KR100639825B1 - 1 1 Pepper CAOXR1 gene derived from Capsicum annuum L. cv. Hanbyul and probing method of plant disease resistance using the same - Google Patents

Abstract

A novel CAOXR1 gene is provided to promote detection of plant disease resistance and development of resistant species by providing a method for determining the defense reaction of plants caused by plant pathogenic bacteria and chemical materials. The CAOXR1 gene encoding plant disease resistance protein derived from Capsicum annum L. cv. Hanbyul includes a nucleotide sequence described as SEQ ID : NO. 1. The method for detecting plant disease resistance comprises the steps of: (a) after isolating RNA from pepper plant inoculated or treated with pathogenic bacteria or chemical materials and electrophoresing the isolated RNA, transitioning it to hybond N+; and (b) reacting the RNA of the step(a) with the CAOXR1 gene probe treated with P^32 to identify differential expression of the CAOXR1.

Description

Oxyredoxetase gene 1 (CAOOR1) derived from Korean red pepper varieties and a method for detecting plant disease resistance using the same {Pepper CAOXR1 gene derived from Capsicum annuum L. cv. Hanbyul and probing method of plant disease resistance using the same}

Figure 1 shows the nucleotide sequence and amino acid sequence of the CAOXR1 gene cloned from Capsicum annuum L. cv. Hanbyul according to the present invention.

Figure 2 Expression of CAOXR1 gene according to the present invention in the time to hanbyeol pepper cultivars inoculated with the virulent strain hayeoteul Ds1 and pepper jeommunuibyeong bacterial strains of non-pathogenic strains Bv5-4a (Xanthomonas campestris pv. Vesicatoria) , non-inoculated healthy and blades Induced results are shown.

Figure 3 shows the results of expression of the CAOXR1 gene according to the present invention according to the time elapsed after the treatment of pepper varieties ethylene, methyl jasmonate, salicylic acid.

Figure 4 shows the expression induction of the CAOXR1 gene according to the present invention according to the elapsed time after the treatment of hydrogen peroxide in the varieties of pepper.

Figure 5 shows the expression induction of the CAOXR1 gene according to the present invention according to the elapsed time after the treatment of methyl viologen in the red pepper varieties.

6 is a cleavage map of the recombinant vector pCAOXR1 into which the CAOXR1 gene is inserted according to the present invention.

Figure 7 shows the results of the pathogenicity test against the bacterial pathogen Pseudomonas Syringe Pasova tomato DC3000 strain in the transgenic Arabidopsis plants overexpressed the CAOXR1 gene according to the present invention.

The present invention relates to a CAOXR1 gene derived from red pepper varieties, and a method for detecting plant disease resistance using the same, and more specifically, from a red pepper varietal ( Capsicum annuum L. cv. Hanbyul), which is known to have resistance to red pepper bacterial spot disease. Screening for plant disease resistance that provides a base sequence and an amino acid sequence by isolating and sequencing the CAOXR1 gene, a new gene related to resistance, and searching for expression of induced resistance during bio / chemical resistance induction. It is about providing a method.

Pepper, which is a representative eggplant with tobacco and tomatoes, is a representative crop that has been used as a spice or seasoning in Korea for a long time. 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 red pepper to induce deciduous leaves and reduce the yield. It is commonly called dermis disease and spot germs, and the red pepper occupies the domestic crops. Considering this, 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 is advisable 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, resistant varieties that are resistant to disease. Finding a way to suppress the disease itself by improving and breeding.

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 calluses, lignin It is a reaction to thickening of cell walls due to the formation of cells, and it 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 corresponding gene clone is secured accordingly. In this sense, it is an urgent challenge to accumulate genetic information of various disease-producing protein (PR protein) that is resistant to plant diseases, thereby facilitating the search for plant disease resistance using the same and further promoting the development of disease resistant varieties of plants. It can be said.

The present invention has been made to solve the above problems of the prior art, an object of the present invention is to participate in the defense response to pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ) which is one of the genes related to disease resistance of plants The CAOXR1 gene encoding the CAOXR1 protein is isolated and translated to provide the nucleotide sequence and the amino acid sequence encoded thereby.

Another object of the present invention is to provide a method for detecting plant disease resistance using the CAOXR1 gene to search for resistance to pathogens or chemicals such as red pepper bacterial spot pattern disease.

Still another object of the present invention is to provide a recombinant vector comprising the CAOXR1 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 Screening was performed to select CAOXR1, and then its nucleotide sequence was determined, and the pepper was inoculated or treated with pathogens or chemicals to achieve the expression of the CAOXR1 gene.

The present invention also provides an amino acid sequence encoded by the CAOXR1 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 The total mRNA obtained from the leaves and the inoculated whole red pepper plants was labeled with dioxygenin to make a probe, and differential hybridization was performed using the cDNA and the probe. A novel plant disease resistance gene derived from a red pepper varietal named CAOXR1 by selecting a gene suspected to be involved in resistance to obtain a clone, selecting the CAOXR1 gene and sequencing its sequence Nucleotide sequence (SEQ ID NO: 1) and the amino acid sequence encoded by it (SEQ ID NO: 2) (See FIG. 1).

Next, the present invention, by using the CAOXR1 protein gene as a probe for a selected organ in a pepper individual inoculated with a pathogenic strain or a non-pathogenic strain to provide a method for detecting disease resistance of the plant consisting of confirming the expression of the CAOXR1 protein. do.

In addition, the present invention, by applying a chemical, and by using the resistance detection method to check whether the expression of the CAOXR1 protein, provides a new resistance induction detection method that can be easier and save time.

Furthermore, the present invention uses a recombinant vector comprising the CAOXR1 gene to prepare a transgenic plant overexpressing the CAOXR1 gene according to the present invention, and to determine whether the transgenic plant to produce disease resistance, having a new disease resistance Provide plants.

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

Example 1: Determination of the base sequence and amino acid sequence of the CAOXR1 gene

The following tests were performed to provide the CAOXR1 protein coding gene sequencing, which is one of the pathogenesis 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 clones obtained, the blast program was used to compare the 5'-terminal sequence with a gene database registered in GenBank to confirm that it is a CAOXR1 protein coding gene, a kind of oxidoredodase, and to the CAOXR1 gene. Named it. The CAOXR1 protein acts as a resistance to various pathogens in addition to pepper bacilli, and has a molecular weight of 40.76992 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 CAOXR1 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 CAOXR1 Gene in Pepper Plants by Pathogens

Using the CAOXR1 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.

After 2, 6, 12 and 24 hours of inoculation, the leaves were sampled to separate RNA, and the isolated RNA was electrophoresed on a gel containing formaldehyde and then transferred to a nylon membrane (Hybond N +).

Next, the CAOXR1 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 CAOXR1 gene in the friendly response after inoculation of pathogenic strains of red pepper bacillus bacteria and incompatible reactions after inoculation with non-pathogenic strains is shown in FIG. 2.

As a result of the above test, it was confirmed that the CAOXR1 gene can be induced by the pathogenic strain and the non-pathogenic strain. However, each expression appeared in a different pattern. That is, in the friendly reaction between the pathogenic strain and the pepper plant, the accumulation of the CAOXR1 gene was observed to increase from 6 hours to 24 hours after the inoculation of the bacterium. The amount increased until later and disappeared afterwards.

Example 3: Induced Expression of CAOXR1 Gene by Chemical Derivatives

In order to find out whether the CAOXR1 gene according to the present invention can be induced by chemical derivatives commonly used for resistance induction and to provide the specific induction method, the following test was performed.

Experimental Example 1.

First, in order to search for derivatives useful for inducing expression of the CAOXR1 gene, resistance induction tests were conducted using ethylene, methyl jasmonate, salicylic acid, hydrogen peroxide and methylbiologen among chemicals known to be involved in the induction of plant disease resistant expression. Was carried out.

Six-leaf pepper plants were treated with 100 μM of methyl jasmonate, 5 mM salicylic acid, 100 μM of hydrogen peroxide, and 100 μM of methylbiologen, sprayed on the front and back of pepper leaves with a double methyl jasmonate. Sealed with a plastic bag. Ethylene was treated at a concentration of 5 μl / L by injection into a closed glass container with plants in a gaseous state.

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 CAOXR1 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, the CAOXR1 gene was strongly induced and expressed in most cases. Based on these results, for ethylene, methyl jasmonate, salicylic acid, hydrogen peroxide and methylbiologen, which strongly induced the expression of the CAOXR1 gene, each inducer was treated and analyzed over time to examine the detailed expression of the CAOXR1 gene. The following test was performed to observe the gene expression pattern.

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.

Ethylene, methyljasmonate, salicylic acid, hydrogen peroxide, methylbiologen treatment to determine the effect of inducing CAOXR1 resistance over time, by treating the ethylene in a gaseous state in a closed glass container with a concentration of 10μl / L Then, 100 μM of methyl jasmonate, 5 mM of salicylic acid, 100 μM of hydrogen peroxide, and 100 μM of methylbiologen were sprayed onto the front and back of the pepper leaves with a sprayer. Plants treated with double methyl jasmonate were sealed with plastic bags. Each plant was harvested after 2, 5, 10, 20 hours.

After extracting RNA from each sample, Northern blotting was performed in the same manner as in Experimental Example 1, and then the expression patterns of CAOXR1 gene after ethylene, methyljasmonate, and salicylic acid treatment were examined. 3 is shown.

As a result of the test, as shown in Fig. 3, the CAOXR1 gene began to accumulate after 2 hours of treatment and its expression amount was maintained up to 24 hours.

Experimental Example 3.

In order to investigate the effect of CAOXR1 resistance induction after treatment with hydrogen peroxide, 100 μM of hydrogen peroxide was evenly sprayed on the front and back of the leaves of red pepper plants, followed by 15 minutes, 30 minutes, 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 time-dependent expression of the CAOXR1 gene after folic acid treatment was examined, the results are shown in FIG.

As a result of the test, as shown in FIG. 4, the CAOXR1 gene began to accumulate from 18 hours after treatment and decreased in expression at 24 hours.

Experimental Example 4.

In order to investigate the effect of CAOXR1 resistance induction after treatment with methylbiologen, 100μM concentration of methylbiologen was sprayed on the front and back of the leaves of pepper plants evenly by spraying, and then 1, 3, 6, 12 and 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 the CAOXR1 gene after folic acid treatment was examined, the results are shown in FIG.

As shown in FIG. 5, the CAOXR1 gene began to accumulate from 6 hours after treatment, peaked at 12 hours, and decreased in expression at 24 hours.

Example 4 Induction of Plant Resistance by CAOXR1 Gene Overexpression

In order to determine whether induces other disease-related genes and increases resistance to bacterial diseases when the CAOXR1 gene is overexpressed in plants, the CAOXR1 gene obtained in Example 1 was introduced into a pBIN35S vector prepared by the reagent company Stratagene. Recombinant vector pCAOXR1 was prepared and a cleavage map is shown in FIG. 6.

The recombinant vector pCAOXR1 thus prepared was introduced into Agrobacterium tumefaciense strain EHA105 by electroporation, and the EHA105 strain having the recombinant vector introduced therein was subjected to a floral dipping method. By overexpressing the CAOXR1 gene by introducing into the Arabidopsis, the following test was performed.

Experimental Example 1.

In order to determine whether the resistance to bacterial disease was increased in plants overexpressing the CAOXR1 gene, Pseudomonas syringe-Pasova tomato DC3000 strain was inoculated into the Arabidopsis plants with the CAOXR1 gene overexpressed and as a control. After harvesting and 5 days after inoculation, the leaves were collected and the number of bacteria per gram was measured. The results are shown in FIG. 7. Where # 1, # 3 and # 4 represent the sample numbers of the plants used in the test.

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

As described above, the present invention isolates the CAOXR1 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 its base sequence and is encoded by By providing the amino acid sequence, it is possible to achieve the effect of being used as a genetic material for labeling the protective response against plant pathogens and molecular breeding of disease-resistant crops.

In addition, the present invention is very useful in the plant breeding industry because it provides an effect of promoting the development of resistant varieties by providing a method for determining the defense response of plants by plant pathogens, chemicals consisting of confirming the differential expression of the CAOXR1 gene It is an invention.

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Claims (6)

An isolated gene CAOXR1 having a nucleotide sequence of SEQ ID NO: 1 encoding a plant disease resistant protein from Capsicum annuum L. cv. Hanbyul. A peptide having the amino acid sequence of SEQ ID NO: 2 encoded by the gene CAOXR1 of claim 1. A recombinant vector pCAOXR1 prepared by inserting the CAOXR1 gene according to claim 1 into pBIN35S. In the disease resistance detection method of plants, RNA was isolated from pepper plants inoculated or treated with pathogens or chemicals, electrophoresed and transferred to a nylon membrane (Hybond N +), and then reacted with the CAOXR1 gene probe of claim 1 treated with ³² P to discriminate the CAOXR1 gene. Comprising the identification of the expression, comprising a disease resistance screening method of the plant. The method of claim 4, wherein The pathogen is characterized in that the pathogenic or non-pathogenic bacteria of red pepper bacterial spot pattern disease, disease resistance detection method of the plant. The method of claim 4, wherein Wherein the chemical is characterized in that any one of ethylene, methyl jasmonate, salicylic acid, hydrogen peroxide and methylbiologen, plant disease resistance detection method.

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