the CAPIP1 gene to search for resistance to pathogens, chemicals and environmental stresses such as pepper bacterial spot pattern disease.

Another object of the present invention is to provide a recombinant vector comprising the CAPIP1 gene and a plant transformed thereby.

The object of the present invention is to isolate the mRNA from the pepper leaf infected by inoculating pepper bacterial spot disease ( Xanthomonas campestris pv. Vesicatoria ) in Capsicum annuum L. cv. Hanbyul and then prepared cDNA library After screening, CAPIP1 was selected and its nucleotide sequence was determined, and pepper was inoculated or treated with any one of pathogens, chemicals, and environmental stresses, and was achieved by examining the expression of the CAPIP1 gene.

The present invention also provides an amino acid sequence encoded by the CAPIP1 gene and a recombinant vector comprising the gene and a plant transformed thereby.

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

First, the present invention is to prepare a cDNA library by separating the mRNA from the red pepper varieties exhibiting hypersensitivity reactions that are resistant to the non-pathogenic strain inoculation of pepper bacterium bacillus bacteria ( Xanthomonas campestris pv.vesicatoria ), pepper plants inoculated with non-pathogenic strains Total mRNA obtained from leaves and healthy red pepper plants without inoculation was labeled with dioxygenin to make a probe, and differential hybridization was performed using the cDNA and the probe for disease resistance. A novel plant disease resistant gene base derived from a red pepper varietal named CAPIP1 by selecting a gene suspected to be involved in obtaining a clone, selecting a CAPIP1 gene and sequencing the nucleotide sequence among the clones. Sequence (SEQ ID NO: 1) and the amino acid sequence encoded by it (SEQ ID NO: 2) (See FIG. 1).

Next, the present invention provides a method for detecting plant disease resistance consisting of confirming the expression of CAPIP1 protein using the CAPIP1 protein gene as a probe for a selected organ in a pepper individual inoculated with a pathogenic strain or a non-pathogenic strain. .

In addition, the present invention, by applying a non-biological induction method other than pathogenic bacteria such as chemicals, physical wounds, environmental stress to the plant, and by using the resistance screening method to check whether the expression of CAPIP1 protein, it is easier It provides a new resistive induction search method that can save time.

Furthermore, the present invention provides a transgenic plant overexpressing the CAPIP1 gene according to the present invention by using a recombinant vector comprising the CAPIP1 gene, and generates environmental stress resistance such as plant disease resistance and high salt and drying of the transgenic plant. By checking whether or not, a plant having new plant disease resistance and environmental stress resistance is provided.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are for illustrating the present invention, the scope of the present invention is not limited by the following examples.

Example 1 nucleotide sequence and amino acid sequence determination of CAPIP1 gene

The following tests were performed to provide the CAPIP1 protein coding gene sequence, which is one of the disease-related proteins for pepper bacterial spot disease, and the amino acid sequence encoded by it.

Capsicum annuum cv.Hanbyul was inoculated with BV5-4a ( Xanthomonas campestris pv.vesicatoria strain BV5-4a), which is a non-pathogenic bacterium of red pepper bacterial spot pattern disease. The leaves were removed from the wet room.

Then, cDNA library was prepared by extracting mRNA from the collected leaf samples, reverse transcripting, and amplifying it with PCR (Polymerase Chain Reaction). Then, individual cDNA clones were prepared from the cDNA library thus prepared and adsorbed on a nylon membrane (Hybond N +), and then the leaves of red pepper plants inoculated with non-pathogenic strains of red pepper bacterial spots, respectively, and healthy red pepper plants not inoculated. The total mRNA extracted from the leaves of was labeled with dioxygenin (dioxygenin) to make a probe, and then differential hybridization was performed.

As a result of hybridization, the genes amplified intensively for mRNA probes of non-pathogenic strain inoculated plants were assumed to be genes related to pathogenic resistance, and clones were obtained.

After sequencing the obtained clones, the CAPIP1 protein coding gene was identified and named CAPIP1 gene by using the blast program to compare its 5'-terminal sequence with a gene database registered in GenBank. CAPIP1 protein acts as a resistance to various pathogens in addition to pepper bacilli, and has a molecular weight of 21.134 kD.

The amino acid sequence inferred from this gene was compared with the amino acid sequence of a protein whose function was not found in Arabidopsis, showing 72% homology. The nucleotide sequence of the identified CAPIP1 gene and the amino acid sequence encoded by the nucleotide sequence are shown in FIG. 1 and summarized by SEQ ID NO: 1 and SEQ ID NO: 2.

Example 2 Induced Expression of CAPIP1 Gene in Pepper Plants by Pathogens

Using the CAPIP1 gene obtained in Example 1 was tested as follows to determine whether the disease resistance can be searched.

In order to specifically set the resistance screening method, Northern Blotting test was performed as follows, using pathogenic strains and non-pathogenic strains of pepper bacterial spot pattern disease, which is most likely to cause resistance.

First, after incubating the pathogenic strain Ds1, which shows a friendly reaction with the plant, and the nonpathogenic strain Bv5-4a, which shows an incompatible reaction, the back of the four-leaf pepper plants 1 and 2 at a concentration of 10 ⁸ cfu / ml. Infiltrated by infiltration using a syringe, and inoculated, and wet-treated at 28 ° C. and 100% relative humidity for 18 hours.

0.5, 1, 2, 6, 12, 18, 24 hours after inoculation, 1, 2 leaves were sampled to separate RNA, and the isolated RNA was electrophoresed on a gel containing formaldehyde, followed by a nylon membrane (Hybond N +). Was transferred.

Next, the CAPIP1 gene obtained in Example 1 was digested with restriction enzymes Eco RI and Xho I, respectively, and only inserted DNA was recovered and labeled with ³² P. Then, the reaction solution [0.25M phosphate buffer, 7% SDS, 1 mM EDTA , 5% Dextran Sulfate] was incubated at 65 ° C. for 16-24 hours to perform hybridization.

The ³² P-labeled DNA probe is attached to the mRNA attached to the nylon membrane, and if the X-ray film is placed on the nylon membrane, the ^32- P labeled DNA photosensitizes the X-ray film. I could recognize it.

By monitoring the amount of ribosomal RNA during the Northern blotting it was confirmed that the same amount of RNA sample was loaded.

As shown in FIG. 2, the expression of the CAPIP1 gene was shown in a friendly reaction after inoculation of pathogenic strains of red pepper bacillus bacteria and incompatible reactions after inoculation with non-pathogenic strains.

As a result, the expression of CAPIP1 gene was similar in both pathogenic and non-pathogenic strains. In the friendly response of pathogenic strains and pepper plants, the CAPIP1 gene was expressed from 30 minutes after inoculation, the amount was most observed after 12 hours, and decreased thereafter, and 30 minutes after incompatibility with non-pathogenic strains. Although the amount was increased after 12 hours and showed a tendency to decrease thereafter, the amount was found to be high in incompatible reactions.

Example 3: Induced Expression of the CAPIP1 Gene by Chemical Derivatives

In order to find out whether the CAPIP1 gene according to the present invention can be induced by chemical derivatives commonly used for resistance induction and to provide a specific induction method, the following test was performed.

Experimental Example 1.

First, to search for derivatives useful for inducing expression of the CAPIP1 gene, ethylene, methyl jasmonate, salicylic acid, BHT, β-aminobutyric acid and hydrogen peroxide and folic acid among chemicals known to be involved in the induction of plant disease resistance expression Resistance induction test was conducted.

Six-leaf pepper plants were treated with 100 μM methyl jasmonate, 5 mM salicylic acid, 20 μM BHT CGA245704, and 20 mM β-aminobutyric acid sprayed on the front and back of the pepper leaves with a double methyl jasmonate treatment. One plant was sealed with a plastic bag. Folic acid was treated by dipping the roots of plants in 100 μM aqueous folic acid solution and spraying the same aqueous solution on pepper leaves. Hydrogen peroxide was treated by the method of vacuum-infiltration with 10 mM hydrogen peroxide on pepper leaves. The concentration was 5 μl / L of silver and treated in a gaseous state into a sealed glass container with plants.

After 18 hours of treatment, the leaves of each plant were harvested, and the leaves of untreated plants were collected together after 18 hours, RNA was extracted from each sample and Northern blotting using the extracted RNA as follows. Was performed.

First, the extracted RNA was electrophoresed on a gel containing formaldehyde and then transferred to a nylon membrane (Hybond N +).

Next, the CAPIP1 gene obtained in Example 1 was digested with restriction enzymes Eco RI and Xho I, respectively, and only inserted DNA was recovered and labeled with ³² P. Then, the reaction solution [0.25M phosphate buffer, 7% SDS, 1 mM EDTA , 5% Dextran Sulfate], and hybridization was performed by incubating at 65-16 hours.

At this time, the DNA probe labeled with ³² P is attached to the complementary mRNA attached to the nylon membrane, and if the X-ray film is placed on the nylon membrane, the ³² P labeled DNA is exposed to the X-ray film, and thus whether it is expressed. You can recognize.

As a result of the test, for most substances, the CAPIP1 gene was strongly induced. Based on these results, for ethylene, methyl jasmonate, salicylic acid, BHT, β-aminobutyric acid, and hydrogen peroxide, which strongly induced the expression of the CAPIP1 gene, each inducer was examined to examine the detailed expression of the CAPIP1 gene. The following test was performed to process the gene expression and observe the gene expression pattern over time.

In each test, it was confirmed that the same amount of RNA sample was loaded by monitoring the amount of ribosomal RNA when performing Northern blotting, and the dry leaf without any material was used as a control.

Experimental Example 2.

In order to investigate the effect of CAPIP1 resistance on a timely basis after ethylene treatment, ethylene was treated by injecting it into a closed glass container containing a plant at a gas concentration of 10 μl / L, and 0.5, 1, 2, 6, 12, The leaves were harvested after 18 and 24 hours.

After extracting RNA from each sample, by performing Northern blotting in the same manner as in Experimental Example 1, the expression pattern of the CAPIP1 gene after ethylene treatment was examined over time, and the results are shown in FIG.

As a result of the test, as shown in FIG. 3, the CAPIP1 gene began to accumulate after 2 hours of treatment, expressed the most at 18 hours, and maintained the expression amount up to 24 hours.

Experimental Example 3.

In order to examine the effect of CAPIP1 resistance on the hour after treatment of methyl jasmonate, 100 μM concentration of methyl jasmonate was sprayed on the front and back of pepper leaves evenly with a sprayer and sealed with a plastic bag, respectively, 0.5, 1 The leaves were harvested after 2, 6, 12, 18 and 24 hours.

After extracting RNA from each sample, Northern blotting was performed in the same manner as in Experimental Example 1, and the time-dependent expression patterns of the CAPIP1 gene after methyl jasmonate treatment were examined, and the results are shown in FIG. 4. It was.

As a result of the test, as shown in FIG. 4, the CAPIP1 gene began to accumulate from 1 hour after treatment and was maintained for 24 hours.

Experimental Example 4.

In order to investigate the effect of CAPIP1 resistance after the treatment of salicylic acid, 5 mM salicylic acid was evenly sprayed on the front and back of the leaves of red pepper plants, respectively, 0.5, 1, 2, 6, 12, 18, 24 hours, respectively. The leaves were then harvested.

After extracting RNA from each sample, by performing northern blotting in the same manner as in Experimental Example 1, the time-dependent expression of CAPIP1 after salicylic acid treatment was examined, the results are shown in FIG.

As a result of the test, as shown in FIG. 5, the CAPIP1 gene began to accumulate after 6 hours of treatment and remained up to 24 hours.

Experimental Example 5.

In order to investigate the effect of CAPIP1 resistance on the basis of the treatment after the treatment of BHT, 10 μM concentration of BHT was evenly sprayed on the front and back of the leaves of pepper plants, respectively, 0.5, 1, 2, 6, 12, 18, 24, respectively. After hours the leaves were harvested.

After extracting RNA from each sample, by performing Northern blotting in the same manner as in Experimental Example 1, the time-dependent expression pattern of CAPIP1 after the BT treatment was examined, the results are shown in FIG.

As a result of the test, as shown in FIG. 6, the CAPIP1 gene began to accumulate after 18 hours of treatment and remained until 24 hours later.

Experimental Example 6.

In order to determine the effect of CAPIP1 resistance after treatment with β-aminobutyric acid, 20-mM concentration of β-aminobutyric acid was evenly sprayed on the front and back of the leaves of red pepper plants, respectively, 0.5, 1, 2, 6, 12 After 18, 24 hours, the leaves were harvested.

After extracting RNA from each sample, Northern blotting was performed in the same manner as in Experimental Example 1, and the expression pattern of CAPIP1 after β-aminobutyric acid treatment was examined. The results are shown in FIG. 7.

As a result of the test, as shown in FIG. 7, the CAPIP1 gene began to accumulate after 6 hours of treatment, accumulated the most after 1 hour, and was maintained up to 24 hours.

Experimental Example 7.

In order to investigate the effect of CAPIP1 resistance on a timely basis after the treatment of hydrogen peroxide, 10 mM hydrogen peroxide was treated on the leaves of pepper plants by vaccinating method, and the leaves after 0.5, 1, 2, 6, 12, 18 and 24 hours, respectively. Was collected.

After extracting RNA from each sample, by performing northern blotting in the same manner as in Experimental Example 1, the time-dependent expression of CAPIP1 after hydrogen peroxide treatment was examined, and the results are shown in FIG. 8.

As a result of the test, as shown in FIG. 8, the CAPIP1 gene was maintained from 18 hours after treatment to 24 hours after accumulation.

Example 4. Induced Expression of CAPIP1 Gene by Environmental Stress

In order to find out whether the CAPIP1 gene according to the present invention can be induced by environmental stress and to provide a specific induction method, the following test was performed.

In each test, it was confirmed that the same amount of RNA sample was loaded by monitoring the amount of ribosomal RNA during Northern blotting, and the dry leaf without any stress was used as a control.

Experimental Example 1.

In order to find out whether the CAPIP1 gene of the present invention can be induced by wound stress and to provide a specific induction method, the following test was performed.

In order to handle the wound stress on the red pepper plants, the leaves of the plants were pricked with needles and wounded throughout the leaves, and the leaves were collected after 0.5, 1, 2, 6, 12, 18 and 24 hours, respectively.

After extracting RNA from each sample, by performing Northern blotting in the same manner as in Experiment 1 of Example 3, the expression pattern of CAPIP1 over time after wound stress treatment was examined, and the results are shown. 9 is shown.

As a result of the test, as shown in FIG. 9, the CAPIP1 gene began to be expressed after 2 hours of treatment and remained up to 24 hours.

Experimental Example 2.

In order to find out whether the CAPIP1 gene of the present invention can be induced by salt stress and to provide a specific induction method, the following test was performed.

In order to investigate the effect of CAPIP1 resistance on a timely basis after treatment with sodium chloride, the salts were treated by soaking the roots of red pepper plants in 200 mM aqueous sodium chloride solution, and then 0.5, 1, 2, 6, 12, 18, and 24 hours later, respectively. The leaves were harvested.

After extracting RNA from each sample, by performing Northern blotting in the same manner as in Experiment 1 of Example 3, the time-dependent expression pattern of CAPIP1 after salt stress treatment, the results are shown in Figure 10 It was.

As a result of the test, as shown in FIG. 10, the CAPIP1 gene was expressed after 2 hours in leaves, and its expression was maintained up to 24 hours.

Experimental Example 3.

In order to find out whether the CAPIP1 gene of the present invention can be induced by cold stress and to provide a specific induction method, the following test was performed.

The leaves were harvested after 0.5, 1, 2, 6, 12, 18, and 24 hours, respectively, while the pepper plants were stored in a refrigerator at 5 ° C. for low temperature treatment.

After extracting RNA from each sample, by performing Northern blotting in the same manner as in Experiment 1 of Example 3, the expression pattern of CAPIP1 over time after the cold stress treatment, and the results are shown in Figure 11 Shown in

As shown in FIG. 11, the CAPIP1 gene was first expressed in the leaves after 6 hours of treatment and maintained until 24 hours.

Example 5 Induction of Resistance of Plants by Overexpression of CAPIP1 Gene

In order to determine whether induction of other disease-related genes, increased resistance to bacterial disease, and increased resistance to environmental stress during overexpression of the CAPIP1 gene in plants, the CAPIP1 gene obtained in Example 1 was used. Recombinant vector pCAPIP1 was prepared by introducing into pBIN35S vector prepared by Stratagene, and a cleavage map thereof is shown in FIG. 12.

The recombinant vector pCAPIP1 thus prepared was introduced into Agrobacterium tumefaciense strain EHA105 through electroporation, and the EHA105 strain into which the recombinant vector was introduced was subjected to a floral dipping method. By overexpressing the CAPIP1 gene by introducing into the Arabidopsis, the following test was performed.

Experimental Example 1.

In order to determine whether resistance to bacterial disease was increased in plants overexpressing the CAPIP1 gene, Pseudomonas syringe-Pasova tomato DC3000 strain was inoculated into the Arabidopsis plants with the CAPIP1 gene overexpressed and wild-type Arabidopsis plants as controls. The leaves were harvested and 3 days after the inoculation, and the number of bacteria per gram was measured. The results are shown in FIG. 13. Where # 7, # 8 and # 9 represent the sample numbers of the plants used in the test.

Test results showed that after 0, 1, 3, 5 days all samples were resistant to bacterial disease compared to wild type.

Experimental Example 2.

In order to determine whether the resistance to environmental stress was increased in plants overexpressing the CAPIP1 gene, the germination rate was obtained by germinating seeds of overexpressed CAPIP1 gene and wild type plants in solid MS medium containing sodium chloride or mannitol. Was measured and the result is shown in FIG.

As a result, plants overexpressing the CAPIP1 gene of the present invention were found to be resistant to sodium chloride and mannitol.

Experimental Example 3.

In order to determine whether the resistance to environmental stress was increased in the plants overexpressed by the CAPIP1 gene, the plants overexpressed the CAPIP1 gene were measured under sodium chloride or dry conditions, and the results are shown in FIGS. 15 and 16.

As a result, the plants overexpressed the CAPIP1 gene of the present invention was found to be resistant to sodium chloride and dry conditions.

In FIG. 15 and FIG. 16, the first column of the vertical column is a wild type plant, and the second column, the third column, and the fourth column represent plant types transformed by the present invention represented by # 7, # 8, and # 9 sample numbers, respectively. In addition, the first row plants in Fig. 15 are treated with 200 mM sodium chloride, the second row plants are treated with 300 mM sodium chloride, and the third row plants are treated with 400 mM sodium chloride. Figure 16 shows the state of the plants 12 days after the drying treatment.

As described above, the present invention isolates the CAPIP1 gene, one of the genes related to plant disease resistance, from the red pepper varieties showing resistance to red pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ) to determine the nucleotide sequence thereof By providing the amino acid sequence, it is possible to achieve the effect of being used as a genetic material for labeling the defense reaction against plant pathogens and molecular breeding of plant disease resistant crops.

In addition, the present invention provides a method for determining the defense response of plants by plant pathogens, chemicals and environmental stress consisting of confirming the differential expression of the CAPIP1 gene plant plant industry It is a very useful invention.

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Claims (9)

Pathogen-derived pepper gene (CAPIP1) of SEQ ID NO: 1 isolated from Capsicum annuum L. cv. Hanbyul. A peptide having the amino acid sequence of SEQ ID NO: 2 encoded by the CAPIP1 gene according to claim 1. A recombinant vector pCAPIP1 prepared by introducing the CAPIP1 gene of claim 1 into a pBIN35S 'vector. A bacterium transformed by the gene of claim 1 or the recombinant vector of claim 3. A transformed Arabidopsis comprising the gene of claim 1. delete In the plant disease resistance and environmental stress screening method, RNA was isolated from pepper plants inoculated with pathogenic and non-pathogenic bacteria of red pepper bacterial spot disease, and electrophoresed and transferred to a nylon membrane (Hybond N +), followed by CAPIP1 gene probe according to claim 1 treated with ³² P. Plant disease resistance and environmental stress screening method comprising the reaction to confirm the differential expression of the CAPIP1 gene. In the plant disease resistance and environmental stress screening method, RNA was isolated from pepper plants treated with ethylene, methyl jasmonate, salicylic acid, BH, β-aminobutyric acid and hydrogen peroxide, electrophoresed and transferred to a nylon membrane (Hybond N +), followed by ³² P treatment. Plant disease resistance and environmental stress screening method comprising the reaction of the CAPIP1 gene probe described in claim 1 to confirm the differential expression of the CAPIP1 gene. In the plant disease resistance and environmental stress screening method, RNA was isolated from the red pepper plants treated with any of the wound stress, salt stress and cold stress, electrophoresed and transferred to a nylon membrane (Hybond N +), and then reacted with the CAPIP1 gene probe according to claim 1 treated with ³² P. Plant disease resistance and environmental stress screening method comprising the identification of differential expression of the CAPIP1 gene.

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