KR20110008530A - Pepper defense-related, mannose-binding lectin (cambl1) gene and disease resistant plants using the same - Google Patents

Abstract

PURPOSE: A pepper mannose-binding lectin1(CaMBL1) and a transgenic plant of disease-resistance obtained using the same are provided to develop species having resistance. CONSTITUTION: A pepper mannose-binding lectin1(CaMBL1) gene is a gene encoding a protein having an amino acid sequence of sequence number 2 and a gene having a base sequence of sequence number 1. A recombinant vector, pBIN35S::CaMBL1 is prepared by inserting CaMBL1 gene to Pbin35S. The plant is treated with salicylic acid or methyl jasmonate.

Description

Pepper defense-related, mannose-binding lectin (CaMBL1) gene and disease resistant plants using the same}

The present invention relates to a pepper mannose-binding lectin 1 gene, CaMBL1 , which is a novel gene related to plant disease defense, and a pathogenic transformed plant using the same, and a method of developing the same. More specifically, the present invention relates to the nucleotide sequence and amino acid sequence of CaMBL1 gene, a novel gene related to plant disease defense, which is strongly expressed in resistance when infected with pathogenic and non-pathogenic strains of red pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ). To detect whether the expression of CaMBL1 protein level in the bio / chemical / physical resistance induction treatment by the differential expression of the resistance-related gene CaMBL1 and the immune response using the antibody produced using the peptide of CaMBL1 It relates to a method of searching for resistance of plants. In addition, the present invention confirms the function of the mannose binding lectin 1 gene for disease resistance by knocking down the expression of the CaMBL1 gene through virus-induced gene silencing (VIGS) in pepper plants. , and relates to the by gwabal current (overexpression), introduced via transformation of the CaMBL1 gene in Arabidopsis (Arabidopsis thaliana) determine plant disease resistance increasing effect of CaMBL1 gene, provides a plant disease-resistant transgenic plant therethrough will be.

The occurrence of plant diseases leads to a significant economic damage by degrading crop production and quality. The program for breeding disease-resistant varieties of peppers and various economic crops in order to minimize the crop production damage caused by these diseases. In order to understand the disease resistance and practical application, studies on resistance mechanisms in plants have been carried out as models of red pepper plants.

     Diseased host plants act on defense mechanisms in a variety of intracellular metabolic pathways, and are determined by the interaction between host plants and phytopathogens in a friendly and incompatible response early in the pathogen infection. This interaction determines whether there is a resistance (defense) reaction that can limit disease progression.

In incompatible interactions, infected plants recognize the invasion of pathogens, and in response to recognition, hypersensitive responses, such as local cell death of the infected site, accumulation of callose, and lignin It shows reactions such as thickening of cell walls due to aging, and produces various kinds of antimicrobial substances such as phytoalexin, and produces defense-related proteins called pathogenesis related (PR) proteins. It is known to cause resistance reactions.

The defense mechanisms of these plants are reported not only in the invasion of pathogens, but also by hormonal stimulation such as salicylic acid and methyl jasmonate, and are reported to be caused by environmental stresses such as physical wounds, salinity and temperature.

Among the defense responses of plants to biological, physical and environmental stresses, in particular, the important role that plants play in the defense against pathogens is the expression of plant disease resistance by the interaction of the plants and pathogens and the signaling associated with them. Path. It has been important to study how genes related to disease defense mechanisms and resistance responses of these plants recognize pathogens and how they respond to the whole plant through the transmission pathways. In this regard, diseases using biological, biochemical, and cell biological methods Research has been done on the function of protective (resistive) genes.

Biological / biochemical / cell biological studies of the function of disease defense (resistance) genes are of high value in studying the physiology and resistance of plants, as well as in the development of resistant varieties through molecular breeding of crops. Can contribute. Involvement of genetic engineering techniques in the molecular breeding technology of crops has been actively conducted in recent years, and the molecular breeding of crops through disease defense (resistance) genes can use foreign genes of other plant species. By enabling breeding technology at the genetic level, it has the advantage of showing more effective breeding effect and maximizing the current breeding technology.

Mannosbinding lectins, on the other hand, contain many plants and are known as carbohydrate-binding proteins and are one of the recently discovered plant lectins. In recent studies, mannose binding lectins are known to be involved in the expression of resistance, and various carbohydrates are known to be involved in various physiological actions such as the interaction between cells and cells, host and pathogens. These findings enable the accumulation of mannose-binding lectin's response to the disease and related genetic information of PR proteins, which can provide an interesting model for signaling the disease in plants. By understanding the biochemical changes, biotechnological plants with enhanced disease resistance can be developed or practically used for molecular breeding as disease-resistant genetic material of plants.

Furthermore, the pepper plants belonging to the eggplant family are crops grown all over the world, including many countries of South America, which are native to Korea, and are the most used seasonings in our diet. They are widely used in processed foods such as spices, kimchi, and kochujang. Among them, it is a representative economic crop with a large amount of cultivation. Domestic annual consumption is 3.5kg per capita dried pepper, consumption is 200,000 tons and the market size is estimated to reach 1 trillion won. Although the domestic red pepper cultivation area is gradually decreasing, the domestic red pepper cultivation area was produced at 53,097 ha in 2006, with 116,915 M / T dry peppers produced. The average yield per 10a is 220kg. This is the second major crop after rice. However, in the developed countries, the importance of red pepper is relatively low, so the research is vulnerable and full investment and research have not been made. In addition, these peppers have been reported a variety of bacterial diseases, fungal diseases, viral diseases, and most seriously reported damage to bacterial plague caused by fungal pestilence, anthrax caused by fungi and bacterial spots caused by bacteria. The damage of these pathogens has resulted in the reduction of the yield and quality of the pepper. To solve these problems, it is recognized that the stabilization of disease-resistant varieties of pepper, considering the stable production, the reduction of the production cost due to the reduction of pesticides and labor force, and the environment ought. In addition, Korea is the only country that uses hybrid technology for the production of red pepper seeds. If molecular genetic breeding technology is introduced to the crop breeding and combined with the existing breeding technology, it is the basis for better breeding of red pepper varieties. Breeding of these superior varieties will not only protect domestic genetic resources in the domestic seed market, but also lead to the development of high value-added genetic resources.

The present invention is to solve the above-mentioned problems in the prior art, the purpose of which is involved in the defense reaction against pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ), pepper mannose bindinlecting one of the genes related to plant disease defense By separating and decoding the CaMBL1 gene encoding the nucleotide sequence information and amino acid sequence information according to it is to be provided.

It is also an object of the present invention to provide a method for detecting resistance of plants using the CaMBL1 gene to search for resistance to plant diseases or chemicals.

Furthermore, an object of the present invention is to introduce a CaMBL1 gene into a plant to produce a transformed plant having resistance to plant diseases.

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 the nucleotide sequence by identifying a novel defense-related gene CaMBL1 isolated from the green pepper varieties exhibiting a hypersensitivity resistant response to non-pathogenic strains of pepper bacterial bacillus bacteria ( Xanthomonas campestris pv.vesicatoria ), This study was performed to investigate the expression of CaMBL1 through biological / chemical treatment and to confirm the plant disease resistance induction function of CaMBL1 gene in red pepper plants and transgenic Arabidopsis thaliana overexpressing this gene.

Accordingly, the present invention (CaMBL1) gene having the nucleotide sequence of a plant disease protective encoding a pepper-derived mannose binding lectin as genes (a) a gene encoding a protein having an amino acid sequence of SEQ ID NO: 2 or (b) SEQ ID NO: 1 To provide.

The present invention also provides an isolated mannose binding lectin protein (CaMBL1) 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 of searching for resistance of a plant, including separating RNA from a plant and confirming differential expression of a CaMBL1 gene or identifying whether CaMBL1 protein is expressed using an antibody of the CaMBL1 protein as a probe. .

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

First, the present invention is to prepare a cDNA library by isolating the mRNA from the cultivars of nocturnal cultivars showing a hypersensitivity resistance to the non-pathogenic strain inoculation of pepper bacterium bacillus bacteria ( Xanthomonas campestris pv.vesicatoria ), leaves of pepper plants inoculated with non-pathogenic strains Probe is labeled with digoxygenin of total mRNA obtained from whole red pepper plants not overinoculated, and differential hybridization is performed using this cDNA and probe to protect against disease (resistance). CaMBL1 protein coding of the red pepper green mine variety named CaMBL1 by selecting a gene suspected to be related to, obtaining a clone, and sequencing the sequencing of the gene identified as the mannose binding lectin ( CaMBL1 ) gene in the clone . Gene base sequence (SEQ ID NO: 1) and amino acid sequence encoded therefrom (SEQ ID NO: Arc 2) (see FIG. 1).

After sequencing the obtained clones according to the present invention, the 5'-terminal sequence is compared with a gene database registered in GenBank using a Blast program to identify CaMBL1 protein coding genes and mannose binding lectins. 1 ( CaMBL1 ) gene. CaMBL1 protein was found to be 55% homologous to mannose binding lectin in beet ( Beta vulgaris ) as compared with amino acid sequence and 48% homologous to amino acid sequence of Arabidopsis. . This gene has a molecular weight of 35kDa that is resistant to various pathogens as well as pepper bacterial spots. The identified CaMBL1 gene nucleotide sequence and the amino acid sequence encoded by the nucleotide sequence are shown in FIG. 1.

In addition, using the antibody of the CaMBL1 protein as a probe provides a method for detecting the resistance of the plant comprising the step of confirming the expression of CaMBL1 protein (see Figures 2 to 4). The method for preparing the antibody of the CaMBL1 protein and the method for confirming the expression of the CaMBL1 protein may use a variety of techniques known in the art to which the present invention belongs (Reference: Andon, NL, Eckert, D., Yates, JR and Haynes, PA 2003. High-throughput functional affinity purification of mannose binding proteins from Oryzasativa . Proteomics 3: 1270-1278).

In addition, the present invention, a non-pathogenic induction method, such as salicylic acid, or methyl jasmonate plant hormonal chemicals to the plant and treated with a non-biological induction method, and using the resistance detection method to determine whether the expression of CaMBL1 protein This provides a new resistance induction method that is easier and saves time.

Next, the present invention provides a method for searching for resistance of a plant, comprising the step of identifying RNA differentially expressing the CaMBL1 gene by separating RNA from the plant subjected to biological / chemical treatment. More specifically, the biological treatment may be pathogens such as pathogenic or non-pathogenic bacteria of pepper bacterial spot pattern disease. Examples of the chemical derivatives include salicylic acid and methyl jasmonate. The CaMBL1 gene expression check may be used in addition to the Northern blot, real-time RT-PCR, microarray method, a variety of methods for measuring the expression of mRNA can be used. Experiments confirmed that CaMBL1 gene according to the present invention is differentially induced expression by phytopathogens , plant hormones or chemical derivatives (see FIGS. 3 and 4). Therefore, the CaMBL1 gene according to the present invention can be usefully used for a method of searching for resistance of plants.

In addition, the present invention determines whether to use a recombinant vector containing the above-CaMBL1 gene production of a typically CaMBL1 gene-specific in pepper plants inhibiting plant, CaMBL1 gene plant disease resistance generation of Arabidopsis thaliana plants with transgenic This can provide materials for new pathogenic transgenic plants and resistant molecular breeding.

More specifically, the present invention also uses the Tobacco Rattle Virus (TRV), which is used in newly developed virus-induced gene silencing (VIGS) technology, to convert the CaMBL1 gene into a TRV2 vector (reference: Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing.Curr. Opin. Plant Biol. 2: 109-113) to provide a recombinant vector TRV2 :: CaMBL1 . The recombinant vector was introduced into the plant to prepare a VIGS plant in which CaMBL1 expression was suppressed, thereby confirming that the disease susceptibility was increased (see FIGS. 13 and 17).

Furthermore, by introducing a recombinant vector (pBIN35S :: CaMBL1 ) comprising the CaMBL1 gene according to the present invention into a plant, a transgenic Arabidopsis plant overexpressed CaMBL1 according to the present invention was confirmed to increase plant disease resistance (FIG. 18 to 23).

Through the pepper plant which disabled the function of the pepper mannose binding lectin and the Arabidopsis plant overexpressing the mannose binding lectin according to the present invention, it was possible to determine whether resistance to phytopathogens was performed. The present invention enables the accumulation of mannose binding lectin's response to disease and related genetic information of pathogenicity related protein (PR protein), thereby providing an interesting model for signaling of diseases in plants, and resistance. By understanding the biochemical changes that cause them, they can be used practically to develop biotech plants that increase disease resistance.

As described above, by completing the present invention, by separating and deciphering the CaMBL1 gene encoding a novel mannose binding leptin protein, which is a plant disease defense-related gene, involved in the defense against the pepper plant bacterial disease, In addition to providing information and corresponding amino acid sequence information, it is possible to provide a method of searching for resistance of plants using antibodies of the genes and proteins, as well as to contribute to the development of resistant plant breeding and transgenic plants.

Therefore, the present invention has an effect that can promote the development of resistant varieties is a very useful invention in the plant breeding industry.

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 CaMBL1  Base / Amino Acid Sequencing of Genes

The following tests were performed to provide mannose binding lectin (CaMBL1) protein coding gene sequences and amino acid sequences deduced therefrom, one of the pathogenicity related proteins (PR protein) against red pepper bacilli.

Capsicum annuum cv. Nockwang was inoculated with red pepper bacterial spot bacterium ( Xanthomonas campestris pv.vesicatoria ), and after 18 hours of wet treatment, the plants which showed hypersensitivity reactions, which were resistant lesions, were taken out of the room and leaves were collected.

Next, cDNA libraries were prepared by extracting mRNA from the leaf samples, reverse transcripting, and amplifying the polymerase chain reaction (PCR). 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 the red pepper plants inoculated with the non-pathogenic strains of the red pepper bacilli, and the healthy red pepper plants not inoculated. Total mRNA extracted from the leaves of the probe was made by labeling with dioxygenin (digoxygenin), and then differential hybridization was performed.

As a result of hybridization, a gene intensively amplified only for mRNA probes of a non-pathogenic strain inoculated plant was assumed to be a gene related to disease defense (resistance), and a clone was obtained.

After sequencing the obtained clones, using the Blast program, the 5'-terminal sequence was compared with the gene database registered in GenBank to identify the CaMBL1 protein coding gene and the mannose binding lectin 1 ( CaMBL1 ) gene. It was named. CaMBL1 protein was found to be 55% homologous to mannose binding lectin in beet ( Beta vulgaris ) as compared with amino acid sequence and 48% homologous to amino acid sequence of Arabidopsis. . This gene has a molecular weight of 35kDa that is resistant to various pathogens as well as pepper bacterial spots. The gene sequence of the identified CaMBL1 and the amino acid sequence encoded by the base sequence is shown in FIG.

Example 2 In Red Pepper Plant by Pathogen Inoculation CaMBL1  Gene Expression Detection Method

In order to confirm the use of the CaMBL1 gene obtained in Example 1 in the resistance search was tested as follows.

[Step 1]

First, in order to specifically presented in the resistive search method, and then culturing the non-pathogenic strain Bv5-4a pepper bacterial spot patterns indicating the pathogen (Xanthomonas campestris pv. Vesicatoria) of the pathogenic strain Ds1 and the incompatible reaction indicating the plants and friendly reaction, 10 ⁸ Inoculate by infiltration using a syringe on the back of the 1st and 2nd leaves of Capsicum annuum cv.Nockwang at the concentration of cfu / ml, and inoculate 28 ℃, 100% relative after inoculation. It was wet-treated for 18 hours under humidity conditions. After 1, 5, 10, 15, 20 and 25 hours of inoculation, RNA was isolated by sampling 1 and 2 leaves, respectively, and the RNA was electrophoresed on a gel containing formaldehyde, followed by a nylon membrane (Hybond N +). Metastasis.

Next, the CaMBL1 gene obtained in Example 1 was labeled using 14-dCTP-biotin and then amplified by PCR (Polymerase Chain Reaction), and then a 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 biotin-labeled DNA probe is attached to the mRNA attached to the nylon membrane. When the X-ray film is placed on the nylon membrane, the biotin-labeled DNA photosensitizes the X-ray film. This made it possible to recognize the expression.

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 experiment are shown in FIG. 2. As a result of the experiment, CaMBL1 gene was expressed in both peppery and non-pathogenic strains. More specifically, in the pathogenic strain, that is, the friendly response, the expression of the CaMBL1 gene increased after 5 hours, and the expression was observed up to 25 hours after showing the highest expression amount at 20 hours. In the non-pathogenic strains exhibiting an incompatible response, the strongest expression was observed at 5 hours and the increased expression level was maintained until 25 hours, and CaMBL1 gene expression was much stronger than the friendly response.

Example 3 by chemical derivative CaMBL1  Induced Expression of Genes

In order to find out whether the CaMBL1 gene of the present invention can be induced by chemical derivatives commonly used for resistance induction and to provide a specific method of conducting the following test, the following test was performed.

[Step 1]

First, in order to search for derivatives useful for inducing expression of the CaMBL1 gene, resistance induction tests were conducted using salicylic acid and methyljasmonate among chemicals known to be involved in the induction of plant disease resistance expression. Six-leaf peppers were used for all experiments. 5 mM salicylic acid, 100 μM of methyl jasmonate was sprayed on the front and back of pepper leaves using a sprayer.

Each plant was harvested after 1,5,10,15,20,25 hours, RNA was extracted from each sample, and Northern blotting was performed using the extracted RNA as follows.

Next, the CaMBL1 gene obtained in Example 1 was labeled with 14-dCTP-biotin and then amplified using PCR (Polymerase Chain Reaction), followed by 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 biotin-labeled DNA probe is attached to the mRNA attached to the nylon membrane. When the X-ray film is placed on the nylon membrane, the biotin-labeled DNA photosensitizes the X-ray film. This made it possible to recognize the expression.

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 experiment are shown in FIGS. 3 and 4, and as a result of the experiment, CaMBL1 gene was observed to accumulate after 5 hours after salicylic acid treatment (FIG. 3). In addition, as observed in FIG. 4, after the treatment of methyl jasmonate, accumulation occurred only in 5 hours and accumulation up to 20 hours was observed.

Example 4 Detection of CaMBL1 Protein Expression in Red Pepper Plants by Pathogen Inoculation and Chemical Derivatives

Experiments were performed by sampling proteins that were treated in the same manner as in Examples 2 and 3 to separate proteins. Next, the CaMBL1 gene obtained in Example 1 was produced in rabbits using peptide synthesis to confirm the expression of CaMBL1 protein. The antibody was prepared by requesting an antibody manufacturer (AbFRONTIER, Korea), and the experiment was performed using the antibody.

First, in order to specifically presented in the resistive search method, and then culturing the non-pathogenic strain Bv5-4a pepper bacterial spot patterns indicating the pathogen (Xanthomonas campestris pv. Vesicatoria) of the pathogenic strain Ds1 and the incompatible reaction indicating the plants and friendly reaction, 10 ⁸ Inoculate by infiltration using a syringe on the back of the 1st and 2nd leaves of Capsicum annuum cv.Nockwang at the concentration of cfu / ml, and inoculate 28 ℃, 100% relative after inoculation. It was wet-treated for 18 hours under humidity conditions. Proteins were extracted by sampling 1 and 2 leaves after 5, 15, 25, and 35 hours of inoculation, respectively. The extracted proteins were electrophoresed using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (HDS). P +). After transfer, expression was confirmed using an antibody (α-CaMBL1) at a dilution ratio of 1: 5,000. When performing the experiment of protein expression it was confirmed that the same amount of protein was loaded by quantifying the amount of protein. The experimental results are shown in FIGS. 2, 3, and 4.

As shown in the lower part of FIG. 2, the CaMBL1 gene was expressed in both the pathogenic and non-pathogenic strains, but the CaMBL1 gene was expressed in the pathogenic and non-pathogenic strains. Expression was confirmed after time and was most strongly expressed at 15 hours in nonpathogenic strains that exhibited incompatible responses. As shown at the bottom of FIGS. 3 and 4, salicylic acid was most strongly expressed at 24 hours with increased expression at 12 hours and no expression of protein levels of methyl jasmonate was observed.

Example 5 Screening Method for CaMBL1 Protein Production and Mannosbinding Activity

In order to investigate the protein function of CaMBL1, an expression product after overexpressing CaMBL1 gene in Escherichia coli, the domains of His-CaMBL1-1 and CaMBL1 from which the CaMBL1 gene obtained in Example 1 and the signal peptide of CaMBL1 were removed His-CaMBL1-2 containing only, His-CaMBL1-3 containing only the c-terminal (c-terminal) portion of CaMBL1 to the pET28a vector (Reference: Andon, NL, Eckert, D., Yates, JR and Haynes) , PA 2003. High-throughput functional affinity purification of mannose binding proteins from Oryzasativa.Proteomics 3: 1270-1278) to prepare a recombinant vector and is shown in FIG.

The recombinant vector was transformed into Escherichia coli strain BL21 and the protein obtained by culturing Escherichia coli containing the recombinant vector was used for the next experiment.

[Step 1]

D-mannose-agarose column (D-mannose-) after sonication of CaMBL1 protein using a buffer for mannose binding assay (50 mM Tris, pH 7.5), 100 mM NaCl, 5 mM CaCl ₂ ) Mannos binding assays were performed using an agarose column. Thereafter, the protein was identified using SDS-polyacrylamide gel electrophoresis. This 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 protein markers and the results are shown in FIG.

[Step 2]

The CaMBL1, His-CaMBL1-1, His-CaMBL1-2, and His-CaMBL1-3 proteins thus bound were transferred to a nylon membrane (Hybond-P) after electrophoresis, and then expressed using anti-His antibodies. . As shown in the lower result of FIG. 5, only His-CaMBL1-1 and His-CaMBL1-2 including CaMBL1 domains were expressed.

Example 6 Location Searching Method for CaMBL1 Using Green Fluorescent Protein

CaMBL1 gene was introduced into a 326GFP vector using a recombinant vector labeled with a green fluorescent protein (GFP) to introduce a CaMBL1 gene into a 326GFP vector to produce a recombinant vector, smGFP :: CaMBL1 . It was. Also CaMBL1 genes of the mannose binding domain, only the introduction of smGFP :: MBL lectin domains (domain) particles shots using DNA transfer using alcohol was introduced into onion plants by (particle bombardment) confocal microscope the position of CaMBL1 (confocal laser search using a scanning microscope (CLSM).

[Step 1]

In order to determine the position where CaMBL1 is expressed in the plant, CaMBL1 labeled with green fluorescence protein was introduced into onion plants using granular DNA transfer and observed using a confocal microscope. 6 shows the result. As a result, in onion plants incorporating only the domains of CaMBL1 and mannose binding lectin, fluorescence green protein is expressed as a plasma membrane, unlike the control in which only the vector is introduced.

[Example 7] Agrobacterium-mediated transient expression method CaMBL1  Effect of Gene Expression on Plants

In order to determine the change of pepper plants when overexpression of CaMBL1 gene in pepper plants by Agrobacterium-mediated transient expression method, pBIN3S vector provided by Stratagene with CaMBL1 gene obtained in Example 1 The recombinant vector pBIN35S :: CaMBL1 was introduced into Agrobacterium tumefaciense strain GV 3101 by electroporation, and the recombinant vector was added to the pBIN35S :: CaMBL1 . Overexpression of the CaMBL1 gene was induced by introducing the GV3101 strain into the pepper plants using an infiltration method using a syringe for each concentration.

[Step 1]

In Fig. 7, 9, and the plant to over-express the CaMBL1 as in 10 of the plants was observed cell death compared to control CaMBL1 is not introduced was observed quickly, especially in high concentrations. In addition, trypan blue staining and ion conductivity were performed to investigate the results of the resistance reaction in these plants. As shown in FIG. 9, the CaMBL1 overexpressed plant was observed to increase cell death as a result of trypan blue staining as compared to the control, and as shown in FIG. 10, the ion conductivity was also increased in the CaMBL1 overexpressed plant. Could.

[Step 2]

To investigate the effect of CaMBL1 overexpression in plants, the expression of other disease-related genes was observed using quantitative RT-PCR. CaBPR1 and CaDEF1, which are known as disease-related genes of pepper plants, could be observed to increase in CaMBL1 overexpressed plants (see FIG. 11).

[Step 3]

To determine the effect of overexpression of CaMBL1 on the production of salicylic acid in plants, salicylic acid was quantified using high performance liquid chromatography (HPLC). As shown in Fig. 12, the plants were harvested and quantified 24 and 48 hours after inoculation, and both free salicylic acid and total salicylic acid were observed to increase in overexpressed plants of CaMBL1 .

Example 8 CaMBL1  Increased disease susceptibility by inhibition of gene expression

In order to determine whether induction of other genes related to disease development and increased susceptibility to disease when CaMBL1 gene expression was inhibited in pepper plants, the newly developed virus-induced gene silencing ( CaMBL1 gene) was obtained. Using the TRV (Tobacco Rattle Virus), which is used in VIGS, virus-induced gene silencing ( CVMB ), CaMBL1 gene was used as 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 :: CaMBL1 . The recombinant vector TRV2 :: CaMBL1 thus prepared was introduced into Agrobacterium tumefaciense strain GV 3101 by electroporation, and the GV3101 strain containing the recombinant vector was then added to the cotyledons during pepper growth. Inhibition of the expression of the CaMBL1 gene was induced by introducing into pepper plants using an infiltration method using a syringe.

[Step 1]

To determine the increased sensitivity to bacterial diseases in plants that inhibited CaMBL1 expression, the pathogenicity of pepper bacterial spot pattern ( Xanthomonas campestris pv.vesicatoria ) in pepper plants and healthy red pepper green varieties in which CaMBL1 gene expression was suppressed. After incubating and inoculating strain Ds1 and non-pathogenic strain Bv5-4a, the plants were harvested at 15 hours from each plant, and RNA was extracted and observed through quantitative RT-PCR.

13-17 show the results after inoculation of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a to pepper plants in which CaMBL1 expression was inactivated by VIGS.

[Step 2]

In order to examine the results of susceptibility reactions in plants in which CaMBL1 expression was suppressed, trypan blue staining and DAB staining were performed. As shown in FIG. 13, inoculated with CaMBL1 expression, the pathogenic strain Ds1 of red pepper bacterial spot pattern disease was inoculated, and thus the incidence of disease was increased, and the susceptibility was increased. After inoculation of non-pathogenic strain Bv5-4a Compared to whole plants, trypan blue staining decreased hypersensitivity apoptosis and DIP staining reduced free radicals.

[Step 3]

On the day 0 and 3 days after inoculation of healthy plants and plants inhibited with CaMBL1 expression to pathogenic strain Ds1 and bacterial non-pathogenic strain Bv5-4a inoculated with CaMBL1 suppressed plants After collecting the 1 cm ² bacteria number of the plant leaves was measured and the results are shown in FIG.

The experimental results compared with the healthy flora in the expression is inhibited, and the plant of CaMBL1 sensitivity is increased in both the pathogenic strain and the non-pathogenic strain Bv5-4a Ds1, particularly by reducing the expression of pathogen resistance genes to CaMBL1 also genes involved in disease resistance It could be seen (see Fig. 15).

[Step 4]

Salicylic acid was quantified using high performance liquid chromatography (HPLC) to investigate the effect of inhibition of CaMBL1 expression on salicylic acid production in plants. As shown in Fig. 17, after inoculating healthy plants and plants with suppressed expression of CaMBL1, inoculated with pathogenic strain Ds1 of red pepper bacterial spot pattern disease and Bv5-4a, which is a non-pathogenic strain, plants were harvested and quantified 24 and 48 hours after inoculation. As a result, both free salicylic acid and total salicylic acid showed decreased free salicylic acid and total salicylic acid compared to whole plants in plants which inhibited the expression of CaMBL1 , indicating that CaMBL1 gene is involved in disease resistance.

Example 9 CaMBL1  Preparation of Transgenic Plants Using Genes CaMBL1  Increased disease resistance due to overexpression of the gene

By introducing the CaMBL1 gene obtained in Example 1 in pBIN3S vector available from Stratagene to investigate as to whether the increase in resistance to induction, and whether the bottle of genes associated with different disease occurs when over expression of CaMBL1 gene in a plant pBIN35S PBIN35S , a recombinant vector thus prepared after the preparation of CaMBL1, was introduced into Agrobacterium tumefaciense strain GV 3101 by electroporation and again contains the recombinant vector GV3101. Was transformed by introducing into Arabidopsis plants through floral dipping method to induce overexpression of CaMBL1 gene, and the following tests were performed.

[Step 1]

In the CaMBL1 gene it is overexpressed plants to investigate whether or not the resistance to bacterial disease increase, was first observed CaMBL1 expression in wild-type Arabidopsis thaliana plant as the CaMBL1 Arabidopsis and control the gene is over-expressed result 18 is shown.

In FIG. 18, # 2, # 4, and # 5 represent the line numbers of the transgenic plants used, and as a result, CaMBL1 was overexpressed in Arabidopsis plants.

[Step 2]

As shown above, inoculated with Pseudomonas syringe Pasova tomato DC3000 and non-pathogenic strain Pseudomonas syringe Pasova tomato DC3000 (ABR-PM1) at a concentration of 10 ⁵ cfu / ml and observed changes of the plant (FIG. 19). In addition, trypan blue staining and DAB staining were performed to investigate the reason why the resistance of the CaMBL1 gene was overexpressed in plants. As a result, as shown in FIGS. 20 and 21, it was observed that CaMBL1 exhibited an increased resistance to bacterial diseases in the overexpressed plants compared to the wild type, and in particular, in FIG. 21, CaMBL1 was detected through trypan blue staining and dip staining. In the plants overexpressed genes, free radicals and peroxidase activity increased in free radicals observed in resistant plants.

[Step 3]

Plants overexpressed CaMBL1 were inoculated with Pseudomonas syringe pesova tomato DC3000 and non-pathogenic strain Pseudomonas syringe pessova tomato DC3000 (ABR-Al-PM). Assays of plants were performed. The leaves were harvested on days 0 and 3 after inoculation to measure the number of 1 cm ² bacteria of the plant leaves and the results are shown in FIG. 20.

As shown in FIG. 20, it was observed that the growth of pathogens in the pathogenic and non-pathogenic strains overexpressed CaMBL1 gene was significantly reduced compared to wild-type plants, thereby having disease resistance.

Example 10 Altanaria Brassica ( Alternaria brassicicola ) After inoculation CaMBL1  Changes in Disease Resistance in Overexpressed Genes and gp1-1 and gp1-2 Plants

The CaMBL1 overexpression Arabidopsis plants and CaMBL1 ohsol log (orthologs) of Arabidopsis thaliana expressing At1g the disablement of the 78,830 genes (knock out) the plants (gp1-1 and gp1-2 system) in the Arabidopsis Alta and Leah beurasi City Tests were performed after inoculation with cola ( Alternaria brassicicola ). More specifically, alternaria brassicicola was dropped on the leaves of Arabidopsis larvae at a concentration of 5 × 10 ⁴ mL ⁻¹ to CaMBL1 overexpressed plants and gp1-1 and gp1-2 plants. ) Inoculated plants were wet treated at 24 ° C.

[Step 1]

In order to observe the resistance to Alternaria brassicicola in each plant, lesion induction and trypan blue staining and Dap staining were performed after inoculation. When the leaves were observed at 5 days after inoculation as shown in FIG. 22, the size of the lesions was not significantly different in the plants overexpressed CaMBL1 compared to the wild type, but gp1-1 and gp1-2 plants showed susceptibility. In addition, as a result of the trypan blue staining, active mycelial growth was observed in gp1-1 plants as shown in FIG.

[Step 2]

In order to test for disease resistance after inoculation of Alternaria brassicicola in each plant, the size of lesions after inoculation was measured.

As shown in FIG. 23, the CaMBL1 overexpressed plants showed distinct resistance in lesion size, but the gp1-1 plants showed increased lesion size.

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. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a view showing the nucleotide sequence and amino acid sequence of the red pepper mannose binding lectin 1 (CaMBL1) gene cloned from the red pepper greenery varieties ( Capsicum annuum L. cv. Nockwang) according to the present invention.

Figure 2 is a chili pepper varieties nokgwang jeommunuibyeong bacterial strains pathogenic strains Ds1 and CaMBL1 expression of genes and proteins in pepper leaves pepper leaves inoculated with the non-vaccinated when inoculated with nonpathogenic strains of Bv5-4a (Xanthomonas campestris pv. Vesicatoria) on It is a figure which shows the result of derivation.

Figure 3 is a diagram showing the results of induction of CaMBL1 gene and protein over time after the treatment of salicylic acid (Salicylic acid) to the red pepper varieties.

4 is a diagram showing the expression of CaMBL1 gene and protein over time after treatment with methyl jasmonate (red pepper) green varieties varieties (methyl jasmonate).

5 shows the results of a mannose binding assay after CaMBL1 protein purification.

6 is a photograph showing the results of subcellular localization in onion plants using the green fluorescent protein (Green fluorescent protein) of CaMBL1.

Figure 7 is a photograph showing apoptosis results after performing a transient assay (transient assay) of CaMBL1 in red pepper cultivation varieties.

8 is a diagram showing the results of confirming the protein expression of CaMBL1 after performing a transient assay (transient assay) of CaMBL1 in red pepper cultivation varieties.

Figure 9 is a photograph showing the results of hypersensitivity cell death (trypan blue staining) after performing a transient assay (transient assay) of CaMBL1 in red pepper cultivation varieties.

10 is a view showing the results of measuring the electrolyte conductivity (ion outflow) of CaMBL1 after performing a transient assay (transient assay) of CaMBL1 in red pepper cultivation varieties.

FIG. 11 is a diagram showing the results of confirming the expression of a disease defense (resistance) related protein after performing transient expression assay of CaMBL1 in red pepper cultivation varieties.

12 is a view showing the results of the changes in salicylic acid after performing a transient assay (transient assay) of CaMBL1 in red pepper varieties.

FIG. 13 is a pepper bacterial spot pattern bacterium of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a ( Xanthomonas) in a plant of cayenne pepper green cultivars whose CaMBL1 gene expression was suppressed using the method of virus-induced gene silencing (VIGS) campestris pv. vesicatoria) is shown the results for the reduction when the inoculation, disease susceptibility is increased susceptibility symptom-up, active oxygen reducing (DAB staining), overactive cell death (trypan blue stained) picture.

FIG. 14 shows pepper bactericidal bacteria of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a ( Xanthomonas) in plants of cayenne pepper cultivars in which CaMBL1 gene expression was suppressed using the method of virus-induced gene silencing (VIGS) campestris pv.vesicatoria ) shows the results of assay for bacterial growth when inoculated.

FIG. 15 shows pepper bacterium bacillus bacteria of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a ( Xanthomonas campestris pv) in plants of cayenne pepper cultivars whose CaMBL1 gene expression was suppressed using virus-induced gene silencing (VIGS) vesicatoria ) is a diagram showing the results of the pattern of changes in gene expression associated with pepper disease (resistance) when inoculated.

16 is a pepper bacterial spot pattern bacterium of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a in a plant of cayenne pepper cultivation in which CaMBL1 gene expression was suppressed using the method of virus-induced gene silencing (VIGS). Xanthomonas campestris pv.vesicatoria ) is a diagram showing the result of observing the reduced expression of CaMBL1 at the protein level when inoculated.

17 is a pepper bacterial spot pattern bacterium of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a in a plant of the cayenne pepper green cultivars whose CaMBL1 gene expression was suppressed using the method of virus-induced gene silencing (VIGS). Xanthomonas campestris pv.vesicatoria ) shows the results of the reduction of salicylic acid when inoculated.

FIG. 18 is a diagram showing the results of overexpression of CaMBL1 gene and expression of resistance-related genes of Arabidopsis in the transgenic Arabidopsis plants which overexpressed Cayenne CaMBL1 gene.

19 shows a bacterial pathogen Pseudomonas syringae pv. Tomato DC3000 and a non-pathogenic strain Pseudomonas syringe Pasova tomato DC3000 (ABR-A-RPM) in a transgenic Arabidopsis plant overexpressing pepper CaMBL1 gene. ) Is a picture of Arabidopsis leaf which is resistant to the disease when inoculated.

20 is a bacterial pathogen Pseudomonas syringae pv. Tomato DC3000 and a non-pathogenic strain Pseudomonas syringe Pasova tomato DC3000 (ABR-ARPM) in a transgenic Arabidopsis plant overexpressing pepper CaMBL1 gene. ) Shows the results of pathogenic expression due to the reduction of bacterial growth when inoculated.

FIG. 21 is a bacterial pathogen Pseudomonas syringae pv. Tomato DC3000 and a non-pathogenic strain Pseudomonas syringe Pasova tomato DC3000 (ABR-ARPM) in a transgenic Arabidopsis plant overexpressing the CaMBL1 gene. ) Is a photograph showing the results of the increase in free radicals (DAB staining) and hypersensitivity apoptosis (trypan blue staining) in plant cells exhibiting pathogenic resistance.

Figure 22 is a CaMBL1 overexpressed Arabidopsis thaliana plants and CaMBL1 ohsol log (orthologs) of Arabidopsis thaliana expressing the disablement of At1g 78830 gene (knock out) the plants (gp1-1 and gp1-2 system) in a mutant Arabidopsis thaliana plant After inoculation with the pathogenic fungus Alternaria brassicicola , it is a photograph showing the results of lesion and trypan blue staining of diseased plant leaves.

Figure 23 is a CaMBL1 overexpressed Arabidopsis thaliana plants and CaMBL1 ohsol log (orthologs) of Arabidopsis thaliana plants At1g the expression of gene 78 830 is disabled (knock out) (gp1-1 and gp1-2 system) in a mutant Arabidopsis thaliana plant A diagram showing the change in lesion size after inoculation of the pathogen fungus Alternaria brassicicola .

<110> Korea University Industrial and Academic Collaboration Foundation <120> Pepper defense-related, mannose-binding lectin (CaMBL1) gene and          disease resistant plants using the same <130> P11-090630-01 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1160 <212> DNA 213 Capsicum annuum L. cv. Nockwang <400> 1 gaaacatgtc tccttcatgg actactacac tttttgtctc tctatttctt ttctcccaaa 60 tattttcttg cattgcccaa gttccagctg aaaacacctt caaattcgtc aacgaaggcg 120 aattaggacc ttatattgtc gaatacagcg ctgattatcg cgttcttcgt atcgccacta 180 ctccgtttca gctctgtttc tacaacacaa cacctaacgc atggacacta gcgttgcgaa 240 tgggcaccgt ccgttctgaa tcactcatgc gttgggtgtg ggaagctaac aggggaaacc 300 ccgttaaaga aaacgccact ttcactttcg ggacagatgg aaatctcgta ttggctgatg 360 ccgacggtcg aattgcatgg caaacaaaca cggccaataa gggcgtgaca gggttcaagt 420 tgttgcctaa tggtaacatg gtgctacatg actccaaggg aaaattcctc tggcagagtt 480 tcaactatcc cacggatact ttactagtgg gccaaacact acgtttgtcc agcccgaata 540 agctcgtgag ccgggcttcg gtgaaaaaca atgcgaacgg gccttatagc ttggtggtgc 600 agccaagatt gtttggtgtc tacaagggga ctaagctaga cgtcgaatta gcttggttca 660 attatggtaa tagcactttg gaatcagtaa aattgaatgc tgggaatcaa aggctgaaat 720 tggattatag attggcaaaa tcaacaacga gaagttcaca tgttatggct tttgtcaatt 780 acaacacaac attgacatat cttagactag aaattgatgg aaatctcaag gcctacactt 840 ttgttcgaga tgaagaagca gatgaattcc gttggaaggt aacttacagc aggatttaaa 900 aaatgttttt cgttaaaagt ttgtgtacac actattatct cagactccga cgttacacga 960 acatgttatt gttgtaagga ttagtgtcca gtataataag tggataaagc gagtgggtcc 1020 acagctagtc caataaatta gttgtaattc ctattttatc ttctgttttc taaagttgta 1080 ttttggttga attaagaatg tattttattt tgatattacc agcattattt ttggattgaa 1140 aaaaaaaaaa aaaaaaaaaa 1160 <210> 2 <211> 297 <212> PRT 213 Capsicum annuum L. cv. Nockwang <400> 2 Met Ser Pro Ser Trp Thr Thr Thr Leu Phe Val Ser Leu Phe Leu Phe   1 5 10 15 Ser Gln Ile Phe Ser Cys Ile Ala Gln Val Pro Ala Glu Asn Thr Phe              20 25 30 Lys Phe Val Asn Glu Gly Glu Leu Gly Pro Tyr Ile Val Glu Tyr Ser          35 40 45 Ala Asp Tyr Arg Val Leu Arg Ile Ala Thr Thr Pro Phe Gln Leu Cys      50 55 60 Phe Tyr Asn Thr Thr Pro Asn Ala Trp Thr Leu Ala Leu Arg Met Gly  65 70 75 80 Thr Val Arg Ser Glu Ser Leu Met Arg Trp Val Trp Glu Ala Asn Arg                  85 90 95 Gly Asn Pro Val Lys Glu Asn Ala Thr Phe Thr Phe Gly Thr Asp Gly             100 105 110 Asn Leu Val Leu Ala Asp Ala Asp Gly Arg Ile Ala Trp Gln Thr Asn         115 120 125 Thr Ala Asn Lys Gly Val Thr Gly Phe Lys Leu Leu Pro Asn Gly Asn     130 135 140 Met Val Leu His Asp Ser Lys Gly Lys Phe Leu Trp Gln Ser Phe Asn 145 150 155 160 Tyr Pro Thr Asp Thr Leu Leu Val Gly Gln Thr Leu Arg Leu Ser Ser                 165 170 175 Pro Asn Lys Leu Val Ser Arg Ala Ser Val Lys Asn Asn Ala Asn Gly             180 185 190 Pro Tyr Ser Leu Val Val Gln Pro Arg Leu Phe Gly Val Tyr Lys Gly         195 200 205 Thr Lys Leu Asp Val Glu Leu Ala Trp Phe Asn Tyr Gly Asn Ser Thr     210 215 220 Leu Glu Ser Val Lys Leu Asn Ala Gly Asn Gln Arg Leu Lys Leu Asp 225 230 235 240 Tyr Arg Leu Ala Lys Ser Thr Thr Arg Ser Ser His Val Met Ala Phe                 245 250 255 Val Asn Tyr Asn Thr Thr Leu Thr Tyr Leu Arg Leu Glu Ile Asp Gly             260 265 270 Asn Leu Lys Ala Tyr Thr Phe Val Arg Asp Glu Glu Ala Asp Glu Phe         275 280 285 Arg Trp Lys Val Thr Tyr Ser Arg Ile     290 295  

Claims (10)

An isolated gene ( CaMBL1) of (a) or (b) which encodes pepper-derived mannose binding lectin as a plant disease defense related gene: (a) a gene encoding a protein having the amino acid sequence of SEQ ID NO: 2; (b) a gene having the nucleotide sequence of SEQ ID NO: 1. An isolated protein having the amino acid sequence of SEQ ID NO: 2 (CaMBL1). A recombinant vector comprising the gene of claim 1. Recombinant vector pBIN35S :: CaMBL1 prepared by inserting the gene of claim 1 into pBIN35S . 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 CaMBL1 gene described in claim 1 resistance detection method of the plant. 7. The method of claim 6, wherein the plant is a plant inoculated with pathogenic or non-pathogenic bacteria of red pepper bacterial spot pattern disease. The method of claim 6, wherein the plant is a plant treated with salicylic acid (Salicylic acid) or methyl jasmonate (Methyl jasmonate). A method for detecting resistance of a plant, comprising the step of checking the expression of CaMBL1 protein using the antibody of claim 2 CaMBL1 protein as a probe. The recombinant vector TRV2 :: CaMBL1 prepared by inserting the CaMBL1 gene according to claim 1 into a Tobacco Rattle Virus 2 (TRV2) vector, which is a vector for virus-induced gene silencing (VIGS).

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