KR100794469B1 - Pepper CaPMEI1 Gene coding a pectin methylesterase inhibitor protein and screening method of plant disease and environmental stress resistance - Google Patents

Abstract

The present invention relates to a CaPMEI1 gene encoding a pectin methyl esterase inhibitor protein, a novel resistance gene specifically induced when infected with pathogenic and non-pathogenic bacteria of red pepper bacterial bacterium ( Xanthomonas campestris pv.vesicatoria ), a bacterial disease of red pepper plants. In addition, the present invention relates to a disease resistance and environmental stress resistance search method using the gene and the production of resistant transformed plants. According to the present invention described above, it is possible to provide a genetic material for labeling the defense reaction of plant pathogens and molecular breeding of environmental stress-resistant plants, and facilitate the search for plant disease resistance.

Pepper, Plant Disease, Resistant, Bacterial Spot Disease, Pectin Methylesterase

Description

 Pepper CaPMEI1 Gene coding a pectin methylesterase inhibitor protein and screening method of plant disease and environmental stress resistance}

1 is a red pepper varieties ( Capsicum annuum L. cv. Hanbyul) is a diagram showing the nucleotide sequence and amino acid sequence of the CaPMEI1 gene according to the invention cloned from,

Figure 2 is a view showing the expression results of CaPMEI1 gene according to the present invention in each organ of the red pepper varieties,

Figure 3 is a pepper bacterial spot pattern bacteria of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a in a special breed of pepper ( Xanthomonas campestris pv. vesicatoria ) is a diagram showing the results of induction of CaPMEI1 gene expression according to the invention in healthy and uninoculated leaves when inoculated,

Figure 4 is a diagram showing the expression induction results of CaPMEI1 gene according to the present invention after treatment with salicylic acid in a special breed of pepper,

5 is a view showing the expression induction results of CaPMEI1 gene according to the present invention after treatment with ethylene in a special variety of pepper, hourly,

6 is a view showing the expression induction results of CaPMEI1 gene according to the present invention after treatment with methyl jasmonate in a special breed of pepper,

7 is a view showing the expression induction results of CaPMEI1 gene according to the present invention after the treatment of abscis acid in a special breed of pepper according to the present invention,

8 is a diagram showing the expression induction results of CaPMEI1 gene according to the present invention after treatment of the wound stress in a special breed of pepper,

9 is a view showing the expression induction results of CaPMEI1 gene according to the present invention after treatment of low-temperature stress in a red pepper varieties,

10 is a diagram showing the expression induction results of CaPMEI1 gene according to the present invention after treatment of dry stress in the red pepper varieties varieties,

11 is a view showing the expression induction results of CaPMEI1 gene according to the present invention after treatment with free radicals in a special breed of pepper,

12 is a vector map of the recombinant vector pET32a :: CaPMEI1 into which the CaPMEI1 gene is inserted according to the present invention;

Figure 13 is a diagram showing the result of purified CaPMEI1 protein in E. coli protein expressed CaPMEI1 gene according to the present invention,

14 is a graph showing the pectin methyl esterase inhibitory activity of CaPMEI1 protein according to the present invention,

15 is a photograph showing the antimicrobial activity of the CaPMEI1 protein according to the present invention against wilt, black rot, gray fungus,

16 is a photograph showing that spore germination of the withered pathogen treated with CaPMEI1 protein according to the present invention is suppressed,

17 is a graph showing that the spore germination rate and the mycelial growth rate of withered germ treated with CaPMEI1 protein according to the present invention are suppressed,

18 is a vector map of the recombinant vector pBIN35S :: CaPMEI1 :: GFP containing the CaPMEI1 gene according to the present invention;

19 is a view showing the results of observing the overexpression of CaPMEI1 gene in the transgenic Arabidopsis plants introduced CaPMEI1 gene according to the present invention,

20 is a photograph of a plant showing disease resistance to a strain of Pseudomonas syringae pv. ,

FIG. 21 is a graph showing the results of pathogenicity test against strains of the strain Monastirs Syringe Pasova tomato DC3000, a bacterial E. coli pathogen, in a transgenic Arabidopsis plant overexpressing the CaPMEI1 gene according to the present invention.

22 is a graph showing the results of resistance assays for 200 mM (millimoles) and 600 mM mannitol of transgenic Arabidopsis plant seeds overexpressing the CaPMEI1 gene according to the present invention.

23 is a graph showing the results of a test for resistance to mannitol of seedling root growth of transgenic Arabidopsis plants overexpressed CaPMEI1 gene according to the present invention,

24 is a photograph showing the results of verifying the survival rate after drying the transgenic Arabidopsis plants and non-transformed control plants overexpressing the CaPMEI1 gene according to the present invention without watering for 14 days,

25 is a graph showing the difference in transpiration rate between the transgenic Arabidopsis plants overexpressing the CaPMEI1 gene and the control plants not overexpressing according to the present invention.

FIG. 26 is a graph showing an increase in germination rate of plants transformed with CaPMEI1 gene according to the present invention in MS medium containing 5 μM (micromolar) concentration and 10 μM concentration of methyl violgen.

27 is a result of quantifying resistance by treating methyl violgen and measuring the content of chlorophyll on the leaves of transgenic Arabidopsis plants overexpressing the CaPMEI1 gene and control plants not overexpressing according to the present invention. Is a graph showing

28 is a photograph showing the results of resistance assay for methyl violgen of seedling growth of transgenic Arabidopsis plants overexpressed CaPMEI1 gene according to the present invention,

FIG. 29 is a graph showing the results of quantifying the resistance by measuring the biomass in the results shown in 28 degrees.

The present invention relates to a pepper gene CaPMEI1 encoding a pectin methyl esterase inhibitor protein, a method for searching for plant disease resistance and environmental stress resistance, and the production of pathogens and environmental stress resistant transformed plants using the same. More specifically, known hanbyeol pepper varieties that have resistance to bacterial jeommunuibyeong pepper (Capsicum annuum L. cv. Hanbyul) isolates and sequencing the CaPMEI1 gene, a new resistance-related gene, and provides the sequencing and amino acid sequences of the genes obtained therefrom, thereby determining whether CaPMEI1 expression of resistance-related genes is expressed in bio / chemical / physical resistance induction treatment. It provides a resistance search method for searching, and relates to the production of recombinant protein using the same and to the production of transformed plants having resistance to pathogens and environmental stress.

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 Korea, and various diseases such as viral disease, bacterial disease, and fungal disease have been reported. Examples include late blight caused by fungi, anthrax caused by fungi, and bacterial spot disease caused by bacteria.

Red pepper bacterial spot disease forms spots on the leaves, stems and fruits of red pepper to cause deciduous leaves and decreases yields. It is commonly called dermatophyte and spot bacteria, and the red pepper accounts for the share of Korean crops. The damage can be said to be 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. It can be said that it is good to find a way to suppress the disease itself through improvement and breeding.

In fact, as part of efforts to reduce the damage caused by pests without using pesticides, programs to cultivate disease-resistant crops have been steadily undertaken for various crops and pathogens including peppers. For the application, studies on resistance mechanisms in disease resistant plants have been conducted on various crops and model plants including pepper.

Once infected with a plant disease, the host plant activates several intracellular metabolic systems and initiates a defense mechanism, which determines the response between the host plant and phytopathogens in the early stages of pathogen infection into friendly and incompatible reactions. This determines whether or not the progress of the disease can be limited.

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 area, accumulation of callose, lignin ( lignin), which is responsible for cell wall thickening, and produces various kinds of antimicrobial substances such as phytoalexin and 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 stimulation such as ethylene and methyljasmonate, 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, induction resistance research has been much faster by the intervention of genetic engineering techniques. In the past, resistance induction treatment has been performed to determine whether resistance is shown after planting and inoculation of plants to check for expression of resistance. While it was necessary to confirm whether or not the gene encoding the resistance-related protein to be induced now can be used to confirm resistance expression 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 a clone of the gene is secured. In this sense, by accumulating the genetic information of various PR proteins that are resistant to plant diseases, In this regard, it is an urgent task to promote the development of plant disease resistant varieties and to promote the development of plant disease resistant varieties.

The present invention is to solve such a problem in the prior art, the purpose of which is pepper bacterial spot pattern ( Xanthomonas campestris pv. vesicatoria ) is involved in the defense response to the CaPMEI1 gene coding for CaPMEI1 protein, one of the genes related to plant disease resistance is isolated and decoded to provide its sequence information and amino acid sequence information accordingly.

In addition, the present invention is to provide a resistance search method for searching for the presence of plant disease resistance and environmental stress resistance such as pepper bacterial spot pattern disease using the CaPMEI1 gene. Furthermore, the object is to produce a plant disease or environmental stress-resistant transgenic plant using the gene.

The above object of the present invention, pepper varieties known to have resistance to bacterial bacterial spot pattern Capsicum annuum cv. Hanbyul) was isolated from the mRNA and then prepared a cDNA library screened to screen the gene of the new resistance-related gene CaPMEI1 and to determine its sequencing, inoculated or treated with pepper pathogens or chemicals of the CaPMEI1 gene It was achieved by examining the expression.

Therefore, the present invention, red pepper varieties ( Capsicum annuum cv. A novel plant disease resistance related protein gene CaPMEI1 having the nucleotide sequence of SEQ ID NO: 1 isolated from Hanbyul) and a protein having the amino acid sequence of SEQ ID NO: 2 encoded by the gene is provided.

The protein is an inhibitory protein that inhibits pectin methylesterase, which is a major enzyme used by plant pathogens to infect plants, and has an antimicrobial activity against phytopathogens.

In another aspect, the present invention provides a recombinant vector and a transformed plant prepared using the gene.

Furthermore, the present invention pathogens, chemicals and the environment after separating the RNA of any one of the stress from the inoculation or a pepper plant treatment was electrophoresed and transferred to nylon membrane (Hybond N +), claim 1, wherein the substrate treated with the ³² P It provides a method for detecting disease resistance and environmental stress resistance of plants, including reacting with CaPMEI1 gene probe to confirm differential expression of CaPMEI1 gene.

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

First, the present invention is pepper bacterium bacillus bacterium ( Xanthomonas campestris pv. vesicatoria ) isolated cDNA libraries from isolates of Korean red peppers which showed hypersensitivity reactions to the non-pathogenic strains of vesicatoria . Probes are labeled with digoxygenin, and differential hybridization is performed using these cDNAs and probes to select genes suspected to be involved in pathogenicity to obtain clones. By sequencing the nucleotide sequence of the gene identified as CaPMEI1 gene in this clone, it was named CaPMEI1 and assigned SEQ ID NO: 1 (see Fig. 1). In another aspect, the present invention provides an amino acid sequence (SEQ ID NO: 2) encoded by the nucleotide sequence of the CaPMEI1 gene according to the present invention (see Fig. 1).

Capsicum Star Varieties ( Capsicum annuum cv. The CaPMEI1 gene isolated from Hanbyul) encodes the CaPMEI1 protein, a type of PR protein that has a protective mechanism against pepper bacterial spot disease. It was observed that the amount was rapidly increased and maintained (see FIG. 3).

It was confirmed through experiments that the CaPMEI1 gene according to the present invention is differentially induced and expressed by various phytopathogens, chemicals or environmental stresses (see FIGS. 3 to 11). Therefore, the CaPMEI1 gene according to the present invention can be used for the disease resistance and environmental stress resistance screening method of plants.

More specifically, the plant disease resistance and environmental stress screening method according to the present invention is isolated from the plant inoculated or treated with pathogens, chemicals or environmental stress, RNA is subjected to electrophoresis and transferred to nylon membrane, and then treated with ³² P And reacting with the CaPMEI1 gene probe to confirm differential expression of the CaPMEI1 gene.

On the other hand, CaPMEI1 protein according to the present invention was confirmed to be a pectin methyl esterase inhibitor protein (see Fig. 14). Pectin methylesterase is a major enzyme used by plant pathogens to infect plants, and very few pectin methylesterase inhibitory proteins with corresponding antibacterial activity have been reported. The antimicrobial activity of CaPMEI1 protein according to the present invention was confirmed through an experiment (see FIGS. 15 to 17).

In addition, the present invention can produce a recombinant vector using a CaPMEI1 gene provided with the base sequence as described above to transform plants, thereby providing a material for resistant molecular breeding through increased disease resistance and environmental stress resistance.

More specifically, the CaPMEI1 gene according to the present invention was transferred to Arabidopsis plants using Agrobacterium to prepare a CaPMEI1 overexpressed transgenic plant, which confirmed plant disease resistance and generation of environmental stress resistance such as active oxygen and drying. (See FIGS. 19-29).

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

Example 1 Base / Amino Acid Sequence Determination of CaPMEI1 Gene according to the Present Invention

The following tests were performed to provide CaPMEI1 protein coding gene sequences and amino acid sequences inferred therefrom, one of the proteins involved in resistance to pepper bacterial spot disease.

Capsicum annuum cv.Hanbyul is a non-pathogenic bacterium of red pepper bacterial spot pattern disease ( Xanthomonas campestris pv. After inoculation with vesicatoria strain BV5-4a) and wet treatment for 18 hours, the plants showing the hypersensitivity reactions, which were resistant lesions, were taken out of the room and the leaves were collected.

Next, cDNA libraries were prepared by extracting mRNA from the collected leaf samples, reverse transcripting, and then amplifying the PCR (Polymerase Chain Reaction).

Next, individual cDNA clones were prepared from the cDNA library thus prepared and adsorbed to the nylon membrane uniformly, and then on the leaves of the red pepper plants inoculated with the non-pathogenic strains of the red pepper bacilli, and the healthy red pepper plants not inoculated. The extracted total mRNA was labeled with dioxygenin (digoxygenin) to make a probe, and then differential hybridization was performed.

As a result of hybridization, the genes amplified only for mRNA probes of plants inoculated with non-pathogenic strains were estimated to be genes related to pathogenic resistance, and clones were obtained.

After sequencing the clones obtained, the presence of the pectin methylesterase inhibitory protein domain was confirmed and named as CaPMEI1 gene by using a blast program to compare its 5'-terminal sequence with a gene database registered in GenBank. .

The amino acid sequences inferred from these genes were compared with those of the DC1.2 protein found in tobacco, Arabidopsis, and Radiata pine, the ripening related protein, and the enzyme (invertase). It shows that it is novel by showing 36% homology.

The nucleotide sequence of the identified CaPMEI1 gene and the amino acid sequence deduced therefrom are shown in FIG. 1 and represented by nucleotide sequence 1 and nucleotide sequence 2, respectively, and submitted as a sequence list.

Example 2 Induced Expression of CaPMEI1 Gene in Pepper Plants by Bacterial Pathogens

Experimental Example 1

First, to examine the expression of CaPMEI1 gene in healthy plants, RNA was isolated from healthy organs (leaves, stems, roots, flowers, green fruits, red fruits) of four-leaf pepper plants, and the isolated RNA was formed of gel containing formaldehyde. After electrophoresis in the phase it was transferred to the nylon membrane (Hybond N +).

Next, the CaPMEI1 gene obtained in Example 1 was digested with restriction enzymes Eco RI and Xho I, respectively, and only the inserted DNA was recovered and labeled with ³² P, which was then reacted with [0.25 M 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.

The results of the above experiments are shown in FIG. 2, and the CaPMEI1 gene shows only strong expression in stem and weak expression in root flower, but cannot be expressed in healthy organs.

Experimental Example 2

After incubating the pathogenic strain Ds1, which has a friendly reaction with plants, and the non-pathogenic strain Bv5-4a, which shows an incompatible reaction, the back of the four-leaf pepper plants 1 and 2 at a concentration of 5 X 10 ⁸ cfu / ml. Infiltrated (Infiltration) using a syringe in the inoculation, and was incubated for 18 hours at 28 ℃, 100% relative humidity conditions.

0.5, 1, 2, 6, 12, 18, 24 hours after inoculation, RNA was isolated by sampling 1 and 2 leaves, respectively, and the RNA was electrophoresed on a gel containing formaldehyde (Hybond N +). Was transferred.

Next, the CaPMEI1 gene obtained in Example 1 was digested with restriction enzymes Eco RI and Xho I, and the 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. 3, the expression of CaPMEI1 gene was shown in the friendly response after inoculation of pathogenic strains of red pepper bacillus bacteria and incompatible reactions after inoculation with non-pathogenic strains. 3 represents the elapsed time from inoculation to capsicum leaf collection.

As a result of the above test, the resistance of red pepper plants to red pepper bacterial spot disease was gradually increased from 1 hour to 18 hours in both pathogenic and non-pathogenic strains and decreased at 24 hours.

Example 3 CaPMEI1 Gene-Induced Expression of the Present Invention by Chemical Derivatives

In order to find out whether the CaPMEI1 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, in order to search for derivatives useful for inducing expression of the CaPMEI1 gene, resistance induction tests were performed using salicylic acid, ethylene, methyl jasmonate, and absic acid (folic acid) among chemicals known to be involved in the induction of plant disease resistance expression. Was carried out.

Six leaf pepper plants were treated with 5 mM salicylic acid, 100 μM methyl jasmonate, and 100 μM folic acid by spraying the leaves on the front and back of the pepper, and the double methyl jasmonate treated plants were sealed with plastic bags. . Ethylene was treated at a concentration of 5 μl / L by injection into a closed glass container with plants in a gaseous state.

Leaves were harvested from each plant after 1, 2, 6, 12, 18, 24 hours of treatment, and untreated plant leaves were harvested together at 2, 18 hours after control, and RNA was extracted from each sample. Northern blotting was performed using the extracted RNA as follows.

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

Next, the CaPMEI1 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, which was then reacted with [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA. , 5% Dextran Sulfate] and incubated at 65 ° C. for 16-24 hours to perform hybridization.

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 is expressed. You can recognize.

As a result of the test, CaPMEI1 gene was strongly induced and expressed in all of the treated materials as shown in FIGS. 3 to 7, but expression was strongly induced only for 1 hour and disappeared thereafter for the treatment of absic acid.

Example 4 CaPMEI1 Gene Induced Expression of the Present Invention by Environmental Stress

In order to find out whether the CaPMEI1 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 when performing Northern blotting, and the dry leaf without any stress was used as a control.

Experimental Example 1

In order to find out whether CaPMEI1 gene according to 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 pierced with needles and wounded throughout the leaves, and the leaves were collected after 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 CaPMEI1 over time after wound stress treatment was examined, and the results are shown. 8 is shown.

As a result of the test, as shown in FIG. 8, the CaPMEI1 gene began to be expressed within a short time after treatment and was maintained for 6 hours.

Experimental Example 2

In order to find out whether CaPMEI1 gene according to the present invention can be induced by low temperature stress and to provide a specific induction method, the following test was performed.

Pepper plants were harvested at 1, 2, 6, 12, 18 and 24 hours, respectively, while chilled plants were kept in a cold room maintained at 4 ° C.

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 CaPMEI1 over time after the cold stress treatment was examined, and the results are shown. 9 is shown.

As a result, as shown in Figure 9, CaMEI1 gene was first expressed in the leaves for 1 hour showed a tendency to maintain up to 20 hours.

Experimental Example 3

In order to find out whether the CaPMEI1 gene according to the present invention can be induced by dry stress and to provide a specific induction method, the following test was performed.

In order to determine the effect of CaPMEI1 resistance by time after drying, soil was removed from the roots of pepper plants, and leaves were collected at 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 time-dependent expression of CaPMEI1 after dry stress treatment was examined, the results are shown in Figure 10 It was.

As a result, as shown in Figure 10, CaPMEI1 gene was strongly expressed in the leaves for 1 hour.

Experimental Example 4

In order to find out whether the CaPMEI1 gene according to the present invention can be induced by hydrogen peroxide and to provide a specific induction method, the following test was performed.

In order to investigate the effect of hydrogen peroxide on the CaPMEI1 resistance over time, 10 mM hydrogen peroxide was sprayed on the leaves of pepper plants, and leaves were collected at 1, 2, 6, 12, 18 and 24 hours, respectively.

After extracting RNA from each sample, Northern blotting was carried out in the same manner as in Experiment 1 of Example 3, and after hydrogen peroxide treatment, the expression patterns of CaPMEI1 by time and concentration were examined. 11 is shown.

As a result of the test, as shown in FIG. 11, the CaPMEI1 gene was most strongly expressed at 1 hour after accumulation and maintained for 18 hours.

Example 5 CaPMEI1 Protein Production and Antimicrobial Activity Assay According to the Present Invention

In order to examine the function of CaPMEI1 protein, which is an expression product after overexpression of CaPMEI1 gene in E. coli, the CaPMEI1 gene obtained in Example 1 was introduced into a pET32a vector provided by Novagen to prepare a recombinant vector pET32a :: CaPMEI1 and map the map. 12 is shown.

The recombinant vector pET32a :: CaPMEI1 thus prepared was transferred to Escherichia coli. coli strain BL21) and purified only the CaPMEI1 protein using Ni-NTA resin among the proteins obtained by culturing E. coli containing the recombinant vector was carried out as follows.

Experimental Example 1

Thioredoxin protein tagged with the pET32a vector (tagged) is expressed together, and then reacted at room temperature with 1 unit of enterokinase (enterokinase) for 16 hours. The CaPMEI1 protein was identified using SDS-polyacrylamide gel electrophoresis prior to purification, after purification and after thioredoxin treatment. The recombinant protein was dissolved in 1 × SDS sample buffer and electrophoresed on 12% SDS-polyacrylamide gel. In order to know the exact size of the protein was assayed using a protein marker and the figure is shown in FIG.

Experimental Example 2

In order to confirm that CaPMEI1 protein has a pectin methylesterase inhibitory activity, it was reacted with an actual pectin methylesterase. The acidity was quantified by a standard curve indicating the amount of NADH as a product after the reaction at 7.8 and 25 degrees, and the results are shown in FIG. 14.

In the reaction with purified recombinant protein, NADH content was found to be lower than that of the control treated with only pectin methylesterase, and the reaction could be measured within 12 minutes. As shown in FIG. 14, the CaPMEI1 protein according to the present invention was confirmed to have a pectin methyl esterase inhibitory effect through this experiment.

Experimental Example 3

To investigate the antimicrobial activity of CaPMEI1 protein, Fusarium , a major pathogen of plants oxysporum f. sp . matthiolae ) , Black pattern germ ( Alternaria brassicicola ), Bacterial fungus ( Botrytis cinerea ) was treated with CaPMEI1 protein by concentration. Antimicrobial activity was shown against the tested pathogens treated at a concentration of 50 μg, which is shown in FIG. 15. In particular, spore germination and mycelial growth were decreased when CaPMEI1 protein was treated with wilts. The photos and graphs showing the results are shown in FIGS. 16 and 17, respectively.

Example 6 Preparation of Transgenic Plants Using CaPMEI1 Gene of the Present Invention and Induction of Disease Resistance of Plants by Overexpression of CaPMEI1 Gene

CaPMEI1 gene obtained in Example 1 was identified as pBIN35S in order to determine whether induction of other pathogenic genes, plant resistance, bacterial resistance, and environmental stress during overexpression of CaPMEI1 gene in plants. The recombinant vector pBIN35S :: CaPMEI1 :: GFP was prepared by introducing into the :: GFP vector and a map thereof is shown in FIG. 18.

The recombinant vector pBIN35S :: CaPMEI1 :: GFP thus prepared was converted to Agrobacterium tumephiens ( Agrobacterium). a test such as tumefaciense strain EHA105) electroporation (electroporation) introducing and dipped again flowers the EHA105 strain that contains the recombinant vector by the (floral dipping) using the following method was expressed over the CaPMEI1 gene by introducing the Arabidopsis thaliana in Was performed.

Experimental Example 1

In order to test the expression level of CaPMEI1 gene in the transgenic plants prepared as described above, Northern blotting was performed in the same manner as in Experiment 1 of Example 3, and as a result, three transgenic plants expressing much CaPMEI1 gene were obtained. A selection was made (see FIG. 19).

Experimental Example 2

To investigate whether the resistance to bacterial diseases was increased in plants overexpressed CaPMEI1 gene, the strains of Pseudomonas syringe-Pasova tomato DC3000 were inoculated on the Arabidopsis plants over-expressed CaPMEI1 gene and control. The leaves were harvested after 2 days and 4 days of inoculation, and the number of bacteria per leaf area was measured. The results are shown in FIG. 21. At this time, the plant lesion photo is shown in FIG. 20. Where # 6, # 8 and # 9 represent the line number of the transgenic plant used for the test.

As a result, as shown in Figure 21, after 0, 2, 4 days all transgenic plants have been shown to have bacterial resistance to bacterial disease by inhibiting bacterial growth compared to wild type.

Example 7 Induction of Environmental Stress Resistance of Plants by Overexpression of CaPMEI1 Gene of the Present Invention

In order to determine whether the resistance to environmental stress is increased in the plants overexpressed CaPMEI1 gene according to the present invention, the results obtained by treatment of dry stress such as mannitol treatment of plants overexpressed CaPMEI1 gene is shown in Figure 22 to 25 and shown in FIGS. 26 to 29 show the resistance to methylbiologen (oxidative stress).

As a result of the test, the plants overexpressed CaPMEI1 gene of the present invention was resistant to dry stress and methylbiologen (oxidative stress) treatment.

As described above, the present invention is completed, pepper bacterial spot disease ( Xanthomonas campestris pv. vesicatoria ), which is involved in the defensive response to vesicatoria, isolates and decodes the CaPMEI1 gene encoding CaPMEI1 protein, one of the genes related to plant disease resistance, to provide sequencing information and amino acid sequence information thereof, and to differentially express CaPMEI1 gene. It is possible to provide a method for detecting plant disease resistance and environmental stress resistance, such as pepper bacterial spot pattern disease consisting of confirming. Furthermore, by using the CaPMEI1 gene according to the present invention will be able to contribute to plant disease resistant and environmental stress resistant plant breeding and transformed plant production.

Furthermore, by separating and functionally analyzing the CaPMEI1 protein according to the present invention, a method of searching for a disease resistance mechanism of a plant, including the analysis method and the antibacterial activity search method, can be provided.

Therefore, the present invention can be said to be a very useful invention in the plant breeding industry because there is an effect that can promote the development of resistant varieties.

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Claims (15)

Capsicum Star Varieties ( Capsicum annuum L. cv. Plant disease resistant protein gene CaPMEI1 having the nucleotide sequence of SEQ ID NO: 1 isolated from Hanbyul). A plant disease resistant protein having the amino acid sequence of SEQ ID NO: 2 encoded by the gene of claim 1. The protein of claim 2, wherein the protein is a pectin methylesterase inhibitor protein. The protein according to claim 2 or 3, wherein the protein has an antimicrobial activity against phytopathogens. A recombinant vector comprising the CaPMEI1 gene of claim 1. The recombinant vector pET32a :: CaPMEI1 prepared by inserting the CaPMEI1 gene into pET32a. The recombinant vector pBIN35S :: CaPMEI1 :: GFP prepared by inserting the CaPMEI1 gene into pBIN35S :: GFP. A plant transformed by the gene of claim 1 or the recombinant vector of claim 5. delete In the search for disease resistance and environmental stress resistance of plants, RNA was isolated from pepper plants inoculated with either pathogenic or non-pathogenic bacteria of red pepper bacterial spot disease, electrophoresed and transferred to a nylon membrane (Hybond N +), and then reacted with the CaPMEI1 gene probe of claim 1 treated with ³² P. To determine the differential expression of the CaPMEI1 gene comprising a plant, pathogen resistance and environmental stress resistance detection method. In the search for disease resistance and environmental stress resistance of plants, CaPMEI1 gene of salicylic acid, ethylene, methyl jasmonate, and then to remove the RNA from the pepper plant treated with any one of the folic acid was electrophoresed and transferred to nylon membrane (Hybond N +), claim 1, wherein the substrate treated with a ³² P probe Comprising the identification of differential expression of CaPMEI1 gene and reacting with, the disease resistance and environmental stress resistance detection method of the plant. In the search for disease resistance and environmental stress resistance of plants, CaPMEI1 according to claim 1, wherein RNA was isolated from the pepper plants treated with any one of wound stress, dry stress, low temperature stress and reactive oxygen stress, electrophoresed and transferred to a nylon membrane (Hybond N +), followed by ³² P treatment. A method for detecting disease resistance and environmental stress resistance, comprising reacting with a gene probe to confirm differential expression of CaPMEI1 gene. A method for analyzing the pectin methyl esterase inhibitory activity of the CaPMEI1 protein by introducing a recombinant vector according to claim 5 into E. coli and reacting with pectin methyl esterase. A method for analyzing the antimicrobial activity of the CaPMEI1 protein by introducing a recombinant vector according to claim 5 into E. coli and then treating the plant pathogen. A method for producing CaPMEI1 protein having antibacterial activity against phytopathogens by introducing a recombinant vector according to claim 5 into E. coli .

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