KR101149109B1 - Disease resistance-related pepper RNA binding protein gene CaRBP1 and transgenic plants using the same - Google Patents

Abstract

The present invention relates to a novel disease resistance-related pepper RNA binding protein gene ( CaRBP1 : Capsicum annuum RNA binding protein 1 , SEQ ID NO: 1) and pathogenic transgenic plants using the same. The present invention also provides a method for detecting disease resistance of a plant using the gene. According to the present invention described above, it is possible to provide a genetic material for labeling the resistance reaction of plant pathogens and molecular breeding of disease-resistant plants, and facilitate the search for plant disease resistance.

Description

Disease resistance-related pepper RNA binding protein gene CaRBP1 and transgenic plants using the same}

The present invention relates to a pepper RNA binding protein gene ( CaRBP1 : Capsicum annuum RNA binding protein 1 ), which is a novel gene related to plant disease resistance, and to a method of searching for biological stress resistance using the same and a disease resistant transgenic plant. More specifically, the present invention relates to the CaRBP1 gene, which is a plant gene resistance-related new gene that is specifically induced when infected with a pathogenic strain and a non-pathogenic strain of pepper bacterial bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ), which is a bacterial disease of pepper plants. The present invention provides a nucleotide sequence and an amino acid sequence according thereto, and relates to a method for detecting disease resistance of a plant by using the same to identify differential expression of resistance-related gene CaRBP1 when inoculating pathogenic or non-pathogenic pathogens. In addition, the present invention inhibits the expression of the CaRBP1 gene using virus-induced gene silencing (VIGS) technology in pepper plants, or Agrobacterium-mediated transient expression of CaRBP1 gene in pepper plants ( Agrobacterium). Induced increased expression of the CaRBP1 gene by introducing it using a mediated transient expression method or by transforming it into Arabidopsis thaliana , a model plant, thereby confirming its role in increasing plant disease resistance. The present invention relates to providing a conversion plant.

Plants are invaded by various pathogens during their lifetime. Plants have various forms of effective defense mechanisms for normal growth and reproduction. These defense mechanisms are important tools for plant survival by enhancing plant resistance. Therefore, identifying mechanisms of defense in vivo that are not known so far not only plays an important role in increasing crop yield economically, but also enables scientific accumulation of new technologies. In particular, identification of the information and function of genes specifically induced in plant defense-related mechanisms can be an important resource for breeding new resistant crops.

As a representative eggplant crop, Capsicum annuum L. is native to South America and is widely grown in the tropics and temperate regions. Although it is not known exactly about the first introduction of red pepper, it has had a great influence on the diet after the introduction of red pepper. In particular, red pepper is one of the most cultivated horticultural crops in Korea and is a very important crop in the agricultural economy. Viruses, bacterial diseases, fungal diseases and the like have been reported variously in these pepper plants, and in fact, the decrease in production due to disease occurs frequently. However, no effective disease resistant crops have been reported to date and are dependent on chemical control for plant protection. Therefore, effective defense-related gene isolation and production of transgenic plants using biotechnological techniques that can compensate for the shortcomings of efforts to develop new resistant crops using traditional breeding are needed to increase farm income and reduce economies of production. It enables the production of high-quality clean crops that are suitable and contributes to the activation of various industries.

On the other hand, most plant genes exist as DNA, and these genes are structurally expressed according to their characteristics or expressed by external stimuli. At this time, the expression of the gene is made from the DNA sequence based on the transcription mechanism in vivo, and the corresponding RNA is made. Later, through the translation process of RNA, it is finally made into protein form and used in various biochemical metabolism in vivo.

In plants, metabolism occurs in vivo through the same mechanism as mentioned. RNA produced during the transcription process is regulated by various RNA binding proteins (RBPs) to control the translation of RNA and protein production. Several RNA binding proteins are synthesized during plant defense-related mechanisms, and these proteins interact with specific RNAs to regulate various defense-related mechanisms. Callose accumulation, increased free radicals, the production of antimicrobial agents such as phytoalexin, hypersensitivity reactions through local cell death, and defense-related protein production. By regulating the RNA produced during the transcription process, normal defense reactions can occur. GRP7, a representative RNA-binding protein known from Arabidopsis as a representative plant, has been reported in recent studies to increase resistance by interacting with effectors of bacterial Pseudomonas syringe pv. Tomato DC3000 .

However, little is known about the RNA-binding proteins induced during defensive reactions in pepper plants. Therefore, the research on RNA binding proteins that regulate defense response by regulating transcription in plants is a very popular field, especially the research on RNA binding proteins involved in plant disease resistance reaction and Demonstrating function plays an important role in understanding the pathogenic mechanisms of pepper plants and contributes to providing a major genetic source for the production of pathogenic transgenic plants.

The present invention is to solve such a prior art problem, the purpose of which is involved in the defensive reaction to pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ), one of the genes related to plant disease resistance pepper RNA binding protein ( By separating and decoding the CaRBP1 gene encoding RNA binding protein1), the base sequence information and amino acid sequence information thereof are provided.

In addition, an object of the present invention is to provide a method for detecting disease resistance of plants using the CaRBP1 gene to search for the presence or absence of resistance to plant diseases.

Furthermore, an object of the present invention is to prepare a transgenic plant having resistance to plant diseases by introducing the CaRBP1 gene into the plant.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

The object of the present invention is to identify a novel resistance-related gene CaRBP1 isolated from the green pepper varieties ( Capsicum annuum cv. Nockwang) that exhibits hypersensitivity resistance to the inoculation of non-pathogenic strains of pepper bacterial bacillus bacteria ( Xanthomonas campestris pv.vesicatoria ). The nucleotide sequence was determined and used to investigate the expression of CaRBP1 through pathogen treatment, and to confirm the plant disease resistance induction function of CaRBP1 gene in pepper plants and transgenic Arabidopsis thaliana overexpressing the gene. Achieved.

Accordingly, the present invention provides a plant disease resistance-related gene, (a) a gene encoding a protein having an amino acid sequence of SEQ ID NO: 2 encoding a pepper-derived RNA binding protein or (b) a gene having a nucleotide sequence of SEQ ID NO: 1 ( CARBP1 ) To provide.

The present invention also provides an isolated RNA binding protein (CaRBP1) having the amino acid sequence of SEQ ID NO: 2.

The present invention also provides a recombinant vector and a transformed plant prepared using the gene.

Furthermore, the present invention provides a method for searching for disease resistance of a plant, including the step of identifying RNA and differential expression of CaRBP1 gene by separating RNA from the plant.

The present invention provides a method of imparting disease resistance to a plant, comprising the step of transforming the plant using a recombinant vector comprising the CaRBP1 gene.

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

First, the present invention pepper bacterial jeommunuibyeong bacterium (Xanthomonas campestris pv. Vesicatoria) of a non-pathogenic strain Bv5-4a to separate the mRNA from nokgwang pepper cultivars showing resistance sensitization reactions to inoculation by making a cDNA library, and inoculated with non-pathogenic strains pepper The total mRNA obtained from the leaves of the plant and healthy red pepper plants not inoculated was labeled with digoxygenin to make a probe, followed by differential hybridization using the cDNA and the probe. Clones that were expected to be related to resistance were selected. By decrypting (Sequencing) The nucleotide sequence of the gene identified as RNA binding protein genes of these clones, peppers nokgwang variety of RNA binding protein (CaRBP1) coding gene sequence called CaRBP1 (SEQ ID NO: 1) and amino acid sequence encoded therefrom (SEQ ID NO: 2) is provided (see FIG. 1).

More specifically, after sequencing the obtained clones according to the present invention, using the Blast program to identify the CaRBP1 protein coding gene by comparing its 5'-terminal sequence with the gene database registered in GenBank and The RNA binding protein ( CaRBP1 ) gene was named. The CaRBP1 protein had an RNA recognition motif typical of RNA binding proteins as a result of comparing amino acid sequences. 87% homology with RNA-binding protein 47 gene (accession no. CAC_01238) in tobacco ( Nicotiana plumbaginifolia ) and 76% at protein level with DNA-binding protein gene (accession no. AAR91698) in tomato ( Lycopericum esculentum ) It can be seen that it is novel (http://blast.ncbi.nlm.nih.gov/Blast.cgi, nucleotide blast program). The gene nucleotide sequence of CaRBP1 identified and the amino acid sequence encoded by the nucleotide sequence are shown in FIG. 1. And to analyze whether the CaRBP1 gene is conserved in other plants was carried out phylogenetic tree (phylogenetic tree) was shown (Fig. 2).

CaRBP1 protein according to the present invention has been confirmed RNA-specific binding activity (see Figs. 3 and 4), located in the nucleus of plant cells (see Fig. 5).

Next, the present invention provides a method for detecting disease resistance of a plant, comprising the step of confirming differential expression of the CaRBP1 gene by separating RNA from the plant subjected to biological treatment. More specifically, the biological treatment may be pathogens such as pathogenic or non-pathogenic bacteria of pepper bacterial spot pattern disease. The CaRBP1 gene expression check may be used in addition to the Northern blot, real-time RT-PCR, microarray method, various methods for measuring the expression of mRNA can be used. It was confirmed by experiment that the CaRBP1 gene according to the present invention is differentially induced expression by phytopathogens (see Fig. 6). Therefore, the CaRBP1 gene according to the present invention can be usefully used for a method for detecting disease resistance of plants.

The present invention also produce resistant plant diseases by producing specific to the production, and Arabidopsis thaliana plants are CaRBP1 gene transgenic for the inhibition plant CaRBP1 gene from pepper plant with a recombinant vector comprising the above-CaRBP1 gene Checking whether it is possible to provide new pathogenic transgenic plants and materials for resistant molecular breeding. Therefore, the present invention also provides a method of imparting disease resistance to a plant, comprising the step of transforming the plant using a recombinant vector comprising the CaRBP1 gene.

More specifically, the present invention relates to a CaRBP1 gene using a TRV2 vector (Baulcombe DC (1999) using a Tobacco Rattle Virus (TRV), which is used for virus-induced gene silencing (VIGS). Fast forward genetics based on virus-induced gene silencing.Curr.Opin.Plant Biol. 2: 109-113) to produce a recombinant vector TRV2 :: CaRBP1 . The recombinant vector was introduced into the plant to prepare a VIGS plant in which CaRBP1 expression was suppressed, and it was confirmed that the disease susceptibility was increased (see FIGS. 9 to 13).

Furthermore, transgenic Arabidopsis plants expressing Agrobacterium- mediated transient expression of a recombinant vector (pBIN35S :: CaRBP1 ) containing the CaRBP1 gene according to the present invention, or overexpressing CaRBP1 according to the present invention. It was possible to confirm the increase in plant disease resistance by preparing (see FIGS. 7, 8, 14, and 15). Therefore, the resistance test for phytopathogens against pepper plants that disabled the function of the pepper RNA binding protein and Arabidopsis plants overexpressing the RNA binding protein according to the present invention was able to determine whether the disease resistance occurred.

As described above, the present invention can accumulate the genetic information of the response of the RNA-binding protein to the disease and its associated pathogenic protein (PR protein), thereby providing an interesting model for signaling of disease in plants. By understanding the biochemical changes that cause resistance, they can be used practically to develop biotechnological plants with enhanced disease resistance.

As described above, by completing the present invention, sequence information is obtained by isolating and deciphering the CaRBP1 gene, which encodes a novel RNA binding protein, which is a plant disease resistance-related gene, which is involved in the defense against pepper bacterial bacterial spot disease. And while providing the amino acid sequence information accordingly, using this gene can not only provide a disease resistance screening method of plants, but also contribute to the resistance plant breeding and production of transgenic plants.

Therefore, the present invention can provide a material for the breeding of disease-resistant plants, and thus has the effect of promoting the development of resistant varieties, which is a very useful invention for the plant breeding industry.

1 is a diagram showing the nucleotide sequence and amino acid sequence of the pepper RNA binding protein ( CaRBP1 ) gene cloned from Capsicum annnuum L. cv. Nockwang according to the present invention,
Figure 2 is a diagram showing the results of systematic analysis using the amino acid sequence of CaRBP1 protein of red pepper green mine varieties,
3 is a diagram showing the results of the CaRBP1 protein expressed in E. coli and purified using Ni-NTA agarose resin in order to measure the RNA binding activity of the CaRBP1 protein according to the present invention,
4 is a diagram measuring the RNA binding activity of CaRBP1 protein according to the present invention,
5 is a view showing the intracellular location of the CaRBP1 protein by fusion of the green fluorescent protein to CaRBP1 protein according to the present invention,
FIG. 6 is a diagram showing the differential expression results of CaRBP1 genes in inoculated and non-inoculated leaves when inoculated with pathogenic and non-pathogenic strains of pepper bactericidal bacterium ( Xanthomonas campestris pv.vesicatoria ) to red pepper varieties.
FIG. 7 shows that apoptosis is induced when CaRBP1 gene is overexpressed in red pepper plants using recombinant vector pBIN35S: CaRBP1 and Agrobacterium tumefaciens strain GV3101 strains, and CaRBP1 protein is overexpressed.
FIG. 8 shows the results of measuring hydrogen peroxide generation and electrolyte leakage due to apoptosis induced by using a recombinant vector pBIN35S: CaRBP1 and Agrobacterium tumefaciens strain GV3101.
9 is a pepper bacterial spot pattern bacterium of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a in a plant of the red pepper cultivation varieties whose CaRBP1 gene expression is suppressed using the method of virus-induced gene silencing (VIGS). Xanthomonas campestris pv.vesicatoria ) is a diagram showing the results of the symptoms and growth of bacteria when inoculated into leaf tissue,
10 is a pepper leaf tissue inoculated with the red pepper bacterium of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a ( Xanthomonas campestris pv. Vesicatoria ) to plants of the red pepper greenery varieties inhibited the expression of the CaRBP1 gene according to the present invention, Shows the results of measurement of hydrogen peroxide generation and electrolyte outflow in
Figure 11 is inoculated with the callus of bacterial pathogenic strains ( Xanthomonas campestris pv. Vesicatoria ) of the pathogenic strain Ds1 and non-pathogenic strain Bv5-4a to the plant of the red pepper greenery varieties inhibited the expression of the CaRBP1 gene according to the present invention, callose ( callose ) ) Is a diagram showing the results of observation of apoptosis due to accumulation and hypersensitivity reactions and reduction of free radical accumulation for the same,
Figure 12 shows the change in salicylic acid accumulation when inoculated with red pepper bacterium of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a ( Xanthomonas campestris pv. Vesicatoria ) to the plant of red pepper green mine varieties inhibited the expression of CaRBP1 gene according to the present invention Shows the result of measuring
Figure 13 shows the effective expression of CaRBP1 when inoculated with the red pepper bacterium of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a ( Xanthomonas campestris pv. Vesicatoria ) to the plant of the red pepper greenery varieties inhibited the expression of CaRBP1 gene according to the present invention Figure showing the results of differential expression of genes related to inhibition and resistance,
14 is a recombinant vector pBIN35S: CaRBP1 and Agrobacterium tumefaciens (Agrobacterium tumefaciens strain GV3101) with the strain, and the CaRBP1 gene over-expression in transgenic Arabidopsis thaliana plants which overexpress CaRBP1 gene, transformed Arabidopsis was overexpressing CaRBP1 gene A diagram showing the results of measuring pathogen growth in plants when inoculating Pseudomonas syringae pv. Tomato DC3000 strain, a bacterial virulent pathogen, from a giant plant.
FIG. 15 is a view showing the results of observing an increase in disease resistance against Hyaloperonospora parasitica Noco2 in transgenic Arabidopsis plants overexpressing the CaRBP1 gene according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1 CaRBP1  Base / Amino Acid Sequencing of Genes

The following tests were performed to provide RNA binding protein protein coding sequence and one amino acid sequence deduced therefrom, which is one of the pathogenic related proteins against red pepper bacterial spot disease.

Capsicum annuum cv.Nockwang was inoculated with red pepper bacterial bacterium ( Xanthomonas campestris pv.vesicatoria ), and after 18 hours of wet treatment, the plants showing resistance to apoptosis, which were resistant lesions, were taken out of the humidity chamber and the leaves were collected. .

Next, cDNA libraries were prepared by extracting mRNA from the leaf samples, reverse transcripting, and amplifying the polymerase chain reaction (PCR). The individual cDNA clones obtained from the cDNA library thus prepared were adsorbed on the nylon membrane (Hybond N +) in the same manner, and then the leaves of the red pepper plants inoculated with the non-pathogenic strains of the red pepper bacterial spot pattern bacteria and the leaves of the healthy red pepper plants. The total mRNA extracted at was labeled with dioxygenin (digoxygenin) to make a probe, and then differential hybridization was performed.

By analyzing the hybridization results, a gene specifically expressed by strongly amplifying the mRNA probe of the plant inoculated with the non-pathogenic strain was estimated as a gene related to pathogenic resistance, and a clone was obtained.

After sequencing the clones obtained, the blast program was used to identify the CaRBP1 protein coding gene by comparing its 5'-terminal sequence with a gene database registered in GenBank and named the RNA binding protein ( CaRBP1 ) gene. Analysis of the amino acid sequence of the CaRBP1 protein revealed an RNA recognition motif that is typical of RNA binding proteins in RNA binding proteins. 87% homology with RNA-binding protein 47 gene (accession no. CAC_01238) in tobacco ( Nicotiana plumbaginifolia ) and 76% at protein level with DNA-binding protein gene (accession no. AAR91698) in tomato ( Lycopericum esculentum ) It can be seen that it is novel (http://blast.ncbi.nlm.nih.gov/Blast.cgi, nucleotide blast program). The gene nucleotide sequence of CaRBP1 identified and the amino acid sequence encoded by the nucleotide sequence are shown in FIG. 1. In order to analyze whether CaRBP1 gene is conserved in other plants, phylogenetic analysis was performed and shown as a phylogenetic tree (FIG. 2).

Example 2 RNA binding activity measurement of CaRBP1 protein

In order to measure the RNA binding activity of the CaRBP1 protein obtained in Example 1 was tested as follows.

[Step 1]

In order to measure the RNA binding activity of CaRBP1 protein, CaRBP1 gene obtained in Example 1 was introduced into pET28a vector (Novagen) and fused with histidine (His-CaRBP1). This fusion protein was expressed using Isopropyl β-D-1-thiogalactopyranoside (IPTG) and then purified using Ni-NTA Agarose resin (Ni-NTA Agarose resin) provided by Invitrogen (Fig. 3). .

[Step 2]

The purified His-CaRBP1 protein was used to react with a ribohomopolymer resine to which each RNA base (poly U, polyG, poly G) was bound to measure RNA binding activity. As shown in Figure 4 CaRBP1 protein showed RNA binding activity. However, it did not show binding activity when reacted with ds DNA or ss DNA resine bound double or single helix DNA. RNA binding activity was measured by Western blot method using histidine antibody and GST antibody (GE Healthcare). GST protein was used as a control.

Example 3 CaRBP1 Protein Location in Plant Cells

The CaRBP1 gene obtained in Example 1 was tested as follows to confirm the position in plant cells.

[Step 1]

In order to confirm the expression position of CaRBP1 protein in plant cells, the green fluorescent (GFP) protein was fused (35S :: CaRBP1: smGFP) to the 3 ′ end of the CaRBP1 gene obtained in Example 1 (FIG. 5). The fusion protein was expressed in onion epidermal cells using a particle bombardment method and observed 24 hours later using a conforcal microscope. As shown in FIG. 5, CaRBP1 protein was observed to be located in the nucleus. In addition, DAPI (4 ', 6-diamidino-2-phenylindole) staining was performed to confirm the presence of CaRBP1 protein in the nucleus.

Example 4 In Red Pepper Plants by Pathogen Inoculation CaRBP1  Gene Expression Detection Method

In order to confirm the method of using the CaRBP1 gene obtained in Example 1 for the resistance search was tested as follows.

[Step 1]

First of all, in order to present a specific resistance screening method, the non-pathogenic strain Bv5-4a exhibiting an incompatible reaction with the pathogenic strain Ds1 of Xanthomonas campestris pv.vesicatoria , which has a friendly reaction with plants, was identified as YN (Yeast- Nutrient). after culturing in a culture medium, 10 ⁸ cfu / ㎖ concentration of 6 pepper nokgwang varieties of leaf stage of 3, were inoculated by using a syringe in the back of the four lobes, inoculation after the (capsicum annuum cv. Nockwang) is 28 ℃, 100% Wet treatment for 16 hours at relative humidity conditions. RNA was extracted by sampling 3, 4 leaves inoculated after 1, 5, 10, 15, 20, 25 hours of inoculation. The RNA thus extracted was electrophoresed on a gel containing formaldehyde and then transferred to a nylon membrane (Hybond N +).

Next, a- ³² P [dCTP] labeled with an isotope was labeled to CaRBP1 gene obtained in Example 1 using Klenow enzyme, and then the reaction solution [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 DNA probe labeled using the isotope binds to the mRNA attached to the nylon membrane (Hybond N +). As a result, when the X-ray film is placed on the nylon membrane (Hybond N +), the isotope is labeled. The resulting DNA photosensitizes the X-ray film, allowing the presence and extent of expression to be determined.

By monitoring the amount of ribosomal RNA during the Northern blotting it was confirmed that the same amount of RNA sample was loaded (loading). The results of the above experiments are shown in FIG. 6. As a result of the experiment, CaRBP1 gene was expressed quickly after inoculation of pathogenic strain and non-pathogenic pepper bacterial spot pattern disease. Since then, the amount of RNA has increased and maintained. That is, in the friendly response, the expression of CaRBP1 gene was maintained at a low level, but in the incompatible reaction, expression was strongly maintained from 5 hours to 25 hours after inoculation.

[Example 5] CaRBP1  Induction of apoptosis-related apoptosis in pepper plants by gene overexpression

Recombinant vector pBIN35S: CaRBP1 prepared by introducing CaRBP1 gene obtained in Example 1 into pBIN35S vector provided by Stratagene in order to determine whether induction of apoptosis for disease resistance when CaRBP1 gene was overexpressed in red pepper plants Agrobacterium tumefaciens strain GV3101 was introduced using an electroporation method and inoculated into red pepper leaves to observe the pathogenicity-related apoptosis changes of pepper plants overexpressing CaRBP1 .

[Step 1]

A recombinant vector overexpressing CaRBP1 (35S: CaRBP1) and the control vector that does not over-expressing the CaRBP1 (35S: 00) the transformant in which Agrobacterium tumefaciens (Agrobacterium tumefaciens strain GV3101) with the absorbance (optical density) 600 ( OD ₆₀₀ ) was inoculated with a syringe at the concentrations of 1.0, 0.5, and 0.1 at the leaf vein border, and the results are shown in FIG. 7. As shown in FIG. 7, apoptosis began to occur at the site of inoculation of Agrobacterium overexpressing CaRBP1 from the second day of inoculation. UV) can be observed. Phenotype on day 6 where apoptosis was observed strongly and CaRBP1 protein was strongly expressed in leaf tissues was confirmed by Western blotting (FIG. 7).

Apoptosis was quantified by measuring the amount of electrolyte flowing out of leaf tissues 24 hours and 48 hours after inoculation. Compared with the control, the electrolyte outflow from the tissues overexpressing CaRBP1 increased significantly (FIG. 8). In addition, the amount of hydrogen peroxide (H ₂ O ₂ ) was quantified using xylenol orange (xylenol orange) to measure the amount of free radicals well known as a signaling material for cell death for disease resistance. As a result, it was observed that the generation of free radicals was very increased at the site inoculated with Agrobacterium strain overexpressing CaRBP1 (Fig. 8).

[Example 6] CaRBP1  Changes in disease resistance by inhibition of gene expression

When in the pepper plant, the expression of CaRBP1 gene is suppressed some bottles produced with genes derived whether and to evaluate the change in the resistance to the disease, exemplary viruses induce CaRBP1 gene obtained in Example 1 in a pepper plant gene of silence ( Using the TRV (Tobacco Rattle Virus), which is used for VIGS, virus-induced gene silencing, the CaRBP1 gene was converted into a TRV2 vector (Bulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing.Curr. Opin. Plant Biol. 2: 109-113) to prepare a recombinant vector TRV2 :: CaRBP1 . The recombinant vector TRV2 :: CaRBP1 thus prepared was introduced into Agrobacterium tumefaciense strain GV 3101 in the same manner as in Example 5, and the transformed GV3101 strain was introduced into the cauliflower of pepper seedlings. By inoculation using the infiltration method, Inhibition of CaRBP1 gene expression was induced during pepper growth.

[Step 1]

In order to confirm the change in the resistance to pathogens in the plants in which CaRBP1 expression was suppressed, the pepper bacterial spot pattern disease was applied to the pepper plants in which CaRBP1 gene expression was suppressed and the green pepper varieties (control) inoculated with TRV: 00 vector. Pathogenic strain Ds1 and non-pathogenic strain Bv5-4a of Xanthomonas campestris pv.vesicatoria ) were inoculated at concentrations of 10 ⁸ , 10 ⁷ and 10 ⁶ cfu ml ^-1 bordering the pepper leaf vein and then inoculated after 3 and 6 days, respectively. Was observed (FIG. 9). And pathogenic strains and non-pathogenic strains were inoculated with 5 x 10 ⁴ cfu ml ^-1 to measure bacterial growth in the infected plant tissues 0, 3 days after inoculation (Fig. 9). Pathogenic and non-pathogenic strains were inoculated with 10 ⁷ cfu ml ^-1, and 12, 24 hours after inoculation, callose accumulation, free radical release and apoptosis associated with the protective reactions of the experimental and control groups were observed. Hydrogen peroxide (H ₂ O ₂ ) generation and electrolyte outflow was measured (Fig. 10) 12, 24 hours after inoculation was observed through aniline blue staining, Dab staining and trypan blue staining (Fig. 11). In addition, inoculated with 10 ⁷ cfu ml ^-1 , 1 and 2 days after inoculation, salicylic acid (SA) was extracted from the infected leaves of the experimental and control groups and quantified by RNA extraction. Real time RT-PCR) and the specific gene inhibitory effect of CaRBP1 was compared with the control plants at the time of bottle inoculation compared to the expression of the disease resistance-related genes (Fig. 12).

As shown in FIG. 9, after the inoculation of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a was observed in the plant inhibited by the expression of CaRBP1 with VIGS, no differential symptoms were observed when the pathogenic strain was inoculated. Inoculation of non-pathogenic strains showed strong local apoptosis due to resistance in control plants, but apoptosis was decreased in plants where CaRBP1 gene expression was suppressed. Similarly, the bacterial growth of pathogenic and non-pathogenic bacteria in infected tissues showed more bacterial growth of non-pathogenic bacteria in plants that inhibited the expression of CaRBP1 compared to the control, but the bacterial growth of pathogenic bacteria did not show any difference. 9). In addition , in plants inhibited with CaRBP1, it was observed that callose accumulation, free radical generation and hypersensitivity apoptosis were significantly reduced for defense reactions occurring when non-pathogenic strains were inoculated (FIGS. 10 and 11). In addition, salicylic acid was significantly reduced when the plant inhibited the expression of CaRBP1 was infected with a non-pathogenic strain (Fig. 12), when the real-time Alti- pishial experiments, when infected with a non-pathogenic strain in a plant inhibited CaRBP1 A specific decrease in the expression of strongly expressed CaRBP1 gene was observed, and expression of resistance related genes such as CaBPR1 and CaDEF1 was decreased (FIG. 13).

Example 7 CaRBP1  Preparation of Transgenic Plants Using Genes CaRBP1  Changes in Disease Resistance in Arabidopsis Plants by Overexpression of Genes

In order to examine whether the resistance to disease during overexpression of CaRBP1 gene in Arabidopsis plants used as a model plant was increased, the recombinant vector prepared by introducing the CaRBP1 gene obtained in Example 1 into a pBIN3S vector provided by Stratagene pBIN35S :: CaRBP1 was introduced into Agrobacterium tumefaciense strain GV 3101 in the same manner as in Example 5, and the GV3101 strain containing the recombinant vector was subjected to a floral dipping method. Induced over-expression of the CaRBP1 gene by transformation into the transformant was tested as follows.

[Step 1]

As described above, the plant overexpressed CaRBP1 was inoculated with the strain Pseudomonas syringae pv. Tomato DC3000, which is a bacterial virulent pathogen, and the growth of bacteria was observed. Bacterial growth was not different in plants overexpressed with CaRBP1 and wild-type control plants not overexpressed (FIG. 14).

In FIG. 14, # 3, # 6, and # 7 indicate the line number of the transgenic plant used.

[Step 2]

CaRBP1 overexpressed plants were inoculated with Hyaloperonospora parasitica Noco2 ( Hyaloperonospora parasitica Noco 2), which causes downy mildew in Arabidopsis; On day 7 after sowing, the cotyledon of overexpressed CaRBP1 and non-overexpressed wild-type control plants was spray-inoculated with Hyeloferonospora parasitica Noco2 at a concentration of 5 x 10 ⁴ spores / ml. After inoculation, it was wet-treated at the temperature of 17 degreeC. As a result, as shown in Figure 15, the increased resistance in transgenic Arabidopsis thaliana plants overexpressing CaRBP1 was observed that bunsaengjagyeong and sporulation of downy mildew Won Kyun suppressed.

Although specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

Attach an electronic file to a sequence list

Claims (8)

An isolated gene having the nucleotide sequence of SEQ ID NO: 1 ( CaRBP1). An isolated protein having the amino acid sequence of SEQ ID NO: 2 (CaRBP1). A recombinant vector comprising the gene of claim 1. A plant transformed by the gene of claim 1 or the recombinant vector of claim 3. Separation of mRNA from the plant to determine the differential expression of the CaRBP1 gene described in claim 1, the disease resistance search method comprising a plant. The method for detecting disease resistance of a plant according to claim 5, wherein the plant is a plant inoculated with pathogenic or non-pathogenic bacteria of red pepper bacterial spot pattern disease. A method of imparting disease resistance to a plant, comprising the step of transforming the plant using a recombinant vector comprising the gene of claim 1.
An isolated gene ( CaRBP1) encoding a pepper-derived RNA binding protein having the amino acid sequence of SEQ ID NO: 2 .

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