KR20110028184A - Disease resistance-related pepper e3 ubiquitin ligase gene caring1 and transgenic plants using the same - Google Patents

Abstract

PURPOSE: An E3 ubiquitin ligase gene(CaRING1) and a disease resistant transgenic plant using the same are provided to search plant disease resistance. CONSTITUTION: A gene CaRING1(Capsicum annuum E3 ubiquitin ligase RING1) encoding Capsicum annuum-derived E3 ubiquitin ligase is a gene of sequence number 1. The protein has an amino acid sequence of sequence number 2. A recombinant vector pBIN35S::CaRING1 contains gene, CaRING1. A detection for disease resistance of a plant is performed by isolating mRNA from a plant transformed by the recombinant vector and confirming different expression of CaRING1 gene.

Description

Disease resistance-related pepper E3 ubiquitin ligase gene CaRING1 and transgenic plants using the same}

The present invention relates to a pepper E3 ubiquitin ligase gene ( CaRing1 : Capsicum annuum E3 ubiquitin ligase RING1 ), which is a novel gene related to plant disease resistance, and a biological stress screening method using the same and a pathogenic transgenic plant. More specifically, the present invention relates to a base sequence of CaRING1 gene, a novel gene related to plant disease resistance, which is induced specifically when infected with pathogenic and non-pathogenic strains of red pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ) The present invention provides an amino acid sequence, and relates to a method for detecting resistance of a plant to identify differential expression of resistance-related gene CaRING1 when inoculating pathogenic or non-pathogenic pathogens. In addition, the present invention using the virus-induced gene silencing (VIGS) technology in pepper plants to inhibit the expression of the CaRING1 gene to confirm the role of CaRING1 gene in pathogenic resistance, and the CaRING1 gene The Agrobacterium- mediated transient expression method and the transgenic expression of Arabidopsis thaliana were introduced into the pepper plants to confirm the effect of increasing the plant disease resistance of the CaRING1 gene. The present invention relates to providing a plant disease resistant transgenic plant.

Plants have various forms of defense-related mechanisms to prevent invasion of external pathogens. These defense-related mechanisms effectively protect plants and identify biomechanical mechanisms that have not been known to date not only play an important role in crop production but also have scientific and technological value. In addition, specific gene information derived for plant defense-related mechanisms can be used as an important resource for breeding new crops.

Red pepper ( Capsicum annuum L. ) is one of the most important crops in the agricultural economy. Virus, bacterial, and fungal diseases have been reported in pepper plants, but no effective disease-resistant crops have been reported. Therefore, the effective isolation of genes related to disease resistance and the production of transgenic plants using biotechnological techniques can reduce production costs and produce high-quality crops for the eco-friendly era, and contribute to the activation of various industries.

Plants respond to a variety of signals to induce the expression of various genes, which are then used in the biochemical metabolism of plants in the form of proteins. Recent studies show that this post-synthesis regulation plays an important role in various metabolisms, and the ubiquitination system is a representative example. The ubiquitination system is an in vivo mechanism that breaks down specific substrates through which plants regulate a variety of defense-related mechanisms, which include pathogen recognition of host cells, hypersensitivity reactions through local cell death, phytoalexin and Such as antimicrobial production, defense-related protein production and the like. In the process of controlling the series of resistance reactions, specific degradation of enzymes or proteins that are unnecessary or inhibiting the defense reactions is important. Such degradation occurs mainly through the ubiquitination system. E3 ubiquitin ligase) allows the degradation of specific proteins by selecting the substrate to be finally degraded. It is known that there are about 1400 E3 ubiquitin lyases in Arabidopsis as a representative model plant, and these genes are expected to regulate not only plant defense-related mechanisms but also physiological processes and biochemical metabolism of various plants, but are still unknown in pepper plants. Few. Therefore, the study of E3 ubiquitin ligase that regulates various metabolism in plants is a very prominent field, especially the study of E3 ubiquitin ligase involved in plant disease resistance reaction. Defining the 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 against pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ), pepper E3 ubiquitin lysine which is one of the genes related to plant disease resistance By separating and deciphering the CaRING1 gene encoding Ez (E3 ubiquitin ligase RING1 ), the nucleotide sequence information and the corresponding amino acid sequence information are provided.

It is also an object of the present invention to provide a disease resistance detection method for plants that detect the presence or absence of resistance to plant diseases using the CaRING1 gene.

Furthermore, an object of the present invention is to produce a transgenic plant having resistance to plant diseases by introducing the CaRING1 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 CaRING1 isolated from the green pepper varieties ( Capsicum annuum cv. Nockwang) exhibiting hypersensitivity resistance to the inoculation of non-pathogenic strains of red pepper bacterial bacillus ( Xanthomonas campestris pv.vesicatoria ). The nucleotide sequence was determined and used to examine the expression of CaRING1 through pathogen treatment, and to confirm the plant disease resistance induction function of CaRING1 gene in pepper plants and transgenic Arabidopsis thaliana overexpressing the gene. Achieved.

Therefore, the present invention provides a gene encoding a protein having an amino acid sequence of SEQ ID NO: 2 encoding pepper-derived E3 ubiquitin ligase as a plant disease resistance related gene, or (b) a gene having a nucleotide sequence of SEQ ID NO: 1 CaRING1 ).

The present invention also provides an isolated E3 ubiquitin ligase protein (CaRING1) 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 disease resistance of a plant, comprising the step of identifying the differential expression of CaRING1 gene by separating RNA from the plant.

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 The total mRNA obtained from healthy red pepper plants not inoculated with dioxygenin was used to make probes, and differential hybridization was carried out using the cDNA and probes to be involved in pathogenicity. Selected genes were selected to obtain clones, and the sequences of the genes identified as E3 ubiquitin ligase ( CaRING1 ) genes were cloned , thereby sequencing the E3 ubiquitin ligase of the red pepper greenery variety named CaRING1 . Izu (CaRING1) protein coding gene sequence (SEQ ID NO: 1) and is encoded therefrom An amino acid sequence (SEQ ID NO: 2) is provided (see FIG. 1).

After sequencing the obtained clones according to the present invention, the 5'-terminal sequence was compared with a gene database registered in GenBank using the Blast program to identify the CaRING1 protein coding gene and E3 ubiquitin This gene was named CaRING1 gene. CaRING1 protein has a RING domain which is typical of E3 ubiquitin ligase as a result of comparison of amino acid sequences, and specifically has a transmembrane domain that plays an important role in determining the position in cells. (See FIGS. 4-7). 65% at the protein level with E3 ubiquitin ligase (Accession no. NP_173506) from Arabidopsis thaliana and 61% at the protein level with zinc finger gene (accession no.ABK56013) from Mustard ( Brassica rapa ) By showing homology, it can be seen that it is novel (http://blast.ncbi.nlm.nih.gov/Blast.cgi, nucleotide blast program). The identified gene nucleotide sequence of CaRING1 and the amino acid sequence encoded by the nucleotide sequence are shown in FIG. 1.

Next, the present invention provides a method for detecting disease resistance of a plant, comprising the step of identifying RNA differentially expressing CaRING1 gene by separating RNA from the biologically treated plant. More specifically, the biological treatment may be pathogens such as pathogenic or non-pathogenic bacteria of pepper bacterial spot pattern disease. The expression of CaRING1 gene may be used in various ways to measure mRNA expression in addition to Northern blot, real-time RT-PCR, microarray method. It was confirmed by experiment that CaRING1 gene according to the present invention is differentially induced expression by phytopathogens (see Figure 3). Therefore, the CaRING1 gene according to the present invention can be usefully used for the disease resistance detection method of plants.

In addition, the present invention determines whether to use a recombinant vector containing the above-CaRING1 gene production of a typically CaRING1 gene-specific in pepper plants inhibiting plant, CaRING1 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 relates to the CaRING1 gene using a TRV2 vector (Reference: Tobacco Rattle Virus), which is used in newly developed virus-induced gene silencing (VIGS) technology. Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing.Curr. Opin.Plant Biol. 2: 109-113) to produce a recombinant vector TRV2 :: CaRING1 . The recombinant vector was introduced into the plant to prepare a VIGS plant in which CaRING1 expression was suppressed, thereby confirming that the disease susceptibility was increased (see FIGS. 11 and 14).

Furthermore, the recombinant vector (pBIN35S :: CaRING1 ) containing the CaRING1 gene according to the present invention was subjected to Agrobacterium- mediated transient expression in pepper plants, or transgenic Arabidopsis plants overexpressed CaRING1 according to the present invention. It was possible to confirm the increase in plant disease resistance by preparing (see FIGS. 8 to 10 and 15 to 16).

To evaluate the resistance of phytopathogens to pepper plants that have disabled the function of pepper E3 ubiquitin ligase and Arabidopsis plants overexpressing E3 ubiquitin ligase according to the present invention. It became. The present invention enables the accumulation of genetic information of E3 ubiquitin ligase in response to the disease and related pathogenic proteins (PR protein) can provide an interesting model for signaling of disease in plants. By understanding biochemical changes that cause resistance, they can be used to develop biotechnological plants that increase disease resistance.

As described above, by completing the present invention, by separating and deciphering the CaRING1 gene, which encodes a novel E3 ubiquitin ligase protein, which is a plant disease resistance-related gene, which is involved in the defensive reaction against pepper plant bacterial disease, which is pepper plant bacterial disease. In addition to providing the sequence information and the corresponding amino acid sequence information, the gene can be used not only to provide a method for detecting disease resistance of plants, but also to contribute to the development of resistant plant breeding and 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.

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 CaRING1  Base / Amino Acid Sequencing of Genes

The following tests were performed to provide E3 ubiquitin ligase RING1 protein coding gene sequences and amino acid sequences deduced therefrom, one of the pathogenic related proteins against red pepper bacterial spot disease.

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). The individual cDNA clones were prepared from the cDNA library thus prepared and adsorbed to the nylon membrane (Hybond N +) regularly, 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 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, the genes amplified intensively for mRNA probes of non-pathogenic strain inoculated plants were assumed to be genes related to pathogenic resistance, and clones were obtained.

After sequencing the obtained clones, using the blast program, the 5'-terminal sequence is compared with the gene database registered in GenBank to identify the CaRING1 protein coding gene and named it as the E3 ubiquitin ligase ( CaRING1 ) gene. It was. Analysis of the amino acid sequence of CaRING1 protein showed a ring domain that is typical of E3 ubiquitin ligase and specific transmembrane domain that plays an important role in determining the position in cells. Had. 65% at the protein level with E3 ubiquitin ligase (Accession no. NP_173506) from Arabidopsis thaliana and 61% at the protein level with zinc finger gene (accession no.ABK56013) from Mustard ( Brassica rapa ) By showing homology, it can be seen that it is novel (http://blast.ncbi.nlm.nih.gov/Blast.cgi, nucleotide blast program). The identified gene nucleotide sequence of CaRING1 and the amino acid sequence encoded by the nucleotide sequence are shown in FIG. 1.

Example 2 In Red Pepper Plant by Pathogen Inoculation CaRING1  Gene Expression Detection Method

In order to confirm the method of using the CaRING1 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, after incubating the non-pathogenic strain Bv5-4a showing an incompatible reaction with the pathogenic strain Ds1 of the red pepper bacterial bacillus pathogen ( Xanthomonas campestris pv.vesicatoria ), which shows a friendly reaction with the plant, 10 ⁸ At the concentration of cfu / ml, the inoculations of the red pepper varieties of Capsicum annuum cv. Nockwang of 6 leaves were inoculated with a syringe. After inoculation, they were incubated for 16 hours at 28 ° C. and 100% relative humidity. Processed. After 1, 5, 10, 15, 20, and 25 hours of inoculation, RNA was isolated by sampling 3 and 4 leaves, and electrophoresed on a gel containing formaldehyde, and then transferred to a nylon membrane (Hybond N +). I was.

Next, the α- ³² P [dCTP] labeled with an isotope was labeled to CaRING1 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 isotopically 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 isotopically labeled DNA is X-ray film. As a result, the expression and degree of expression were recognized.

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. 3. As a result of the experiment, the CaRING1 gene was started to express quickly after inoculation of the pathogenic strain and the non-pathogenic pepper bacterial spot pattern disease, and its expression was strong and time-sensitive in the pepper plants inoculating the non-pathogenic strain. As time progressed, expression increased. That is, in the friendly response, the expression of CaRING1 gene reached a peak after 1 hour and then rapidly decreased, but in an unfriendly response, the expression gradually increased from 1 hour to 25 hours.

Example 3 CaRING1 Protein Location in Plant Cells

In order to confirm the migration in the plant cell of the CaRING1 gene obtained in Example 1 was tested as follows.

[Step 1]

In order to confirm the movement of CaRING1 protein in plant cells, fluorescein (CaRING1: smGFP) was expressed on onion epidermal cells by fusion of the green fluorescent protein at the 3 'end of the CaRING1 gene obtained in Example 1 and then confocal. (conforcal) was observed using a microscope. At this time, in order to elucidate the function of the transmembrane domain, which is thought to play an important role in intracellular positioning of CaRING1 gene, the 5 'end (amino acids 1-55 of SEQ ID NO: 2) containing this domain was fused with the green fluorescent protein. The same experiment was carried out by fusion of (CaRING1ΔRING: smGFP) and the 3 ′ terminus (amino acids 56-197 of SEQ ID NO: 2) without this domain with the green fluorescent protein (CaRING1ΔTM: smGFP). As a result, it was observed that CaRING1 protein migrates to the cell membrane as shown in FIG. 4, and at the same time, it was found that the transmembrane domain was required to determine the intracellular location of the CaRING1 protein.

Example 4 Determination of E3 ubiquitin ligase activity of CaRING1 protein

The function of the CaRING1 protein obtained in Example 1 was tested as follows to measure E3 ubiquitin ligase activity.

[Step 1]

The CaRING1 gene obtained in Example 1 for the E3 ubiquitin la this rise measuring the activity of CaRING1 protein pMAL c4x vector (New Engliand biolabs) monohydrate binding protein (maltose binding protein, MBP) end is introduced to and was fusion (MBP-CaRING1 ). In order to elucidate the role of the ring domain in the E3 ubiquitin ligase activity, a portion having no ring domain (MBP-CaRING1ΔRING) (amino acids 1-78 of SEQ ID NO: 2) and a portion having a ring domain (MBP- CaRING1ΔTM) (amino acids 79-197 of SEQ ID NO: 2) and proteins mutated with ring domains (MBP-CaRING1C110S, MBP-CaRING1H131Y) were similarly fused to maltose binding proteins. The mutated proteins MBP-CaRING1C110S and MBP-CaRING1H131Y are mutated amino acid sequences essential for E3 ubiquitin ligase activity by determining the structure of the ring domain, and MBP-CaRING1C110S is the amino acid of SEQ ID NO: 2. Cysteine, the 110th amino acid, is serine, and MBP-CaRING1H131Y is a mutant protein in which the histidine, the 131th amino acid, of the amino acid of SEQ ID NO: 2 is substituted with tyrosine. The fusion protein as described above was expressed using Isopropyl β-D-1-thiogalactopyranoside (IPTG), and the protein was purified using amylose resin (amylose resin) provided by New England BioLab.

[Step 2]

The purified MBP-CaRING1 protein was used to react with urea (E1, E2, ATP, ubiquitin) required for the ubiquitin system to measure E3 ubiquitinase activity for 3 hours at 30 ° C. As observed in FIG. 6, the CaRING1 protein exhibited E3 ubiquitin ligase activity in the ubiquitation system and was active only when all elements required for the ubiquitation system were present. E3 ubiquitin ligase activity was measured by Western blotting method using ubiquitin antibody and MBP antibody. The fusion protein MBP was used as a control.

[Step 3]

To elucidate the role of the ring domain of CaRING1 protein in E3 ubiquitin ligase activity, purified deletion mutant variants (MBP-CaRING1ΔRING, MBP-CaRING1ΔTM) and site directed mutants variants (MBP-CaRING1C110S, MBP-CaRING1H131Y). ) Was used to measure E3 ubiquitin ligase activity. As observed in FIG. 7, it was found that a ring domain is essential for E3 ubiquitin ligase activity. Western blot used the same method as in step 2.

Example 5 CaRING1  Apoptosis-related Apoptosis Changes of Pepper Plants by Gene Overexpression

When in the pepper plant is CaRING1 gene is overexpressed In order to examine whether the increase in apoptosis for resistance to disease, by introducing CaRING1 gene obtained in Example 1 in pBIN35S vector available from Stratagene pBIN35S: after producing the CaRING1 recombinant Vector, pBIN35S: CaRING1 was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation method and inoculated into red pepper leaves to change the pathogenicity-related apoptosis of pepper plants overexpressing CaRING1 . Was observed.

[Step 1]

Agrobacterium tumefaciens strain GV3101 transformed with a recombinant vector overexpressing CaRING1 (35S: CaRING1 ) and a control vector overexpressing CaRING1 (35S: 00) absorbance (optical density) 600 ( OD ₆₀₀ ) was inoculated at the concentrations of 1.0, 0.5, and 0.1 at the leaf vein of the pepper leaves, and the results are shown in FIG. 8. As a result, apoptosis was observed at the inoculated sites of Agrobacterium overexpressing CaRING1 on the 4th and 7th day of inoculation, respectively, and at this time, the accumulation of phenolic compounds UV) can be observed. In order to quantify apoptosis, the electrolyte flowing out of the leaf tissue was measured 20 and 40 hours after inoculation, respectively. Compared with the control group, the electrolyte leaked from the tissues overexpressing CaRING1 was significantly increased (FIG. 9). Trypan blue staining of the leaves inoculated at different concentrations along the leaf vein resulted in increased cell death compared to the control group. (FIG. 10). Dab staining {3,3'-diaminobenzidine (DAB) staining} was performed to determine the amount of free radicals known as pathogenic signaling material three days after inoculation and the results are shown in FIG. As a result, it was observed that active oxygen accumulation strongly occurred at the site inoculated with Agrobacterium strain overexpressing CaRING1 .

Example 6 CaRING1  Changes in disease resistance by inhibition of gene expression

In order to determine whether induction of other genes related to disease development and increased susceptibility to disease when CaRING1 gene expression was suppressed in pepper plants, the newly developed virus-induced gene silencing ( CaRing1 gene) obtained in Example 1 Using the TRV (Tobacco Rattle Virus), which is used in VIGS, virus-induced gene silencing ( CARRING1 ), the CaRING1 gene was transferred to 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 :: CaRING1 . Thus prepared recombinant vector TRV2 :: CaRING1 was introduced into the Agrobacterium tumefaciense strain GV 3101 by electroporation, and again the GV3101 strain containing the recombinant vector was introduced into pepper seedlings. Inhibition of CaRING1 gene expression was induced by inoculation by infiltration using a syringe into the pepper plants.

[Step 1]

In order to investigate the changes in the resistance to bacterial diseases in plants with suppressed CaRING1 expression, pepper plants with red pepper varieties inoculated with the green pepper varieties inoculated with TRV: 00 vector as a control and the pepper plants inhibited with the expression of CaRING1 gene ( Xanthomonas pathogenic strains Ds1 and non-pathogenic strains of Bv5-4a ^{campestris pv. vesicatoria) 10 8,} 10 7, 10 6 were inoculated to a concentration of cfu ml ^-1 of 0, after three days were observed for disease symptoms in inoculated leaves (Fig. 11 ), Inoculated with 5 × 10 ⁴ cfu ml ⁻¹ to measure bacterial growth in infected plant tissues at 0 and 3 days after inoculation (FIG. 12). Also, inoculated with 10 ⁷ cfu ml ^-1 , 12 and 24 hours after inoculation, salicylic acid (SA) was extracted from infected leaves of experimental and control groups, and RNA was extracted to determine real-time Alti-Phisal (real). time RT-PCR) showed a difference in the expression of pathogen-related genes compared with the control plants at the time of inoculation with the specific gene inhibitory effect of CaRING1 (FIG. 13). After inoculation with non-pathogenic strain Bv5-4a at 10 ⁷ cfu ml ^-1 , the active oxygen release and apoptosis associated with the hypersensitivity reactions in the experimental and control groups were determined 12, 24 hours after the inoculation, respectively. It was observed by measuring the electrolyte outflow according to (Fig. 14).

As shown in FIG. 11, the symptom was observed after inoculation of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a in the plant inhibited by the expression of CaRING1 with VIGS. Progress was observed and apoptosis was observed in the control plants on day 3 when the non-pathogenic strains were inoculated, but local apoptosis was decreased in the plants inhibited by CaRING1 expression. As a result of measuring the bacterial growth of pathogenic and non-pathogenic bacteria in infected tissues, more bacterial growth was observed in plants in which CaRING1 expression was suppressed compared to the control (FIG. 12). Accumulation of salicylic acid was particularly reduced when CaRING1- inhibited plants were infected with non-pathogenic strains, and real-time Alti- Phisal experiments showed that the inhibition of pepper defense-related genes appeared effectively in plants inhibited by CaRING1. And expression of resistance related genes such as CaBPR1, CaDEF1, CaPO2 was reduced (FIG. 13). In addition, as observed in FIG. 14, it was observed that free radical accumulation and apoptosis were significantly reduced for the hypersensitivity reaction of non-pathogenic strain inoculation in the plants inhibited by CaRING1 (FIG. 14).

Example 7 CaRING1  Preparation of Transgenic Plants Using Genes CaRING1  Changes in Disease Resistance of Arabidopsis Plants by Overexpression of Genes

To investigate whether the resistance to disease during overexpression of CaRING1 gene in Arabidopsis plants used as model plants was increased, the CaRING1 gene obtained in Example 1 was introduced into the pBIN3S vector provided by Stratagene to provide pBIN35S :: CaRING1 . After preparation, the recombinant vector pBIN35S :: CaRING1 thus prepared was introduced into Agrobacterium tumefaciense strain GV 3101 by electroporation, and again, the GV3101 strain containing the recombinant vector was submerged ( Induced overexpression of CaRING1 gene by transformation into Arabidopsis plants by floral dipping method was performed as follows.

[Step 1]

As described above, the strains overexpressed CaRING1 were inoculated with the strain Pseudomonas syringae pv. Tomato DC3000, which is a bacterial virulent pathogen, and the growth and symptoms of the bacteria were observed. As shown in FIG. 15, it was observed that bacterial growth was inhibited in CaRING1 overexpressed plants as compared to wild type, and the symptom was observed by inoculation at a concentration of 10 ⁷ cfu ml ⁻¹ .

In FIG. 15, # 5, # 13, and # 16 represent line numbers of the transgenic plants used.

[Step 2]

CaRING1 overexpressed plants were inoculated with Hyaloperonospora parasitica Noco2 ( Hyaloperonospora parasitica Noco 2), which causes downy mildew in Arabidopsis; The cotyledon of CaRING1 overexpressed and wild-type non-overexpressed cotyledon was sprayed with Hyaloferonospora 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 FIG. 16, it was observed that the resistance to transgenic Arabidopsis plants overexpressing CaRING1 suppressed the conidia disease and spore formation of the Bacillus fungi , and the resistance of the plants overexpressed CaRING1 to fungal disease Cell level observations of were performed via aniline blue staining. As a result, it was observed that mycelial growth was suppressed in the plants overexpressed CaRING1 gene in the control group.

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 diagram showing the nucleotide sequence and amino acid sequence of the pepper E3 ubiquitin ligase ( CaRING1 ) gene cloned from the pepper greenery varieties ( Capsicum annuum L. cv. Nockwang) by the present disease.

Figure 2 is a view showing the expression results of CaRING1 gene in each organ of the red pepper varieties.

Figure 3 shows the results of induction of the expression of CaRING1 gene in red pepper leaves and inoculated red pepper leaves when inoculated with the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a of the red pepper bacteriophage ( Xanthomonas campestris pv.vesicatoria ) The figure which shows.

Figure 4 is a diagram showing the intracellular location of the mutant protein to confirm the role of the transmembrane domain in the intracellular location of CaRING1 and CaRING1 fusion of the green fluorescent protein.

5 is a diagram showing the results of the expression of CaRING1 and mutant proteins in E. coli and purified using amylose resin in order to measure the E3 ubiquitin ligase activity of CaRING1 protein.

6 is a diagram measuring the E3 ubiquitin ligase activity of CaRING1 protein.

7 is a diagram measuring the E3 ubiquitase activity of CaRING1 and mutant proteins to confirm the role of the ring domain in the E3 ubiquitin ligase activity of CaRING1 protein.

8 is a photograph showing the result of apoptosis induced when CaRING1 gene is overexpressed in pepper plants using the recombinant vector pBIN35S: CaRING1 and Agrobacterium tumefaciens strain GV3101 strain.

9 is a diagram showing the results of measuring electrolyte leakage due to apoptosis induced by using the recombinant vector pBIN35S: CaRING1 and strain.

10 is a photograph showing the results of observing the accumulation of reactive oxygen induced by recombinant vector pBIN35S: CaRING1 and strain through Dap staining and apoptosis through trypan blue staining.

FIG. 11 is a pepper bacterial spot pattern bacterium of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a ( Xanthomonas) in a plant of red pepper green mine varieties whose CaRING1 gene expression was suppressed using the method of virus-induced gene silencing (VIGS) campestris pv.vesicatoria ) is a photograph showing the increased disease susceptibility when inoculated.

Figure 12 shows the bacterial growth in infected pepper leaf tissues when inoculated with the pepper bacterial strain of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a ( Xanthomonas campestris pv.vesicatoria ) to the plants of the pepper green mine varieties inhibited CaRING1 gene expression The figure shown.

FIG. 13 shows the effect of the change in salicylic acid accumulation and the effect of CaRING1 when inoculated with the causative strain Ds1 and non-pathogenic strain Bv5-4a of red pepper bacterium ( Xanthomonas campestris pv. Vesicatoria ) in the red pepper cultivated varieties whose expression of CaRING1 gene was suppressed. Figures showing the results of expression difference between expression inhibition and resistance related genes.

FIG. 14 shows a decrease in apoptosis due to hypersensitivity reactions and activity for the inoculation of pepper pathogen Bacteria ( Xanthomonas campestris pv.vesicatoria ) of the non-pathogenic strain Bv5-4a to the plant of the pepper greenery varieties in which CaRING1 gene expression was suppressed. It is a figure which shows the result of having measured oxygen accumulation.

15 is a recombinant vector pBIN35S: CaRING1 and Agrobacterium tumefaciens (Agrobacterium tumefaciens strain GV3101) when Pseudomonas is bacterial heukban pathogen in transgenic Arabidopsis thaliana plants that overexpressed the CaRING1 genes using the strains ringge lost bar tomato DC3000 (Pseudomonas syringae pv. tomato DC3000) is a diagram showing the results of observation of pathogen growth and symptoms in plants during strain inoculation.

16 is a diagram showing the results of pathogenic resistance to Hyaloperonospora parasitica Noco2 in transgenic Arabidopsis plants overexpressing the CaRING1 gene.

<110> Korea University Industrial and Academic Collaboration Foundation <120> Disease resistance-related pepper E3 ubiquitin ligase gene          CaRING1 and transgenic plants using the same <130> P11-090820-01 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 985 <212> DNA 213 Capsicum annuum L. cv. Nockwang <400> 1 ctaagctctg gatacttctt acttatttag tttgaagctt ttttttcctc tctctatttt 60 caatatgagt agtgatgatc ctttacataa cgccggcggc caccttaagc cgtcaagtac 120 accgtcgccg gctggggatt caactacgcg ccgtgtggac tccgacttcg tcgtcgtcct 180 ggcggcgctt ctctgtgcac tcatttgcgt tctcggcctt gttgccgttg ctcgctgcaa 240 ctggatccgt cggatctccg gaagtattgc cgggaattca gcgtttgcat cagctccggc 300 gaataaaggt ttgaagaaga aggtactcaa gtctcttccg aagttcaatt acggagctga 360 acacgctgat aaattctctg aatgtgcaat ttgcttagcg gaatttgcag tcggagagga 420 aattcgtgtg ttgccgcagt gtggacatgg ttttcatgtt ggatgtattg atacttggtt 480 aggttcgcac tcttcctgtc cttcttgccg gtcgattttg gtcgttacga gatgtcataa 540 gtgcggtgag ttgccggtag ctagctcctc atcgtcgtcg tcatctgcgg cggcgaccgg 600 agctgggacg gaaagccgat taccgagaaa ttatcatgta aatgcatttc tgccgtaaat 660 ctcttaggta ttaattattt gattaggctg tgtggatatg ttaattaagt actgttaagt 720 gtaatctctg tatgtttaac gtgtatatcc ttccagctct actgtatcag caattaggat 780 tacagaaaag tgaaaactac tgtttggatt agtgttgtat tactgtttgg attaatgtga 840 atttgtattt aattaaaaaa aatgatcaca agttttaaat tttcaagaaa aaagaaaaca 900 aaagaaagag taaaaaaata ttgtatagga gataatagat aaaatgatag ataaaaaagg 960 ggccactgtg tgcggcccgc tcgaa 985 <210> 2 <211> 197 <212> PRT 213 Capsicum annuum L. cv. Nockwang <400> 2 Met Ser Ser Asp Asp Pro Leu His Asn Ala Gly Gly His Leu Lys Pro   1 5 10 15 Ser Ser Thr Pro Ser Pro Ala Gly Asp Ser Thr Thr Arg Arg Val Asp              20 25 30 Ser Asp Phe Val Val Val Leu Ala Ala Leu Leu Cys Ala Leu Ile Cys          35 40 45 Val Leu Gly Leu Val Ala Val Ala Arg Cys Asn Trp Ile Arg Arg Ile      50 55 60 Ser Gly Ser Ile Ala Gly Asn Ser Ala Phe Ala Ser Ala Pro Ala Asn  65 70 75 80 Lys Gly Leu Lys Lys Lys Val Leu Lys Ser Leu Pro Lys Phe Asn Tyr                  85 90 95 Gly Ala Glu His Ala Asp Lys Phe Ser Glu Cys Ala Ile Cys Leu Ala             100 105 110 Glu Phe Ala Val Gly Glu Glu Ile Arg Val Leu Pro Gln Cys Gly His         115 120 125 Gly Phe His Val Gly Cys Ile Asp Thr Trp Leu Gly Ser His Ser Ser     130 135 140 Cys Pro Ser Cys Arg Ser Ile Leu Val Val Thr Arg Cys His Lys Cys 145 150 155 160 Gly Glu Leu Pro Val Ala Ser Ser Ser Ser Ser Ser Ser Ala Ala                 165 170 175 Ala Thr Gly Ala Gly Thr Glu Ser Arg Leu Pro Arg Asn Tyr His Val             180 185 190 Asn Ala Phe Leu Pro         195  

Claims (10)

An isolated gene ( CaRING1) of (a) or (b) which codes for pepper-derived E3 ubiquitin ligase protein as a plant disease resistance 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 (CaRING1). A recombinant vector comprising the gene of claim 1. Recombinant vector pBIN35S :: CaRING1 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 CaRING1 gene of claim 1 characterized in that the disease resistance detection method of the plant. 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. Recombinant vector TRV2 :: CaRING1 prepared by inserting the CaRING1 gene of claim 1 into a Tobacco Rattle Virus 2 (TRV2) vector, which is a vector for virus-induced gene silencing (VIGS). In the recombinant vector group for intracellular positioning of the CaRING1 protein as described in claim 2, the recombinant vector CaRING1: smGFP, wherein the CaRING1 gene as described in claim 1 is introduced into a p326GFP vector, and the transmembrane domain of the protein as described in claim 2 Recombinant vector CaRING1ΔRING: smGFP which introduced the 5 'end including the base sequence coding into a p326GFP vector and the 3' end which does not contain the base sequence which codes the transmembrane domain of the protein of Claim 2 were introduce | transduced into the p326GFP vector Recombinant vector group comprising one recombinant vector CaRING1ΔTM: smGFP. In the protein group for measuring the E3 ubiquitinase activity of the CaRING1 protein as described in Claim 2, MBP fusion to the maltose binding protein (MBP) after introducing the CaRING1 gene as described in Claim 1 into the pMAL-c4x vector Of the MBP-CaRING1ΔTM, the protein of claim 2, introduced into the pMAL-c4x vector and then fused to the maltose binding protein (MBP) MBP-CaRING1ΔRING fused to maltose-binding protein (MBP) after introducing a portion containing no nucleotide sequence encoding ring domain into the pMAL-c4x vector, each of the 110th amino acid of amino acid of SEQ ID NO: 2 The proteins MBP-CaRING1C110S and MBP-CaRING1H131Y substituted with this serine or mutated with amino acids of ring domain wherein histidine, the 131th amino acid of amino acid of SEQ ID NO: 2, is substituted with tyrosine Protein group.

Images