KR100796164B1 - Plant membrane receptor protein gene camrp1 from pepper and environmental stress resistant transgenic plants using the same - Google Patents

Abstract

A plant membrane receptor protein gene CaMRP1 derived from pepper is provided to confer resistance against environmental stresses including methyl jasmonate and high salt on plants by overexpressing the gene, so that the gene is useful for producing and screening transgenic plants having environmental stress resistance. A plant membrane receptor protein gene CaMRP1 derived from pepper(Capsicum annuum L. cv. Hanbyul) has the nucleotide sequence of SEQ ID NO:1, wherein the plant membrane receptor protein has the amino acid sequence of SEQ ID NO:2. An environmental stress resistant transgenic plant is produced by inserting the plant membrane receptor protein gene CaMRP1 into pBIN35S to prepare a recombinant vector, and transforming a plant such as Arabidopsis thaliana with the recombinant vector.

Description

Plant membrane receptor protein gene CaMRP1 from pepper and environmental stress resistant transgenic plants using the same}

1 is a red pepper varieties ( Capsicum) according to the present invention annuum L. cv. Cloning from one Hanbyul) CaMRP1 A diagram showing the nucleotide sequence of the gene (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) of the protein encoded by the gene,

Figure 2 is a diagram showing the expression results of CaMRP1 gene according to the present invention in each organ of the red pepper varieties,

Figure 3 shows the results of inducing the expression of CaMRP1 gene according to the present invention in healthy and non-inoculated leaves after inoculation of the pathogenic strain Ds1 and non-pathogenic strain Bv5-4a causing the bacterial bacterial spot disease in a special breed of pepper Is a diagram representing

Figure 4 is a diagram showing the expression results of CaMRP1 gene expression according to the present invention after the treatment of wound stress in a special breed of pepper,

5 is CaMRP1 according to the present invention A vector map of the recombinant vector pBIN35S :: CaMRP1 with the gene inserted therein,

6 is CaMRP1 according to the present invention CaMRP1 in Transgenic Arabidopsis Plants Overexpressing Genes A diagram showing the results of observing gene overexpression,

7 is CaMRP1 according to the present invention A diagram showing the results of a test for resistance to methyl jasmonate in seedling growth of transgenic Arabidopsis plants overexpressed genes,

8 is CaMRP1 according to the present invention This figure shows the results of quantifying resistance by treating methyl jasmonate and measuring the content of chlorophyll to seedlings of transgenic Arabidopsis plants overexpressing genes and control plants not overexpressing.

9 is CaMRP1 according to the present invention A diagram showing the results of resistance test for methyl jasmonate of seedling root growth of transgenic Arabidopsis plants overexpressed genes,

10 is CaMRP1 according to the present invention A diagram showing the results of a test for resistance to sodium chloride (NaCl) in seedling growth of transgenic Arabidopsis plants overexpressed genes,

11 is CaMRP1 according to the present invention This figure shows the result of quantifying resistance by treating sodium chloride (NaCl) and measuring the content of chlorophyll to seedlings of transgenic Arabidopsis plants overexpressing genes and control plants not overexpressing genes.

12 is CaMRP1 according to the present invention Transgenic Arabidopsis plants over-expressed genes and non-transformed control plants were treated with 400 mM sodium chloride to verify the survival rate against salt.

The present invention relates to a CaMRP1 gene, which is a specific induction-related gene for pepper bacterial spot disease, and a protein encoded by the gene sequence. In addition, the present invention relates to a plant disease and environmental stress resistance detection method using the gene and to the production of environmental stress resistant transformed plants. More specifically, the present invention is a red pepper varieties ( Capsicum) known to have resistance to red pepper bacterial spot pattern disease annuum L. cv. Hanbyul) isolated and sequencing the CaMRP1 gene, a novel resistance-related gene, to provide its sequencing and amino acid sequence, thereby leading to the pathogenesis-related gene CaMRP1 during bio / physical resistance induction. It provides a resistance search method for searching for expression and a transformed plant having resistance to environmental stress using the gene.

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. The causes of the disease are largely caused by biological factors such as viruses, bacteria, and fungi and external environmental stress. These include biological factors such as late blight caused by fungal bacteria, anthrax caused by fungi, bacterial spots caused by bacteria, and damage caused by abiotic factors such as drought and lack of water due to high salinity. Can be mentioned.

Red pepper bacterial spot disease forms spots on the leaves, stems, and fruits of red pepper to induce deciduous leaves and decreases yields. It is commonly called deodorant disease, spot germs, and the proportion of red pepper in Korean crops. In view of this, the damage is serious, and the increase in soil salinity due to pollution and the use of excessive fertilizers is a major factor in crop harvest. In particular, chemical control using pesticides to control pepper bacterial spot disease has been done, and its toxicity has been damaging ecosystems and polluting soil and water quality.

In the future, it may be desirable to use a method of reducing pesticide use as much as possible by appropriately mixing physical / biological control measures in consideration of the environment and conditions in which plants are located, and furthermore, disease resistance based on studies on resistance to disease and environmental stress. And it may be desirable to find a way to suppress the disease itself through improvement and breeding of environmentally resistant 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 being conducted. It has been studied for decades. To understand the disease resistance and flame resistance and its practical application, various crops and model plants have been applied.

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 disease progression 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 site, accumulation of callose, lignin It is a kind of cell wall thickening, 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 protective signaling mechanisms are transmitted not only by the protective protein but also by chemical substances produced in plants such as ethylene and methyl jasmonate, which are important signaling agents in response to environmental stress such as physical wounds, salinity and temperature. It is reported to appear.

In particular, proteins that control stress against high salt include polyamine, sugar alcohol, glycine-betaine, and proline, and channel proteins present in cell membranes. Or H + -ATPase is reported to be involved in osmotic pressure regulation. Although many proteins and genes encoding them, which play an important role in promoting flame resistance and signaling, have been actively studied, the pathways related to signal transduction systems and ion homeostasis in the field are still unknown.

This resistance, induced by substances transmitted by biological, physical, or environmental stresses to plants, is not only the ability of plants to defend against pathogens, but also their ability to remain constant in order to survive harmful environments. Contributes to the beginning of a systematic signaling pathway. These inductive resistances are interesting and of practical value as a model for signal transduction, as well as those that work to develop genetically engineered plants or promote plant resistance genetic mechanisms by understanding the biochemical changes that cause resistance and resistance. It can be used to develop protective chemicals. Eventually, these studies will lead to the development of more resistant and resistant varieties to achieve high quality, high yield targets.

Therefore, it is an urgent challenge to accumulate genetic information of various pathogenic related proteins (PR proteins) that are resistant to plant diseases, thereby facilitating a search method using the same.

The present invention is to solve the above problems of the prior art, the purpose of which is pepper bacterial spot disease ( Xanthomonas campestris pv. as involved in the defense response to vesicatoria) CaMRP1 coding for a protein of the CaMRP1 specific reaction related genes controlling plant diseases By separating and deciphering a gene, the nucleotide sequence information and the amino acid sequence information encoded by the gene are provided.

Another object of the present invention is to provide a transgenic plant using the gene.

It is also an object of the present invention to provide a plant disease and environmental stress resistance search method for plants to search for the presence of resistance to pathogens and environmental stress using the gene.

The above object of the present invention, red pepper varieties ( Capsicum) showed hypersensitivity reaction by inoculation of pepper bacterial spot pattern annuum cv. Hanbyul) isolated the mRNA and then prepared a cDNA library, screening it to select a new pathogenic gene CaMRP1 and determine the base sequence, and inoculated or treated with pepper pathogens or environmental stress to express the CaMRP1 gene Achieved by examining whether or not.

Therefore, the present invention is a pepper varieties known to have resistance to pepper bacterial spot pattern ( Chassium varieties) annuum cv. Cell membrane receptor-like protein gene CaMRP1 and the amino acid sequence of SEQ ID NO: 2 encoded by it, which is a new pathogenesis-related gene isolated from Hanbyul). .

The present invention also provides a method for detecting disease resistance and environmental stress resistance of a plant to search for expression of induction resistance during resistance induction treatment using the gene.

Furthermore, the present invention provides a recombinant vector and a transgenic plant using the gene.

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

First, the present invention, pepper bacterial spot pattern germ ( Xanthomonas campestris pv. vesicatoria ) was isolated from the red pepper cultivars that showed the hypersensitivity response to the non-pathogenic strain inoculation. Differential hybridization is performed to select genes suspected to be involved in disease resistance, obtain clones, and sequence sequences of genes identified as cell membrane receptor-like protein coding genes among these clones. Gene Named CaMRP1 and assigned SEQ ID NO: 1 (see FIG. 1).

The red pepper varieties ( Capsicum annuum cv. CaMRP1 isolated from Hanbyul) The gene encodes a cell membrane receptor-like protein, which is a kind of PR protein that has a defense against pepper bacterial bacterial spot disease.

The amino acid sequence (SEQ ID NO: 2) encoded by the nucleotide sequence of the gene is shown in FIG. 1 (see FIG. 1).

CaMRP1 of the present invention is a protein having a signal base sequence at the amino terminal end and a transmembrane helix structure at the carboxyl end, and is believed to be involved in the recognition of the host's early defense response when plant pathogens invade. In addition, the inventors of the present invention have produced a transgenic plant using the gene for the first time (see FIG. 6). It was confirmed to have a high level of resistance (see FIGS. 7-12).

Therefore, the present invention provides a pathogenic and environmental stress resistance screening method for checking whether the pathogenic strain or non-pathogenic strain, the induction or expression of the gene CaMRP1 represented by the nucleotide sequence of SEQ ID NO: 1 in plants inoculated or treated with environmental stress do.

In other words, plants resistant to disease show various defense reactions when invading pathogens, and the induced expression of pathogenic proteins can be regarded as molecular markers indicating plant resistance. It was confirmed through experiments that the pathogenic protein gene CaMRP1 according to the present invention is differentially induced and expressed by phytopathogens or environmental stresses (see FIGS. 3 and 4). Therefore, the gene CaMRP1 according to the present invention can be used in a method for searching for plant disease and environmental stress resistance of plants.

The phytopathogenic bacteria may be pathogenic bacteria or non-pathogenic bacteria of red pepper bacterial spot pattern disease, and the environmental stress is a wound.

Furthermore, the present invention can provide a material for resistant molecular breeding through an increase in resistance by preparing a recombinant vector and transforming Arabidopsis using the gene of CaMRP1 provided with the nucleotide sequence as described above. The Arabidopsis transformation method may use a method known in the art to which the present invention belongs. For example, a flower immersion method using Agrobacterium can be used.

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 Sequence and Amino Acid Sequence Determination of CaMRP1 Gene according to the Present Invention

The following tests were performed to provide the nucleotide sequence of CaMRP1 protein coding gene according to the present invention, which is one of the proteins specifically induced against red pepper bacterial spot disease, and the amino acid sequence deduced therefrom.

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

Then, cDNA library was prepared by extracting mRNA from the collected leaf specimens, reverse transcripting, and amplifying the PCR (Polymerase Chain Reaction).

Individual cDNA clones were prepared from the cDNA library thus prepared and adsorbed to nylon membranes, and then total mRNA extracted from the leaves of pepper plants inoculated with the non-pathogenic strains of the red pepper bacterial spot bacteria and the leaves of healthy pepper plants not inoculated Was labeled with dioxygenin (digoxygenin) to make a probe, and then differential hybridization was performed.

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

After sequencing the clones obtained, a blast sequence was used to compare the nucleotide sequence with a gene database registered in GenBank. A cell membrane receptor-like protein having a signal base sequence at the amino terminal and a transmembrane helix at the carboxyl terminal. Check that it is CaMRP1 Named gene (see Figure 1).

The amino acid sequence inferred from this gene is 66% homologous to the amino acid sequence of the tobacco membrane receptor protein of tobacco and 20-30% homology with that of rice or Arabidopsis receptor protein. (See Figure 1).

The base sequence of the identified CaMRP1 gene is represented by SEQ ID NO: 1, the amino acid sequence encoded by the CaMRP1 gene is represented by SEQ ID NO: 2 and shown in Figure 1 and submitted as a sequence list.

Example 2 CaMRP1 in Pepper Plants Caused by Bacterial Pathogens Induced Expression of Genes

[Step 1]

First, CaMRP1 in healthy plants To examine gene expression, RNA was isolated from healthy organs (leaves, stems, roots, flowers, green berries, red berries) of four-leaf pepper plants, and the RNA was electrophoresed on a gel containing formaldehyde. Transfer to nylon membrane (Hybond N +).

Next, CaMRP1 obtained in Example 1 The genes were digested with restriction enzymes Eco RI and Xho I, respectively, and the inserted DNA was recovered and labeled with ³² P. Then, the reaction solution [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% dextran sulfate (Dextran Sulfate) )] Was hybridized 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 experimental results are shown in Figure 2 CaMRP1 The gene could not find expression in all healthy organs.

[Step 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. Inoculated by injecting with a syringe, and inoculated in a wet state at 28 ° C. and 100% relative humidity for 18 hours.

After 1, 5, 10, 15, 20, and 25 hours of inoculation, RNA was isolated by sampling 1 and 2 leaves, respectively, and the transferred RNA was electrophoresed on a gel containing formaldehyde and then transferred to a nylon membrane (Hybond N +). I was.

Next, CaMRP1 obtained in Example 1 The genes were 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 (Dextran Sulfate) Incubation 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 processed.

As such, CaMRP1 was observed in friendly reactions after inoculation of pathogenic strains of red pepper bacterial bacillus and incompatible reactions after inoculation with non-pathogenic strains. Expression of the gene is shown in FIG. In Figure 3, the number represents the elapsed time from inoculation to pepper leaf collection.

As a result of the above test, as shown in FIG. 3, the resistance of pepper plants to pepper bacterial spot disease was gradually increased and decreased from 1 hour to 10 hours after the infection of both pathogenic and non-pathogenic strains. The expression for non-pathogenic bacteria was strong.

Example 3 CaMRP1 due to Environmental Stress Induced Expression of Genes

CaMRP1 of the invention In order to find out whether the gene 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 samples were processed by monitoring the amount of ribosomal RNA during Northern blotting, and the dry leaf without any stress was used as a control.

[Step 1]

CaMRP1 of the invention In order to find out whether the gene 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 0.25, 0.5, 1, 5, 10, 15, 20, and 25 hours, respectively.

After extracting RNA 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, CaMRP1 obtained in Example 1 The genes were digested with restriction enzymes Eco RI and Xho I, respectively, and only 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] ] 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.

By performing northern blotting as described above, the expression pattern of CaMRP1 over time after wound stress treatment was examined, and the results are shown in FIG. 4.

Test results, as shown in Figure 4, CaMRP1 The gene begins to express quickly after treatment and rapidly decreases within 1 hour.

Example 4 CaMRP1 Preparation of Transgenic Arabidopsis Using Genes

CaMRP1 in Plants CaMRP1 obtained in Example 1 to determine whether induction of other disease-related genes, response to bacterial disease and increased resistance to environmental stress during gene overexpression The recombinant vector pBIN35S :: CaMRP1 was produced by introducing the gene into the pBIN35S vector, and a map thereof is shown in FIG. 5.

Thus prepared recombinant vector pBIN35S :: CaMRP1 Agrobacterium tumefaciens ( Agrobacterium By introducing via electroporation (electroporation) methods to tumefaciense strain EHA105) introducing EHA105 strain back into the recombinant vector in Arabidopsis thaliana using the method flower dipping (floral dipping) and over-expressing CaMRP1 gene (see Fig. 6 The following test was carried out.

CaMRP1 in Transformed Plants In order to test the expression level of genes, the results of examining through ALTI-PSI (RT-PCR) are shown in FIG. 6. At this time, in the CaMRP1 gene it is overexpressed plants CaMRP1 Gene expression was observed, resulting in CaMRP1 Three transgenic plants expressing genes were selected. In order to evaluate the same RNA level, the gene AtUBQ having a constant expression level of Arabidopsis was used. The same amount of RNA was reacted by controlling the expression level of AtUBQ.

Example 5 CaMRP1 Induction of Environmental Stress Resistance in Plants by Gene Overexpression

CaMRP1 obtained in Example 4 To determine whether resistance to environmental stress was increased in plants overexpressed genes, CaMRP1 7, 8, and 9 show results obtained by treating stresses such as jasmonate treatment of plants overexpressed genes, and showing resistance by treating sodium chloride (salt stress). , 12 is shown.

Test result, CaMRP1 of the present invention Plants overexpressed genes have been shown to be resistant to stress from methyl jasmonate and sodium chloride treatment.

As described above, the present invention has been completed, red pepper bacterial spot pattern disease ( Xanthomonas campestris pv. as involved in the occurrence of the disease vesicatoria) CaMRP1 encoding a plant cell membrane receptor-like protein By separating and deciphering the gene to provide its sequence information and corresponding amino acid sequence information, CaMRP1 Providing a method for detecting resistance of plants by identifying the differential expression of genes, and the method and environment for the production of environmental stress resistant transgenic plants by confirming resistance to stress caused by methyl jasmonate and high salt treatment of plants overexpressed CaMRP1 gene It also provides a way to explore stress tolerance. In addition, by separating and functionally analyzing the CaMRP1 protein according to the present invention induced by the pathogen, a method of searching for a signaling mechanism for resistance to environmental stress, including the analysis method, can be provided.

Therefore, the present invention can achieve the effect that can be used as a genetic material for labeling the protective response to plant pathogens and environmental stress and molecular breeding of environmental stress resistant crops. Furthermore, the present invention is a very useful invention in the plant breeding industry because it has the effect of promoting the development of resistant varieties.

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Claims (9)

Capsicum Star Varieties ( Capsicum annuum L. cv. Hanbyul) cell membrane receptor-like protein gene CaMRP1 having the nucleotide sequence of SEQ ID NO: 1. A cell membrane receptor-like protein having the amino acid sequence of SEQ ID NO: 2 encoded by the gene of claim 1. A recombinant vector comprising the gene of claim 1. The recombinant vector of claim 3, wherein the recombinant vector is prepared by inserting the CaMRP1 gene into pBIN35S. A bacterium transformed by the gene of claim 1 or the recombinant vector of claim 3. Arabidopsis transformed by the gene of Claim 1 or the recombinant vector of Claim 3. 7. The Arabidopsis vulgaris of claim 6, wherein the transformed Arabidopsis is resistant to methyl jasmonate and sodium chloride. delete delete

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