KR101047619B1 - Calmodulin gene CMC1 associated with pepper disease resistance and transformed plants using the same - Google Patents

Abstract

The present invention relates to a pepper calmodulin (CaCaM1) gene (SEQ ID NO: 1), which is a novel gene associated with plant disease resistance, and a pathogen resistant transgenic plant using the gene. The present invention also provides a method for searching for biotic and abiotic stress resistance in plants using the gene. According to the present invention described above, it can be used as a marker of the defense reaction of plant pathogens, can provide a genetic material for molecular breeding of disease-resistant and environmental stress-resistant plants, can facilitate the search for plant disease resistance, etc. have

Pepper, Plant Disease, Resistant, Bacterial Spot Disease, CaCaM1, Calmodulin

Description

Pepper disease resistance-related Calmodulin gene CaCaM1 and transgenic plants using the same}

The present invention is red pepper varieties ( Capsicum annuum L. cv. Nockwang) Calmodulin gene CaCaM1 , a novel gene related to plant disease resistance isolated from plants ( Capsicum annuum Calmodulin 1 ) and methods of screening for biotic and abiotic stress in plants and for producing disease resistant transgenic plants. More specifically, the present invention is to isolate the cDNA clone containing the CaCaM1 gene, a new gene related to plant disease resistance isolated from the red pepper cultivation varieties, sequencing the base sequence and amino acid sequence of the CaCaM1 gene obtained here The present invention provides a resistance search method that searches for differential expression of a gene during bio / chemical / physical resistance induction treatment using the sequence. In addition, the present invention CaCaM1 Recombinant vector with the gene (pBIN35S: CaCaM1) and Agrobacterium tumefaciens (Agrobacterium tumefaciens strain GV3101) with the strain, transient expression in the pepper plant (transient expression) method and Arabidopsis (Arabidopsis thaliana ) relates to the confirmation of plant pathogenic activating function of CaCaM1 gene through transgenic expression in plants, thereby providing a transgenic plant.

Pepper, which is a representative eggplant plant with tobacco and tomatoes, is one of the most used vegetables in our diet, and 65% of the entire farms depend on farm income through pepper production, which is a major economic crop.

In pepper plants, viral diseases, bacterial diseases, fungal diseases, and the like have been reported in various ways, and chemical control methods using pesticides have been mainly used until now to control these diseases. However, the excessive use of pesticides has caused the toxicity to destroy ecosystems and threaten human survival. In particular, in Korea, interest in the regulation of pesticide use has been continuously increasing since joining the OECD in 1996. Therefore, in the future, finding ways to improve and breed disease-resistant varieties is necessary for sustainable agriculture.

On the other hand, when the plant is infected with a pathogen, the host plants is to activate defense mechanisms by operating the in metabolic system a number of cells, the calcium in such defense mechanism ions (Calcium ion, Ca ^{2 +),} lipid (lipid), cyclic nucleotide It is known that a cyclic nucleotide and the like serve as a second messenger for transmitting an invasion signal. Especially Ca ^{2 +} has been reported to increase during pathogen invasion, the activity of the vacuoles (vacuole), ER (endoplasmic reticulum) are rapidly ejected from the cell organelles, such as the cytoplasm (cytoplasm) calmodulin (Calmodulin, CaM). However, a number of calmodulin isoforms exist in plants, and they are reported to play different roles for various stresses, and their function is responsible for the defense mechanisms of plants that respond to various stresses. In addition to helping to understand this, it can also contribute to breeding resistant varieties.

The present invention is to solve such a problem in the prior art, the purpose of which is pepper bacterial spot pattern disease ( Xanthomonas campestris pv. Calcica ( CaCaM1 : Capsicum ), a gene related to plant disease resistance, is involved in the defense against vesicatoria annuum Calmodulin 1 ) is isolated and translated to provide the sequence information and the amino acid sequence information accordingly.

In addition, the present invention is to provide a resistance search method for searching for the presence of resistance to plant diseases, chemicals or environmental stress using the CaCaM1 gene. Furthermore, the aim is to produce a plant disease or environmental stress resistant transgenic plant using this gene.

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, red pepper varieties ( Capsicum annuum cv. Nockwang) Pepper Bacterial Spot Disease ( Xanthomonas) campestris pv. After inoculation of vesicatoria ), the new resistance-related calmodulin gene CaCaM1 isolated from the inoculated pepper leaves was identified to determine its base sequence, and the presence of CaCaM1 was expressed through pathogens, chemicals, and environmental stress treatment. And pepper plants and transgenic Arabidopsis vs. Arabidopsis thaliana ) was achieved by confirming the plant disease resistance induction function of the CaCaM1 gene.

Therefore, the present invention is red pepper varieties ( Capsicum annuum L. cv. Nockwang) and isolated calmodulin gene CaCaM1 having the nucleotide sequence of SEQ ID NO: 1 related to plant disease resistance and isolated calmodulin protein (CaCaM1) having the amino acid sequence of SEQ ID NO: 2.

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

Furthermore, the present invention is to isolate the RNA from the plant inoculated or treated with any one of the pathogens, chemicals and environmental stress by identifying the differential expression of CaCaM1 gene by Northern blot analysis, plant resistance, chemicals or environmental stress Provide a resistive search method.

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

First, the present invention is pepper bacterium bacillus bacterium ( Xanthomonas campestris pv. vesicatoria ) was isolated from mRNAs of nocturnal cultivars that showed hypersensitivity resistance to inoculation of non-pathogenic strains of vesicatoria , and cDNA libraries were prepared. Total mRNAs obtained from leaves and healthy pepper plants inoculated with non-pathogenic strains were deoxygenated. Probes are labeled with digoxygenin and differential hybridization is performed using the cDNA and the probe to select clones that are thought to be involved in pathogenicity to obtain clones. By sequencing the nucleotide sequence of the gene identified as heavy calmodulin gene, the calmodulin protein coding gene nucleotide sequence (SEQ ID NO: 1) and amino acid sequence encoded therefrom of the red pepper verdure variety named CaCaM1 ( SEQ ID NO: 2) is provided (see FIG. 1).

Combining four calcium ions typical of results calmodulin gene was analyzed and the amino acid sequence of CaCaM1 protein according to the invention were found to have the ^{(Calcium ion, Ca 2 + -binding} ) region, tomato (Solanum lycopersicum and the calmodulin gene (accession no. AK246240) were 89% at the level of nucleotides ( Capsicum) annuum) by the calmodulin gene (accession no. AF108889) than denotes homology of 88% at the nucleotide level was found to be novel (http://blast.ncbi.nlm.nih.gov/Blast.cgi, nucleotide blast program). The gene base sequence of the identified CaCaM1 and the amino acid sequence encoded by the base sequence are shown in FIG.

Next, the present invention provides a method for detecting resistance of plants consisting of confirming whether CaCaM1 protein is expressed by applying selected organs and biological or abiotic induction methods in a red pepper plant and performing northern blots. More specifically, the present invention consists of confirming whether CaCaM1 protein is expressed by using the CaCaM1 protein gene as a probe for a selected organ in a red pepper inoculated or treated with any one of pathogens, chemicals and environmental stresses. It also provides a method of searching for resistance of plants.

The pathogen may be a pathogenic or non-pathogenic bacterium of red pepper bacterial spot disease, the chemical may be salicylic acid, methylzasmonic acid, abscis acid, or hydrogen peroxide, and the environmental stress may be dry, wounded or cold.

It was confirmed through experiments that the CaCaM1 gene according to the present invention is differentially induced and expressed by various phytopathogens , chemicals or environmental stresses (see FIGS. 3 to 10). Therefore, the CaCaM1 gene according to the present invention can be used for a method for detecting resistance of plants.

In addition, the present invention is the CaCaM1 By using a recombinant vector containing a gene to determine whether the plant disease resistance of pepper plants and transgenic Arabidopsis plants can be produced, a new method for producing a disease-resistant transformed plant and a material for breeding resistant molecules can be provided. More specifically, the present invention provides a transient expression in a pepper plant using a recombinant vector (pBIN35S :: CaCaM1 ) (FIG. 11) and an Agrobacterium tumefaciens strain GV3101 strain containing a CaCaM1 gene. ) Method and Arabidopsis ( Arabidopsis) thaliana ) Confirming the plant pathogenic activating function of the CaCaM1 gene through transgenic expression in plants (see FIGS. 12 to 19), thereby providing a transgenic plant.

As described above, by completing the present invention, pepper bacterial spot disease ( Xanthomonas) is a pepper plant bacterial disease campestris pv. CaCaM1 , which encodes a calmodulin protein, one of the genes involved in plant disease resistance, which is involved in the defense against vesicatoria ) By separating and deciphering genes, the sequence information and amino acid sequence information are provided, and at the same time, the gene provides a method for screening plant disease resistance and environmental stress resistance as well as contributing to the production of resistant plant breeding and transgenic plants. It became possible to plan the effect.

The present invention enables the accumulation of gene information of the reaction to the disease of calmodulin and related disease-associated protein (PR protein) can provide an interesting model for signaling of the disease of plants, and resistance By understanding the biochemical changes that result, it may be practically used to develop genetically engineered plants that promote disease resistance or to develop plant protection chemicals that work to promote the plant's genetic resistance. 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. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

[ Example  One] CaCaM1 Gene base sequence and amino acid sequence determination

Calmodulin ( CaM1 : Capsicum) is one of the pathogenic proteins related to red pepper bacterial spot disease . annuum Calmoudulin1 ) The following tests were performed to provide protein coding gene sequences and amino acid sequences deduced therefrom.

Capsicum pepper varieties ( Capsicum annuum cv. Nockwang ( Xanthomonas Red Pepper) campestris pv. vesicatoria ) were inoculated and treated for 18 hours in a wet place , and then the plants showing the sensitizing apoptosis, a resistant lesion, were taken out of the room and the leaves were collected.

Next, cDNA libraries were 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 membrane (Hybond N +) constantly, and then the leaves of the red pepper plants inoculated with non-pathogenic strains of the red pepper bacterial spot pattern bacteria and the healthy red pepper plants not inoculated. Total mRNA extracted from the leaves was labeled with dioxygenin (digoxygenin) to make a probe, and then differential hybridization was performed.

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

After sequencing the clones obtained, the CaCaM1 protein coding gene was identified and named as a CaCaM1 gene by comparing its 5'-terminal sequence with a gene database registered in GenBank using a blast program. The CaCaM1 protein according to the present invention was found to have four calcium ion (Calcium ion, Ca ^{2 +} -binding) sites, which are typical of calmodulin gene, and tomato ( Solanum). 89% at the nucleotide level than calmodulin gene (accession no. AK246240) of lycopersicum), peppers (Capsicum a calmodulin gene (accession no. AF108889) than at the nucleotide level, indicates a homology of 88% of annuum) was found to be novel (http://blast.ncbi.nlm.nih.gov/Blast.cgi, nucleotide blast program). The gene base sequence of the identified CaCaM1 and the amino acid sequence encoded by the base sequence are shown in FIG.

[ Example  2] Red pepper in red pepper plants by pathogen inoculation CaCaM1  How to Explore Expression of Genes

In order to confirm the method of using the CaCaM1 gene obtained in Example 1 for the resistance search was tested as follows.

[Step 1]

First, in order to present a specific resistance screening method, Xanthomonas pepper bacterium, which shows a friendly reaction with plants, campestris pv. vesicatoria) of the pathogenic strain Ds1 and fire After incubation the non-pathogenic strain Bv5-4a represents the reaction-friendly, 10 pepper nokgwang ⁸ varieties of 4 leaf stage in a concentration of cfu / ㎖ (Capsicum annuum cv. Nockwang) infiltrated (Infiltration) using a syringe on the back of the first and second leaves, and after the inoculation was subjected to a wet treatment for 18 hours at 28 ℃, 100% relative humidity conditions.

After 1, 6, 12, 18, 24 and 48 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, the CaCaM1 gene obtained in Example 1 was labeled with 14-dCTP-biotin and then amplified by PCR (Polymerase Chain Reaction), 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 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.

In this way, friendly reactions after inoculation of pathogenic strains of red pepper bacillus bacteria and incompatible reactions after inoculation with non-pathogenic strains The expression pattern of CaCaM1 gene is shown in FIG. 3. In Figure 3, the number represents the elapsed time from inoculation to pepper leaf collection.

As shown in FIG. 3, the CaCaM1 gene was expressed as a fast response after inoculation against the pathogenic and non-pathogenic strains as a resistance of the pepper plants to pepper bacterial spot disease. More specifically, the red pepper plants inoculated with non-pathogenic strains showed a strong and long-lasting expression. In other words, CaCaM1 in a friendly reaction Although the expression of the gene reached its peak at 1 hour, it rapidly decreased, but in an incompatible reaction, the expression peaked at 1 hour after inoculation and expression was maintained until 12 hours.

[ Example  3] by chemical derivatives CaCaM1  Induced Expression of Genes

In order to find out whether the CaCaM1 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 the expression of CaCaM1 gene, salicylic acid, methyljasmoic acid, abscisic acid, Resistance induction test was conducted using a small number of peroxide (H ₂ O ₂ ). Six-leaf peppers were used for all experiments.

Salicylic acid was 5 mM, methylzasmonic acid was 100 μM, abscisic acid was 100 μM, hydrogen peroxide was 10 mM and sprayed on the leaves of pepper, and treated with methylzasmonic acid after sealing.

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 CaCaM1 gene obtained in Example 1 was labeled with 14-dCTP-biotin, and then amplified using PCR (Polymerase Chain Reaction), which was then reacted with [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA. , 5% Dextran Sulfate] was incubated at 65 ° C. for 16-24 hours to perform hybridization. The biotin-labeled DNA probe is attached to the mRNA attached to the nylon membrane (Hybond N +). When the X-ray film is placed on the nylon membrane (Hybond N +), the biotin-labeled DNA is Since the X-ray film is photosensitized, it is 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 above experiments are shown in FIGS. 4 to 7, and as a result of the experiment, the CaCaM1 gene was induced from 1 hour after salicylic acid treatment, and the expression was maintained strongly until 10 hours, and gradually decreased thereafter (FIG. 4). After treatment with methylzasmonic acid, expression was induced at 1 hour and then expression was decreased (FIG. 5). After treatment with abscic acid and hydrogen peroxide, expression was gradually increased to 25 hours. It was possible (FIGS. 6 and 7).

[ Example  4] due to environmental stress CaCaM1  Induced Expression of Genes

In order to find out whether the CaCaM1 gene of the present invention can be induced by environmental stress and to provide a specific induction method, the following test was performed.

In each test, it was confirmed that the same amount of RNA sample was loaded by monitoring the amount of ribosomal RNA when performing Northern blotting, and a healthy leaf that was not subjected to any stress was used as a control.

[Step 1]

In order to find out whether the CaCaM1 gene of the present invention can be induced by dry stress and to provide a specific induction method, the following test was performed.

In order to investigate the CaCaM1 resistance induction effect after drying, pepper plants were harvested at 1, 5, 10, 15, 20, and 25 hours after the dry stress treatment.

After extracting RNA from each sample, by performing Northern blotting in the same manner as in step 1 of Example 3, the time- dependent expression of CaCaM1 after the dry stress treatment was examined , the results are shown in Figure 8 .

As shown in FIG. 8, the CaCaM1 gene was found to increase in expression at 1 hour but then decreased in expression.

[Step 2]

In order to find out whether the CaCaM1 gene of the present invention can be induced by wound stress and to provide a specific induction method, the following test was performed.

In order to handle the wound stress on the red pepper plants, the leaves of the plants were pierced with needles and wounded throughout the leaves, and the leaves were collected after 0.25, 0.5, 1, 5, 10, 15, 20, and 25 hours, respectively.

After extracting RNA from each sample, by performing Northern blotting in the same manner as in step 1 of Example 3, the expression pattern of CaCaM1 gene over time after wound stress treatment was examined, and the results are shown. 9 is shown.

As a result, as shown in Figure 9, the CaCaM1 gene peaked at 0.5 to 1 hours after treatment, after which the accumulation amount decreased.

[Step 3]

In order to find out whether the CaCaM1 gene of the present invention can be induced by cold stress and to provide a specific induction method, the following test was performed.

In order to determine the CaCaM1 resistance induction effect after the low-temperature treatment, pepper plants were treated at 4 ℃ and leaves were collected at 1, 5, 10, 15, 20 and 25 hours, respectively.

After extracting RNA from each sample, by performing Northern blotting in the same manner as in step 1 of Example 3, the time- dependent expression of CaCaM1 after cold stress treatment, and the results are shown in Figure 10 .

Test results, as shown in Figure 10, CaCaM1 The gene began to express at 1 hour and appeared to be the most strongly expressed at 20 hours.

[ Example  5] CaCaM1  Preparation of Transgenic Plants Using Genes CaCaM1  Changes in Disease Resistance of Red Pepper Plants Caused by Gene Overexpression

In order to investigate whether the CaCaM1 gene was overexpressed in red pepper plants to induce other genes related to disease development and to increase disease resistance, the CaCaM1 gene obtained in Example 1 was introduced into a pBIN3S vector provided by Stratagene. PBIN35S :: CaCaM1 was prepared (FIG. 11).

The recombinant vector pBIN35S :: CaCaM1 was prepared as Agrobacterium tumefaciens ( Agrobacterium). Tumefaciense strain GV 3101) was introduced through electroporation and inoculated to pepper leaves of GV3101 strain containing the recombinant vector to observe the disease resistance change of pepper plants overexpressing CaCaM1 gene. The test was performed.

[Step 1]

A recombinant vector overexpressing CaCaM1 (35S: CaCaM1) and the control group that does not over-expressing the CaCaM1 vector (35S: 00) the transformant in which Agrobacterium tumefaciens (Agrobacterium using Tumefaciense strain GV 3101 was shaken in Yeast-Peptone (YEP: yeat extract 10g, peptone 10g, sodium chloride 5g / L) medium, and the strain suspension concentration was measured using a spectrophotometer (optical density 600, OD). ₆₀₀ ) to 0.5, 0.2, 0.1, 0.05, and inoculated with the leaf vein of the pepper leaves, respectively, and the results are shown in Figure 12.

As shown in Figure 12, was observed that the cell death that occurs in inoculated with Agrobacterium (Agrobacterium) overexpressing the inoculation day 4, CaCaM1 portion (first picture of FIG. 12), where the phenol in the peripheral region Castle The accumulation of phenolic compounds could be observed under an ultra violet (UV) lamp (second picture in FIG. 12).

In order to observe the apoptosis at the site of inoculation, the chlorophyll of the pepper leaf was decolorized with ethanol, and it was more clearly observed that the site of the apoptosis was browned (third photo of FIG. 12). On day 1 after inoculation, DAB staining (3,3'-diaminobenzidine staining) was performed to determine the amount of free radicals known as pathogenic signaling materials. The results are shown in the fourth photograph of FIG. 12. As a result, it was observed that the accumulation of free radicals that occur strongly Agrobacterium (Agrobacterium) strains overexpressing CaCaM1 from one injection site.

[Step 2]

In order to observe the expression of resistance-related genes in CaCaM1 overexpressed pepper leaves, plants were harvested, RNA was extracted, Alti-pisal (RT-PCR) was performed, and the results are shown in FIG. 13. The increase in the expression of CaCaM1 CaCaM1 leaves inoculated with Agrobacterium Terry Stadium (Agrobacterium) overexpressing such as 13 and reported as disease resistance genes CaBPR1 (Capsicum The expression of annuum basic pathogenesis-related protein 1), CaPR10 ( Capsicum annuum pathogenesis-related protein 10), and CaPOA1 ( Capsicum annuum ascorbate peroxidase 1) were also increased (Choi et al., 2007 Plant Physiology Vol. 145). 890-904p).

[Step 3]

CaCaM1 is one part of the pepper leaves to observe an increase if the disease resistance in overexpressed pepper leaf overexpressing CaCaM1 or (35S: CaCaM1) that do not overexpress (35S: 00) Agrobacterium inoculation Leeum (Agrobacterium) and the other side After inoculation with red pepper bacterial spot pathogen was determined whether resistance. The results are shown in FIG.

As shown in Figure 14, the pepper leaves overexpressed CaCaM1 was observed to weaken the symptoms, it was observed that the growth rate of the bacteria is reduced. Therefore, it was observed that the resistance to red pepper bacterial spot germ was increased in CaCaM1 overexpressed red pepper leaves.

[ Example  6] CaCaM1  Disease Resistance Changes in Arabidopsis Plants by Overexpression of Genes

Model to investigate than treat the increase in the resistance to the induced and whether the bottle of genes associated with different disease occurs when over expression of CaCaM1 gene in Arabidopsis thaliana plants used in the plant or not, a CaCaM1 gene obtained in Example 1 in the Stratagene PBIN35S: CaCaM1 was prepared by introducing into a provided pBIN35S vector (FIG. 11).

The recombinant vector pBIN35S :: CaCaM1 was prepared as Agrobacterium tumefaciens ( Agrobacterium). Tumefaciense strain GV 3101) was introduced by electroporation and transformed by introducing the GV3101 strain containing the recombinant vector into Arabidopsis plants through floral dipping to induce overexpression of the CaCaM1 gene. After the test was carried out as follows.

[Step 1]

In order from the CaCaM1 gene is overexpressed plants Learn whether the resistance to bacterial disease increases, the resistance-related genes of CaCaM1 and Arabidopsis thaliana plants from the wild-type Arabidopsis thaliana plants as the CaCaM1 gene is overexpressed Arabidopsis thaliana and control PR1, PDF1 .2, were examined the expression of the gene RD29a shown in Figure 15 the results.

# 1, # 2, and # 3 in FIG. 15, # 4, # 5 is a trait indicates the system number of the transgenic plants, the experimental results CaCaM1 gene overexpression transgenic Arabidopsis plants only CaCaM1 genes and disease resistance related PR1 use , PDF1 .2 was expressed.

[Step 2]

When inoculated with Pseudomonas strain ringge lost bar tomato DC3000 in a plant CaCaM1 the over expression as described above and observed changes in the plant. In addition, DAB staining and trypan blue staining were performed to investigate the reason for the resistance in plants overexpressed CaCaM1 gene.

As a result, as shown in Fig. 16, CaCaM1 over-expressed plants showed resistance to bacterial diseases compared to wild type, DAB staining results and trypan blue staining (trypan blue staining) of FIG. Results Through CaCaM1 In the overexpressed genes, free radicals and apoptosis were observed in resistant plants. In addition, it could be confirmed through Northern blotting that the expression of resistance-related gene PR1 is increased compared to the control.

[Step 3]

Hyaloperonospora ( Haloperonospora ), a hyalperonospora parasitica that causes downy mildew in the Arabidopsis, in plants overexpressed with CaCaM1 After inoculation with parasitica Noco 2), changes in disease resistance were observed. Hyaloferonospora parasitica Noco2 was sprayed at a concentration of 5 × 10 ⁴ spores / ml during the cotyledon period of overexpressed CaCaM1 and non-overexpressed wild type control plants. After inoculation, it was wet-treated at the temperature of 17 degreeC. As a result, the resistance was increased in transgenic Arabidopsis plants overexpressing CaCaM1 , and it was observed that spore formation of the fungal pathogen was suppressed, and the results are shown in FIG. 18.

[Step 4]

In order to observe at the cellular level the resistance to fungal disease of plants overexpressed CaCaM1 , DAB staining and trypan blue staining were performed and the results are shown in FIG. 19. As a result, the increase in free radicals and apoptosis reactions observed in resistant plants were observed in plants overexpressing the CaCaM1 gene. However, it was observed that free radicals and apoptosis did not occur in wild-type control plants, that is, plants not overexpressing CaCaM1 .

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.

1 is a pepper verdure varieties ( Capsicum) by the present onset annuum L. cv. A diagram showing the nucleotide sequence and amino acid sequence of the pepper calmodulin 1 ( CaCaM1 ) gene cloned from Nockwang ),

Figure 2 is a diagram showing the expression results of CaCaM1 gene in each organ of the red pepper varieties,

Figure 3 is red pepper bacterial spot pattern bacteria ( Xanthomonas) in pepper plants campestris pv. vesicatoria ) is a diagram showing the results of induction of CaCaM1 gene expression in inoculated and non-inoculated leaves when inoculated with pathogenic strains and non-pathogenic strains,

4 is a diagram showing the results of inducing the expression of CaCaM1 gene over time after the treatment of salicylic acid (Salicylic acid) to pepper plants,

5 is a diagram showing the results of inducing the expression of CaCaM1 gene over time after the treatment of red pepper plants with methyljasmonic acid (Methyjasmonic acid),

6 is a view showing the results of inducing the expression of CaCaM1 gene over time after the treatment of abscisic acid in pepper plants,

7 is a view showing the results of inducing the expression of CaCaM1 gene over time after the treatment of hydrogen peroxide (H ₂ O ₂ ) to pepper plants,

8 is a view showing the results of inducing the expression of CaCaM1 gene over time after the dry stress treated pepper plants,

9 is a view showing the results of inducing the expression of CaCaM1 gene over time after the treatment of the wound stress on pepper plants,

10 is a view showing the results of inducing the expression of CaCaM1 gene over time after the cold stress treatment on pepper plants,

Figure 11 is CaCaM1 gene pepper and Arabidopsis ( Arabidopsis thaliana ) is a diagram showing an arrangement map of the recombinant vector pBIN35S: CaCaM1 for overexpression in plants,

12 is a recombinant vector pBIN35S: CaCaM1 and Agrobacterium tumefaciens strain GV3101 strain using overexpression of CaCaM1 gene in red pepper plants to induce a resistance reaction including reactive oxygen generation and hypersensitive cell death Is a diagram showing the result,

13 is a diagram showing the results of confirming the expression of the defense-related genes of pepper plants through the RT-PCR when over-expressing CaCaM1 gene in pepper plants ( CaBPR1 : Capsicum annuum basic pathogenesis - related protein 1 , CaPR10 : Capsicum annuum pathogenesis-related protein 10 , CaPOA1 : Capsicum annuum ascorbate peroxidase 1 ),

Figure 14 shows bacterial spots on pepper leaves overexpressing the CaCaM1 gene in pepper plants ( Xanthomonas campestris pv. vesicatoria ) is a view showing the result of increased resistance to,

15 is a recombinant vector according to the present invention pBIN35S: CaCaM1 and Agrobacterium tumefaciens (Agrobacterium tumefaciens strain GV3101) with the strain, and the CaCaM1 gene expression over in transgenic Arabidopsis thaliana plants that overexpressed the CaCaM1 gene, resulting in Arabidopsis PR1 ( Pathogenesis - realted) protein 1) and PDF1 .2 (Defensin - liken protein ) is a diagram showing the results of observing the increase in the expression of the gene,

FIG. 16 shows Pseudomonas cyringe Pasova tomato DC3000 ( Pseudomonas) , a bacterial E. coli pathogen, in transgenic Arabidopsis plants overexpressing the CaCaM1 gene syringae pv. tomato DC3000) is a graph showing the results of assaying pathogen growth in plants and photographs of plants showing disease resistance to strains,

17 shows the increase in free radicals and sensitizing apoptosis after bacterial E. coli inoculation in transgenic Arabidopsis plants overexpressing the CaCaM1 gene, respectively, by DAB staining and tryptophan blue staining. At this time, it is a view showing the result that the expression of PR1 is increased,

18 shows downy mildew, Hyaloperonospora , in transgenic Arabidopsis plants overexpressing the CaCaM1 gene. parasitica ) is a diagram showing the results of disease resistance to,

19 shows downy mildew, Hyaloperonospora in transgenic Arabidopsis plants overexpressing the CaCaM1 gene. parasitica ) shows the result of DA accumulation and trypan blue staining, which are accompanied by accumulation of active oxygen and hypersensitivity apoptosis when pathogenic resistance to parasitica appears.

<110> Korea University Industrial and Academic Collaboration Foundation <120> Pepper disease resistance-related Calmodulin gene CaCaM1 and          transgenic plants using the same <130> P11-080729-01 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 687 <212> DNA 213 Capsicum annuum cv. Nockwang <400> 1 tctttccaaa aataaaaaca agtagtacaa cttatcttgt atttgaaatt ctagaagaaa 60 atggcagatc agttgactga tgaccagatc tctgagttta aggaagcatt tagcttgttt 120 gataaggatg gagatggttg catcactact aaggagcttg ggactgtgat gaggtcgctg 180 ggacagaacc cgactgaagc tgaactccag gacatgataa acgaggtgga tgcagatggt 240 aatggaacca tcgacttccc agagtttcta aacctcatgg ccaagaagat gaaggataca 300 gattccgagg aggagctgaa agaggcattc agggttttcg acaaggatca aaatggcttc 360 atctcggctg ctgagcttcg ccatgtgatg actaaccttg gtgagaagct cactgatgaa 420 gaagtcgatg agatgattag ggaagcggat ttcgatggtg atggtcaaat taactatgaa 480 gagtttgtta aggtcatgat ggccaagtaa tccaccaccc tcttgttaaa agtttaagtt 540 tagacttgtt aaaatttgtg aaaaaatgcc aaaaaatgtt tcattggata ggatgtgctt 600 agtataattt atgtttttac catctctaga tgtattggac cttgaatgaa tgtaatgttt 660 tattgtaaaa aaaaaaaaaa aaaaaaa 687 <210> 2 <211> 149 <212> PRT 213 Capsicum annuum cv. Nockwang <400> 2 Met Ala Asp Gln Leu Thr Asp Asp Gln Ile Ser Glu Phe Lys Glu Ala   1 5 10 15 Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly Cys Ile Thr Thr Lys Glu              20 25 30 Leu Gly Thr Val Met Arg Ser Leu Gly Gln Asn Pro Thr Glu Ala Glu          35 40 45 Leu Gln Asp Met Ile Asn Glu Val Asp Ala Asp Gly Asn Gly Thr Ile      50 55 60 Asp Phe Pro Glu Phe Leu Asn Leu Met Ala Lys Lys Met Lys Asp Thr  65 70 75 80 Asp Ser Glu Glu Glu Leu Lys Glu Ala Phe Arg Val Phe Asp Lys Asp                  85 90 95 Gln Asn Gly Phe Ile Ser Ala Ala Glu Leu Arg His Val Met Thr Asn             100 105 110 Leu Gly Glu Lys Leu Thr Asp Glu Glu Val Asp Glu Met Ile Arg Glu         115 120 125 Ala Asp Phe Asp Gly Asp Gly Gln Ile Asn Tyr Glu Glu Phe Val Lys     130 135 140 Val Met Met Ala Lys 145  

Claims (9)

Capsicum pepper varieties ( Capsicum annuum L. cv. An isolated calmodulin gene ( CaCaM1 ) having a nucleotide sequence of SEQ ID NO: 1 from Nockwang ). An isolated calmodulin (CaCaM1) protein having the amino acid sequence of SEQ ID NO. CaCaM1 as described in claim 1 Recombinant vector comprising a gene. Recombinant vector pBIN35S :: CaCaM1 prepared by inserting the CaCaM1 gene according to claim 1 into pBIN35S . A plant transformed by the gene of claim 1 or the recombinant vector of claim 3. The plant of claim 5, wherein the plant is pepper or Arabidopsis. RNA isolated from plants inoculated with pathogenic or non-pathogenic bacteria of red pepper bacterial spot disease by Northern blot analysis as described in claim 1 CaCaM1 A method for searching for resistance of a plant, comprising the step of confirming the expression of a gene. CaCaM1 gene as described in claim 1 by RNA isolated from plants treated with salicylic acid, methyljasmoic acid, abscisic acid and hydrogen peroxide (H ₂ O ₂ ). Searching for resistance of the plant comprising the step of confirming the expression of. A method of detecting resistance of plants, comprising the step of confirming the expression of CaCaM1 gene according to claim 1 by Northern blot analysis by separating RNA from plants treated with any one of wound stress, dry stress and cold stress.

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