KR20120005876A - Calysethc R1 gene derived from red pepper plants involved in the defense reaction of plants and its use - Google Patents

Abstract

The present invention relates to a pepper plant-derived CalecRK1 gene involved in plant defense reaction and its use, and more particularly to CaLecRK1 ( Capsicum annuum lectin receptor kinase1 ) derived from pepper plant having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2. It's about genes.
According to the present invention, CaLecRK1 , which has been rarely studied until now, was found to perform CaLecRK1 through a virus-induced gene silencing (VIGS). When inhibiting the expression of CaLecRK1 of the present invention using VIGS, it was confirmed that viral envelope protein expression was increased and hypersensitive resistance (HR) of the infected site was reduced, thereby improving varieties resistant to disease and The breeding method can regulate the expression of the CaLecRK1 gene to provide an infection-resistant pepper plant. Therefore, it is expected that the CaLecRK1 gene can be used to produce transformed plants with increased plant defense response.

Description

CaLecRK1 gene from hot pepper concerned in defense response of plants and use approximately}

The present invention relates to CaLecRK1 gene derived from red pepper plants involved in the defense reaction of plants, and more particularly, CaLecRK1 derived from red pepper plants having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 ( Capsicum annuum lectin receptor kinase1 ) gene.

Plants have developed various defense mechanisms to survive local biological and abiotic stresses. Plant defense responses generally include orchestrated transcriptional activation of multiple genes and the accumulation of secondary metabolites, often the activation and systemic acquisition of hypersensitive responses (HR). Accompanied by the development of systemic acquired resistance [Ryals et al., 1996 The Plant Cell 8: 1809-1819; Dangl and Jones, 2001 Nature 411: 826-833. When these elicited reactions occur quickly and equally during a given plant-pathogen interaction, the plant becomes resistant to disease. In susceptible plants, the onset of the defense mechanism responds more slowly to pathogen infection. Thus, the timely recognition of invading microorganisms and the induction of protective responses quickly and effectively seem to make a major difference between resistant and susceptible plants [Yang et al., 1997 Genes Dev. 1; 11 (13): 1621-39.

Pepper, which is a representative eggplant plant, is a representative crop that has been used as a spice or spice for a long time in Korea, and various diseases such as viral disease, bacterial disease, and fungal disease have been reported. And anthracnose caused by fungi and bacterial spot disease caused by bacteria. Until now, chemical control methods using pesticides have been mainly used to control these plant diseases against peppers. However, due to its toxicity, not only pathogens but also organisms in surrounding ecosystems have been killed in large quantities to destroy ecosystems and pollute soil and water quality.

Capsicum annuum L. cv. Bukang) is infected by tobacco mosaic virus and many other viruses and expresses various genes in response to it. Among them, PMMoV-P ₀ ( Pepper mild mottle virus strain P ₀ ) causes a defensive response such as a hypersensitive response.

Accordingly, the present inventors have made diligent research to overcome the problems of the prior arts, and as a result, the expression was increased during the virus treatment using a microarray to find genes present in the upper part in the defense reaction caused by the interaction of pepper and nasal cavity. The present invention was completed by selecting CaLecRK1 genes, which have not been studied much and are likely to exist at the top.

Therefore, the main object of the present invention is to provide CaLecRK1 gene and its protein involved in resistance to plant infection in pepper plants.

Another object of the present invention to provide a composition for increasing plant infection resistance using the CaLecRK1 gene and its protein.

According to an aspect of the present invention, the present invention provides pepper lectin receptor phosphorylation 1 ( Capsicum) having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 annuum lectin receptor kinase1 ; CaLecRK1 ) gene.

In the present invention, the nucleotide sequence may be any nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, preferably characterized in that the base sequence of SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides Calicec receptor 1 ( CaLecRK1 ) protein having an amino acid sequence of SEQ ID NO: 2. The full-length cDNA of the CaLecRK1 protein encodes a polypeptide of 701 amino acids.

According to another aspect of the present invention, the present invention provides a recombinant vector introducing the Calicectin receptor 1 ( CaLecRK1 ) gene. The recombinant vector into which the CaLecRK1 gene is introduced is shown in FIG. 3.

By using the CaLecRK1 gene of the present invention, a recombinant vector for transforming a plant can be prepared. For the preparation of the recombinant vector, a general vector designed to be expressed in a plant, but not including a general binary vector, a cointegration vector, or a T-DNA site may be used. In particular, the binary vector that can be used in the present invention may be any binary vector containing BR and BL of T-DNA capable of transforming plants when present with the Ti plasmid of Agrobacterium ( A. tumefaciens ). PGAG series, pCG series, pCIT series, pGPTV series, pBECK2000 series. BiBAC series and pGreen series vectors can be used.

In addition, the expression vector is an antibiotic resistance gene such as a hygromycin resistant gene, a herbicide resistance gene such as bar gene, GUS, GFP (as a selection factor for effectively selecting a plant to which the CaLecRK1 gene of the present invention is introduced). It may include label genes such as green fluorescent protein (LUX) and luciferase (LUX), and may include a promoter for producing RNA sequences of genes of interest in plant cells, and structural DNA bases having information of genes of interest. And at least one structure consisting of a 3 ′ nontranslated DNA sequence of an RNA base sequence and the like in a plant cell. In addition, the vector may further include a gene encoding a reporter molecule (eg, luciferase and β-glucuronidase).

According to another aspect of the present invention, the present invention can provide a plant transformed with the recombinant vector introduced CaLecRK1 gene.

According to an aspect of the present invention, the present invention provides a composition for increasing infection resistance of a plant containing the expression inhibitor of the CaLecRK1 gene as an active ingredient.

When the composition of the present invention is administered to a plant with weakened disease resistance, the expression of its own genes may be reduced due to genetic or environmental factors, thereby inducing a rapid recovery of the plant with reduced disease resistance. Above the dose, the dose effect can lead to a steady state, ie higher disease resistant effect than the wild type.

In the composition of the present invention, the CaLecRK1 gene expression inhibitor may be siRNA (short interfering RNA) or shRNA (short hairpin RNA) using RNA interference (RNA interference) of the CaLecRK1 gene, but preferably CaLecRK1 It is preferable that the gene is a viral vector for virus-induced gene silencing (VIGS).

The term 'VIGS' is an experimental method using a plant defense mechanism that recognizes and degrades double strand RNA inevitably generated by the replication of an RNA virus. VIGS relies on the homology of gene sequences during virus invasion. When a plant gene is introduced into a viral vector and inoculated into a plant, the expression of the transgene is inhibited by a mechanism similar to or after gene transcription. It is a way to analyze the function.

The viral vector for VIGS that can be used in the present invention may be a viral vector for VIGS such as tobacco rattle virus (TRV), tobacco mosaic virus (TMV), potato virus X (PVX) and tomato golden mosaic virus (TGMV), but is preferred. In particular, the tobacco rattle virus (TRV) vector is not suitable for the study of genes related to plant development because it does not induce the phenotype of bleaching, leaf distortion and cell necrosis of other vectors and growing point. It is preferable because it can overcome. Infection with TRV vectors only results in mild symptoms and can be infected at growing points, making it suitable for genetic research related to plant development. The viral vector for VIGS of the present invention is characterized in that pTRV2 :: CaLecRK1 disclosed in FIG. The pTRV2 :: CaLecRK1 vector is prepared by inserting CaLecRK1 ( 2204-2360 bp) into a multiple cloning site (MCS) existing between vector sites of coat protein (CP) and transcriptional terminators (T). In the vector shown in Figure 3, LB is Left border, Rb is Right border, 35S is CaMV 35S promoters, T is transcriptional terminators, RdRp is RNA-dependent RNA polymerase, MP is movement protein, 16K is 16k protein, CP is coat protein , MCS represents multiple cloning sites.

The term 'RNAi' is a method of post-transcriptional gene regulation conserved in many eukaryotes. RNAi is induced by short double-stranded RNA (dsRNA) molecules present in cells (Fire et al. (1998), Nature 391: 806-811). These short dsRNA molecules, also called siRNAs, are separated into single strands and then bind to RNA-induced silencing complexes (RISCs) to cleave targeted mRNAs or inhibit translation [Elbashir SM et al. (2001), Genes Dev, 15: 188-200.

In addition, when the expression inhibitor is siRNA, the siRNA polynucleotide sequence may be operably linked to an expression control sequence, wherein the operably linked gene sequence and the expression control sequence comprise a selection marker and a replication origin together. It can be included in one expression vector. The term “operably linked” may be genes and expression control sequences linked in such a way as to enable gene expression when the appropriate molecule is bound to the expression control sequences. The "expression control sequence" refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular host cell. Such regulatory sequences include promoters for carrying out transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation.

In the present invention, the plant infection (infection) is PMMoV-P ₀ ( Pepper mild mottle virus strain P ₀ ) is characterized by.

More specifically, in the present invention, as a reverse genetic approach, a TRV-based VIGS method was used, wherein the suppression of CaLecRK1 gene was significantly higher than that of control plants when PMMoV -P ₀ was treated in plants. The accumulation of PMMoV-P ₀ CP gene is shown. In addition, when PMMoV -P ₀ was treated in plants that inhibited the CaLecRK1 gene, the number of lesions due to the hypersensitive response was significantly reduced compared to plants that did not inhibit the CaLecRK1 gene. These results indicate that the CaLecRK1 gene is involved in the resistance response in plants, and demonstrates that the CaLecRK1 gene can be very useful as a gene that can enhance the resistance of peppers with many pests.

The composition according to the present invention may further comprise a stabilizer or excipient generally used in plant preparations as an active ingredient in addition to the CaLecRK1 gene, CaLecRK1 protein or recombinant vector. Specifically, by making a CaLecRK1 protein as a preparation and administering it directly to a plant, or by cloning the CaLecRK1 gene into a known plant expression vector, a recombinant vector is made, and then transformed into a plant through Agrobacterium ( A. tumefaciens ), By expressing the CaLecRK1 protein in plants, thereby increasing the expression of genes involved in expression, it can ultimately increase the resistance of pepper plants.

In addition, the composition is characterized by imparting resistance to plant infection (infection). The plant infection can be caused, for example, from PMMoV-P ₀ pathogens and the like.

As described above, according to the present invention, CaLecRK1 , which has not been studied so far, was found to perform CaLecRK1 through a virus-induced gene silencing (VIGS). When inhibiting the expression of CaLecRK1 of the present invention using VIGS, it was confirmed that viral coat protein gene expression is increased and hypersensitive reponse (HR) at the infected site is reduced, thereby improving the disease-resistant varieties and CaLecRK1 as a method of breeding The expression of the gene can be regulated to provide an infectious pepper plant. Therefore, it is expected that the CaLecRK1 gene can be used to produce transformed plants with increased plant defense response.

1 is a schematic diagram illustrating the principle of performing 5 'Rapid Amplication of cDNA Ends (RACE).
Figure 2 shows the schematic diagram of the virus-induced gene silencing (VIGS) principle used in the present invention.
3 is a diagram showing a recombinant vector pTRV2 :: CaLecRK1 into which the CaLecRK1 gene is inserted.
Figure 4 shows the quantitative change in the expression of the cacn14085 gene according to wound stress (wound stress) treatment by RT-PCR.
Figure 5a shows the change in lesion incidence due to hypersensitivity response when the virus is treated to plants which inhibited the cacn14085 gene and not to the plant, and Figure 5b shows the change in the expression of the gene in the plants. -Quantified by PCR.
Figure 6 shows the agarose gel band of the gene obtained after 5 'RACE.
Figure 7 shows the domain structure of the CaLecRK1 gene.
Figure 8 shows the alignment of the amino acid sequence of another LecRK and CaLecRK1 of Arabidopsis (Arabidopsis thaliana).
9 shows the results of phylogenetic analysis of the CaLecRK1 gene.
Figure 10 shows the quantitative change in expression of genes involved in signaling after viral infection by RT-PCR.
Figure 11 shows the transcriptional levels of genes involved in salicylic acid signaling, genes involved in methyl jasmonate (MeJA) signaling and genes involved in ethylene signaling in virus-infected plants after inhibition of CaLecRK1 gene. Is shown by performing semiquantitative RT-PCR.
Figure 12 shows that after infecting plants with and without inhibiting the CaLecRK1 gene, the plants were observed and the transcript levels of the CaLecRK1 gene, PMMoV -P ₀ CP (coat protein), were performed semi-quantitatively by RT-PCR. It is shown.
FIG. 13 shows that PMMoV -P ₀ amplification was quantified using RT-PCR in plants that inhibited and inhibited CaLecRK1 gene, and then the CaLecRK1 gene and PMMoV -P ₀ CP (coat) after infection with the virus. protein) expression level is shown by performing semi-quantitative RT-PCR.
Figure 14 after the infection of the virus in the plant that is not inhibited and the plants that suppresses CaLecRK1 gene, CaLecRK1 gene using the result, and semi-quantitative RT-PCR was observed by staining the plant with trypan blue, PMMoV-P ₀ CP It shows the expression level of (coat protein).
FIG. 15 shows the results of staining the plants with DAB after inhibiting CaLecRK1 gene and plants without inhibiting the virus, and using CaLecRK1 gene, PMMoV -P ₀ CP (coat) using semi-quantitative RT-PCR. protein) expression level.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example  One. DNA Microarray

Expression Analysis Capsicum oligonucleotide annuum 300 K were carried out using the oligonucleotide microarray (GreenGene Biotech). The microarray was designed from 29,580 unigene of red pepper. The direction of 24,417 genes in the unigene is known, and the direction of 5,163 genes is unknown. Each gene was designed redundantly with 6 probe 2 blocks, and the 6 probes were designed in sense and anti-sense directions, respectively. A total of 350,000 probes were designed. The average size of the probe is 60-nt with adjustment at 75-85 ° C. temperature. This microarray was manufactured by GreenGene Biotech.

Total RNA was prepared from pepper leaves treated with PMMoV-P ₀ and control buffer for 12, 24, 36 and 48 hours. Superscript Double-Stranded cDNA Synthesis kit (Invitrogen, USA) was used for the synthesis of double stranded cDNA, and 40 μl (1 OD) of Cy3-9mer was added to 1 μg of double stranded cDNA for the synthesis of C3-labeled target DNA fragments. Mixed with primers (Sigma-Aldrich, USA). The microarrays were then scanned with a Genepix 4000B (Axon Instruments) preset with 5 μm resolution and Cy3 signals. The measured signal was digitized and analyzed by Nimblescan (Nimblegen, USA).

Example  2. Plant cultivation and pathogen inoculation

Capsicum used in the experiment of the present invention annuum L. cv. Bukang) is resistant to PMMoV-P ₀ used in Example 1.

First, the pepper pepper plants were incubated at 23 to 26 ° C. with a photoperiod of 16 hours day and 8 hours night. Approximately 6 weeks old plants were used for pathogen inoculation and nucleic acid extraction. PMMoV-P ₀ is for tobacco ( N. tabacum cv. Samsun) were infected and expanded. Sap of pepper leaves containing PMMoV-P ₀ was obtained by crushing infected pepper leaves in virus inoculation buffer (0.1 M Tris-HCl pH 8.0, 1% sodium sulfate). Then, in order to inoculate the plants, the sap containing the virus was inoculated on the surface of the pepper leaf mixed with an appropriate amount of carborundum. Mock-inoculated plants were treated only with inoculation buffer and carborundum. To observe the tissue response, the bottoms of the two pepper leaves were inoculated with PMMoV-P ₀ sap, and 10 days after the inoculation, the tops of the untreated pepper leaves were harvested.

Example 3. Extraction of Total RNA

To make the pepper leaves into powder form, they were ground with liquid nitrogen. The pepper leaf powder was transferred into 2 ml of RNA extraction buffer (0.2 M Tris-HCl pH 8.0, 0.4 M LiCl ₂ , 25 mM EDTA pH 8.0 and 1% SDS). Phenol saturated with 2 ml of water was added to a tube containing pepper leaf powder and buffer, and the tube was vigorously stirred. The tube was then centrifuged at 14,000 x g for 15 minutes at 4 ° C. The supernatant was precipitated with ethanol and the resulting pellet was washed with 70% ethanol (DEPC-treated) and then dissolved in 4 M LiCl ₂ . After reprecipitation thereafter, the pellet was dissolved in DEPC-treated distilled water to obtain total RNA.

Example  4. RT - PCR  And semiquantitative ( semiquantitative ) RT - PCR  analysis

First, cDNA was synthesized using 5 μg of total RNA, oligo (dT) primer or random primer and superscript reverse transcriptase (Promega, USA). Semiquantitative RT-PCR was performed as described in Liu et al. (2002).

Gene-specific primers are shown in Table 1 (Tables 1-1 and 1-2) below. CaActin gene was used as internal standard for RNA quantification in RT-PCR. Each PCR product was separated by 1% agarose gel electrophoresis and visualized with ethidium bromide under ultraviolet light. The concentration of fragments amplified by RT-PCR was analyzed and quantified using Quantity one (Ver.4.3, Bio-Rad, USA) and Multi Gauge (Ver.3.0, Fujifilm, Japan).

Table 1-1. Gene Specific Primer for RT-PCR Analysis]

Table 1-2. Gene Specific Primer for RT-PCR Analysis]

Example  5. 5 ′ RACE On by CaLecRK1 cDNA Isolation and Genetic Analysis

Fragments of the CaLecRK1 gene were obtained through the cDNA library. To isolate CaLecRK1 whole cDNA clones, pepper RNA was isolated from plants infected with PMMoV -P ₀ . cDNA was synthesized using the cDNA amplification kit (Clontech, USA) according to the manufacturer's instructions. As shown in FIG. 1, CaLecRK1 was used to perform Rapid Amplication of cDNA Ends (5′RACE). Two gene specific primers (GSP1; 5'-GGCCTGTTGATGCTGAGAACCCAAAGTA-3 'and GSP2; 5'-TGGTGTTGAACCATTTTTCCCCATT-3') corresponding to the highly conserved sequence of the kinase domain for PCR amplification in conjugation with adapter primers Was used. PCR amplification was carried out in 30 seconds at 94 ° C, 30 seconds at 68 ° C and 30 seconds at 72 ° C. PCR products were inserted into TA cloning vectors (Invitrogen) and inserts were sequenced (see http://dna.macrogen.com/eng/ ). Structural domain predictions were performed using SMART [see http://smart.embl-heidelberg.de ] and Pfam [see http: /. Pfam.sanger.ac.uk] databases. TMHMM [see http://www.cbs.dtu.dk/services/TMHMM ], SOSUI [see http://bp.nuap.nagoya-u.ac.jp/~sosui/ ] to predict membrane potential domains Was used. Prediction of other domains was done using InterProScan [see http://www.ebi.ac.uk/Tools/InterProScan/ ].

Example 6. Virus-induced gene silencing (VIGS)

VIGS (Virus-induced gene silencing) is a kind of RNA-mediated plant defense mechanism against the virus acts on the principle shown in FIG. VIGS is a type of post-transcriptional gene silencing (PTGS), characterized by post-transcriptional, RNA turnover, and nucleotide sequence-specific characteristics. Has As a VIGS vector, plant genes are cloned into PVX (Potato virus X) amplicon or TRV (Tobacco rattle virus), and in the present invention, Liu et al., (2002) virus-induced gene silencing in tomato. The pTRV1 and pTRV2 vectors reported in Plant J. 31 (6) were used.

PCR amplifies the 2204-2360 bp region, 3′UTR region of the CaLecRK1 cDNA obtained in Example 5, and the MSC (CP) between the coat protein (CP) of the pTRV2 vector containing a part of the TRV genome and transcriptional terminators (T) multiple cloning site) (Ratcliff et al., 2001). For Agro-infiltration, pTRV1, pTRV2 and recombinant plasmid pTRV2 :: CaLecRK1 were prepared and then Agrobacterium via freeze-thaw method [An et al., 1988]. Tumefaciens GV3101 was transformed. The recombinant plasmid pTRV2 :: CaLecRK1 was prepared by linking CaLecRK1 , a target gene, to the Eco RI restriction site of pTRV2 (FIG. 3). Each transformed Agrobacterium Tumefaciens were incubated overnight at 28 ° C. in YEP containing 50 μg / ml kanamycin and 50 μg / ml rifampicin. The cultured cells were precipitated by centrifugation (3000 rpm, 15 minutes, 4 ° C.), suspended in an infiltration buffer (20 mM citric acid, 2% sucrose and 200 μm acetosyringone pH 5.2), followed by OD ₆₀₀ = 0.3 The concentration was adjusted with the infiltration buffer to have a value. PTRV1 and pTRV2 or pTRV2 :: CalecRK1 were respectively mixed at a ratio of about 1: 1.5, and then injected into a pepper cotyledon germinated at 18 ° C. and 30% humidity using a 1 ml syringe without a needle.

Example  7. sequence  Alignment and phylogenetic ( phylogenetic ) analysis

Domain sequences and standard length proteins of CaLecRK1 and its homologues in Arabidopsis were aligned by the ClustalW method, and phylogenetic analysis was performed by the Megalign Program (Intelligenetics, USA).

Example  8. DAB (3,3'- diaminobenzidine ) dyeing

Hydrogen peroxide accumulation was visualized by DAB staining [thordal Christensen H et al., 1997].

First, TRV2 and CaLecRK1 VIGS plant leaves were infected with PMMoV -P ₀ . Three days after infection, infected leaves were dissected and soaked in 0.1% DAB solution. Then, after 20 minutes vacuum infiltration, the sample was incubated at room temperature for 20 hours. After the stain was stopped, chlorophyll was removed by boiling with 96% (v / v) ethanol for 10 to 40 minutes. Afterwards, brown spots, which are reaction characteristics of DAB and H ₂ O ₂ , were analyzed.

Example  9. Trypan blue ( trypan blue ) dyeing

Dead plant cells in plant tissue were visualized by staining with trypan blue.

First, TRV2 and CaLecRK1- VIGS plant leaves were infected with PMMoV-P ₀ . Three days after infection, infected plant leaves were stained with trypan blue (10 g phenol, 10 ml glycerol, 10 ml lactic acid, 10 ml distilled water and 0.02 g trypan blue). Infected plant leaves were boiled with staining solution for 10 minutes and restained overnight with chloral hydrate.

Result 1.PMMoV-P ₀  Identification of Protein Kinases Regulated by Inoculation

To identify protein kinases increased following PMMoV-P ₀ treatment, expression analysis was performed with a Capsicum annuum 300 K microarray (GreenGene Biotech, Korea). Total RNA was prepared from pepper leaves treated with PMMoV-P ₀ and control buffer for 12, 24, 36 and 48 hours. Then we obtained information about 34748 genes in response to PMMoV-P ₀ . Of these genes, 434 genes encoding protein kinases were isolated, showing 38 protein kinases that increased expression more than three times in response to PMMoV-P ₀ of the isolated genes (Table 2). Among the protein kinases with increased expression, lectin protein kinase (clone ID: cacn14085 ) was selected for the study because the gene belongs to a gene encoding a kinase such as a receptor, and its function is mostly unknown. As a result of microarray, cacn14085 increased the expression by 4.32 times or more.

Table 2-1. Protein Kinase Expression Changes]

Table 2-2. Protein Kinase Expression Changes]

Result 2.PMMoV-P ₀  In response to inoculation cacn14085 Expression pattern of

To observe whether Cacn14085 transcription is induced in response to PMMoV -P ₀ inoculation, pepper leaves were infected with PMMoV -P ₀ , and total RNA was added to PMMoV-P ₀ and control buffers 12, 24, 36 and It was extracted from the pepper leaves treated for 48 hours. Mock inoculation was used as a control. Six hours after inoculation, cacn14085 transcription accumulated in both mock and virus infection treatment groups. In addition, RT-PCR was performed to confirm the expression of cacn14085 gene under wound stress. RT-PCR measurement results are shown in FIG. 4. It was confirmed that the cacn14085 gene is expressed after 2, 6, and 12 hours in response to wound stress (FIG. 4A). This indicates that the accumulation of early cacn14085 transcription in both mock and viral infection treatment groups is due to a response to wound stress. PMMoV-P ₀ 24 and 48 hours after inoculation, cacn14085 in virus-infected pepper leaves compared to the mock treatment group The gene was strongly induced (FIG. 4B). These results indicate that cacn14085 during the hypersensitive response (HR) to PMMoV -P ₀ infection. It means that the gene is specifically expressed. Expression of the CaPR1 gene was observed as a positive control for PMMoV -P ₀ inoculation. Strong expression of the CaPR1 gene was observed 24 and 48 hours after inoculation (FIG. 4B).

Result 3. PMMoV - P ₀ From induced cell death cacn14085 -control( silencing ) effect

We obtained some sequences of the cacn14085 gene from the EST clones. The cacn14085 gene sequence is 1754 bp in length, and includes 3'UTR, kinase domain and some lectin domain. In order to confirm the function of the gene in the defense response to PMMoV -P ₀ , a plant was prepared in which the cacn14085 gene was suppressed. Tabacco rattle virus (TRV) -based virus-induced gene silencing (VIGS) was used to inhibit the expression of the cacn14085 gene [Liu et al., 2002]. The 3'UTR 157 bp fragment of the cacn14085 gene was used for VIGS, which fragment was synthesized via specific primers. Specific primers for the gene are as follows.

cacn14085  gene

Forward; 5'-TAGCAGCATACATATTACCAACA-3 '

Reverse; 5'-TGCAGCCCAAAAGAAAA-3 '

One month after performing VIGS on the cacn14085 gene, the pepper leaves were inoculated with PMMoV-P ₀ , and four days after the inoculation, the pepper leaves were observed. Observation results are shown in FIG. 5. As shown in FIG. 5A, the number of hypersensitive lesions (HR-lesion) was significantly reduced in the group inhibited cacn14085 as compared to the TRV2 vector group. Supersensitive lesions appear as a protective response mediated by L-genes against PMMoV-P ₀ . In particular, 39 hypersensitive lesions appeared in the TRV2 control group, and the expression of the PR1 gene was also strongly induced. In addition, different gene expression patterns and the number of lesions were observed in plants that inhibited cacn14085 . Plant 1 had 41 supersensitive lesions, plant 2 had 39, plant 3 had 3-4, and plant 4 had 1-2 hypersensitive lesions. Transcription levels of cacn14085 were not inhibited at all in plant 1, 54% in plant 2, 66% in plant 3, and 73% in plant 4. cacn14085 As the level of inhibition of the gene increases, the number of hypersensitive lesions decreases further. In addition, the expression level of CaPR1 (FIG. 5B ) was also cacn14085 The level of inhibition decreased with increasing.

Result 4. Using 5'-RACE CaLecRK1 Isolation of full-length cDNA

To obtain a full-length cDNA of cacn14085 , two primers corresponding to the conserved sequence of some lectin domains (GSP1; 5′-GGCCTGTTGATGCTGAGAACCCAAAGTA-3 ′ and GSP2; 5′-TGGTGTTGAACCATTTTTCCCCATT-3 ′) were synthesized. 'Rapid amplification of cDNA ends (RACE) was performed as shown in FIG. As a result of 5'RACE, as shown in FIG. 6, the present inventors obtained a band of 950 bp agarose gel, and then sequenced the band to further find a cacn14085 gene of 679 bp. It was obtained in the full-length cDNA of cacn14085, and named the gene to CaLecRK1 (Capsicum annuum Lectin receptor kinase1) . The nucleotide sequence of the CaLecRK1 cDNA is 2433 bp (SEQ ID NO: 1) and includes an open reading frame (ORF) encoding a polypeptide of 701 amino acids (SEQ ID NO: 2).

Result 5. CaLecRK1 ( Capsicum annuum Lectin receptor kinase1 Sequence analysis

Protein sequencing of CaLecRK1 using the Blast tool revealed that CaLecRK1 is an N-terminal extracellular lectin domain (amino acid sequences 72 to 301), a membrane potential domain ( inos acid sequences 318 to 338) and a C-terminal cytoplasmic protein kinase domain (amino acid sequence 376). 578), it was confirmed that it includes three major domains (Fig. 7). The C-terminal protein kinase domain comprises an ATP-binding site and a Ser / Thr protein active site. Other LecRK and amino acid alignment of CaLecRK1 of Arabidopsis (Arabidopsis thaliana) are shown in Fig. CaLecRK1 phylogenetic analysis revealed that CaLecRK1 was 60% identical at the amino acid level to At5g06740 (GeneBank Accession no. NM_120757), the Arabidopsis Lectin receptor kinase (FIG. 9).

Outcome 6. Expression of Defense Signal Components CaLecRK1 Influence of

To identify expression patterns of genes involved in signaling in response to PMMoV-P ₀ , we performed RT-PCR.

Cachi1 , which encodes chitinase, is a component of a protein involved in pathogenesis, CaGlu1 is a glutamic acid synthesis gene, CaOSM1 and CaOSML , which encodes an osmosensory MAPK, CaLOX1 and CaLOX2 is lipoxygenase and (lipoxygenase) gene, CaPDF2 .2 is a peptide depot Millar claim (deformylase) gene, CaPR2, CaPR4 and CaPR5 are components that encode proteins involved in etiology, CaRIN4 is an RPM1 INTERACTING PROTEIN 4 gene, CaNDR1 is a GPI (glycosylphatidylinositol) anchor gene present in the cell membrane, CaNPR1 is a non- expressor of PR1, CaPAD4 is PHYTOALEXIN DEFICIENT 4 gene, CaCPR5 is CONSTITUTIVE EXPRESSION OF PR gENE 5, CaCOI1 is CORONATINEINSENSITIVE1 gene, CaJAZ1 is JASMONATE-ZIM-DOMAINPROTEIN1 gene, CaCTR1 is CONSTITUTIVETRIPLERESPONSE1 gene, CaEIN3 is ETHYLENE-INSENSITIVE 3 gene, CaERF is ERF (ethylene response factor) gene, CaHSTF is a heat shock transcription factor ( HSTF ) gene, elF3 and elF4E are elongation factor genes.

As a result of measuring gene expression, Cachi1 , CaGlu1 , CaLOX1 , CaPDF2.2 , CaPR2 , CaPR4 , CaPR5 , CaNPR1 , CaPAD4 , CaCOI1 , CaJAZ1 , CaCTR1 , CaEIN3 , CaERF , And CaHSTF genes increased expression in response to PMMoV -P ₀ . Gene expression analysis for wound treatment was performed as a control (FIG. 10).

Then, in order to observe the effects of CaLecRK1 genes at the molecular level, after which the inoculated plants and plant TRV2 vector (control) inhibited the CaLecRK1 to PMMoV-P _0, and inoculated 24, 48, 72 and 96 hours of semi-quantitative RT- PCR was performed to observe. As shown in FIG. 11A , CaLecRK1 gene is inhibited in each pepper leaf observed at the time. At 24, 48, 72 and 96 hours after virus infection, CaLecRK1 genes were inhibited by 30%, 70%, 92% and 78% in red pepper leaves which inhibited CaLecRK1 compared to TRV2 control.

Also PMMoV-P ₀ after inoculation, as the reduction in ultra-sensitive reactions observed in pepper leaves were suppressed CaLecRK1 (result 3), the inventors have found that genes involved in signaling in then inoculated with the PMMoV-P ₀ CaLecRK1 inhibition was pepper leaves Their expression level was measured using semiquantitative RT-PCR. First, genes involved in signaling SA ( caPAD4 , CaNPR1 , CaPR1 , CaPR2 , CaPR4 , CaPR5 , CaPR10 ) were measured. As a result of the measurement, as shown in FIGS. 11B to 11K, genes involved in SA signaling were suppressed in CaLecRK1 -suppressed pepper leaves except CaPAD4 gene. Genes involved in MeJA (Methyl jasmonate) signaling ( CaJAZ1 , CaCOI1 ) were also inhibited in CaLecRK1 inhibited pepper leaves . However, CaEIN3 , a gene involved in ethylene signaling, was not inhibited in red pepper leaves that inhibited CaLecRK1 . These results indicate that CaLecRK1 gene regulates the expression of the majority of genes involved in signaling in response to PMMoV -P ₀ .

Result 7. CaLecRK1  Restraint PMMoV - P ₀  For infection CP ( coat protein A) increase in level of warrior

For in resistance to PMMoV-P ₀ to observe the effect of CaLecRK1 gene, using the _{PMMoV-P 0 CP (coat protein} ) gene primers was performed semi-quantitative RT-PCR. Accumulation of the PMMoV-P ₀ CP gene is used as an indicator of viral proliferation.

First, PMMoV -P ₀ was inoculated into CaLecRK1 inhibited plants and TRV2 vector plants (control), and then the plants were observed 72 hours after virus infection. As a result, inhibition of CaLecRK1 expression and reduction of HR lesions were observed in plants that inhibited CaLecRK1 . Thereafter, the proliferation of PMMoV-P ₀ was confirmed by semi-quantitative RT-PCR using the CP gene. As shown in FIG. 12, when the expression of the CaLecRK1 gene was suppressed by 55%, the accumulation of PMMoV-P ₀ CP gene transcription was increased by 5 times in CaLecRK1 inhibited plants compared to the TRV2 control group. Therefore, it was confirmed that inhibition of CaLecRK1 resulted in increased transcription of PMMoV -P ₀ CP gene.

In addition, CaLecRK1 for the systemic spreading of PMMoV -P ₀ . In order to confirm the inhibitory effect, we infected the lower part of plants that inhibited and uninhibited CaLecRK1 with PMMoV -P ₀ , and after 10 days of infection, the PMMoV-P ₀ CP gene at the top of each plant not infected with the virus. The accumulation of was compared using RT-PCR. RT-PCR was performed with primers of PMMoV-P ₀ CP gene. As a result of RT-PCR, PMMoV-P ₀ CP was observed to accumulate more in CaLecRK1 inhibited plants compared to TRV2 plants (Fig. 13 (A)). Then, semi-quantitative RT-PCR was performed to confirm the inhibition of CaLecRK1 and PMMoV -P ₀ CP accumulation. Transcription of the PMMoV -P ₀ CP gene was accumulated more than four times in plants inhibiting CaLecRK1 (FIG. 13B).

PMMoV-P ₀ The protective response mediated by CaLecRK1 following inoculation was observed by trypan blue staining and DAB staining.

First, plants inhibiting CaLecRK1 and TRV2 plants (control) were infected with PMMoV-P ₀ . Three days after infection, the plants were stained with trypan blue and DAB. The staining can identify dead tissue or dead cells. As a result of observing plants after staining, as shown in FIG. 14A, cell death was lower in plants inhibiting CaLecRK1 than in TRV2 plants (control). Then, CaLecRK1 inhibition and accumulation of PMMoV -P ₀ CP gene from the plants were confirmed by performing semiquantitative RT-PCR. As a result of RT-PCR, cell death was reduced when the CaLecRK1 gene was 78% inhibited, resulting in a 2.7-fold increase in the accumulation of PMMoV-P ₀ CP gene transcription (FIG. 14B).

In addition, H ₂ O ₂ , a molecule formed when ROS (reactive oxygen species) is released during an oxidative burst, was visualized by staining with DAB. As a result, it can be seen that H ₂ O ₂ production is lower in CaLecRK1 inhibited plants than in TRV2 plants (FIG. 15 (A)). Thereafter, CaLecRK1 inhibition and accumulation of PMMoV -P ₀ CP gene from the plants were confirmed by semiquantitative RT-PCR. As a result, when the CaLecRK1 gene was 80% inhibited, the transcription of the PMMoV -P ₀ CP gene was increased five times (Fig. 15 (B)).

Based on the above results, CaLecRK1 seems to be required for plant resistance to PMMoV-P ₀ through the regulation of programmed cell death (PCD) and H ₂ O ₂ production.

<110> Korea University Industrial & Academic Collaboration Foundation <120> CaLecRK1 gene from hot pepper concerned in defense response of          plants and use pretty <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 2433 <212> DNA <213> Artificial Sequence <220> <223> Capsicum annuum lectin receptor kinase 1 <400> 1 acatgggcat gattgtgttt gtcacttcca attttttatt ttacacaatc ccaacgtgct 60 ttaaaaacct tctttttatc aaccctccat gtatcttcct ctcataatcc tcacttctct 120 atacttcatt tcactacatg aaaacaaaaa aatgaaattc ttgacaccca aaatcatcag 180 tatcttcata tttttctctt gtatacaatc catatcacaa gccaagatca aaaaatttga 240 caaacaatat ggtgatcctt ttgatcatac atatactccc atatttgaaa tcaaacatcc 300 tgcacaaatc agcaacctag ctcttcaaat caccccagac accgcgtctt ctgcttatca 360 aatgttcaat aactcaggtc gaatcctgtt gaaacgacca ttcagattgt gggatagtag 420 tcatgatgac ggagttgagg atctttcaag gttggcgtct ttcaacactt cttttttagt 480 aaacatttac aggccaaaaa atgacacacc agctgaagga ttggcattct tgatttgtcc 540 tgatttagac ctgccaaaaa acagtcaggg ccagtactta ggcctgacaa atagtactac 600 tgatggcgac gcttccaaca ggattatcgc ggttgagcta gacacgttca agcaagattt 660 tgacatcgat gacaaccaca ttggaattga tttacacagt atagattcta ttaaatcgga 720 gtcactgact ccgcatggaa ttcaactagc accaataggt gcaagatttt acaacatttg 780 gatacaatat gacggaatca agaaagtact tgatgtgtac attgttgaac aaatggggaa 840 aaatggttca acaccaccta gaccaaatga tccaatatta acacacaatc ttgatttaag 900 aaaatttgta aatcaagaat catactttgg gttctcagca tcaacaggcc atttcaatca 960 gttgaattgt gtgttgagat ggaatttaac agttgaatat tttcaagaaa agaatcatgg 1020 tttgataatt ggattaggtg ttggtgtacc tatagtagtt gtgttaatga ttttgtttgg 1080 gtattttggt tacttttatt ataagaaaaa aaggggtgat aggtcacaat ctaatatatt 1140 gggtgcatta aagagtttac ctggtatgcc tagagatttt gagtttaaag aattgaaaaa 1200 agctactaat aattttgatg aaaaaaataa acttggtgaa ggtggatatg gagttgttta 1260 caaaggcaat ttggttgatg aaaaattgga aattgcagtg aagtggtttt ctagagaaag 1320 tatcaaaggt gaagatgatt tcttggctga gttgacaatt atcaatcgtt taaggcataa 1380 acatcttgtc aaattacttg gatggagcca taagcatgga aagctactac ttgtttatga 1440 gtacatgcca aatggtagcc tagacaaaca tctcttctca gcgccagata aagaaccact 1500 cagctggtgc gtccgctaca acattgtatc aggcgtcgcg tcagccctgc actatctgca 1560 caatgagtac gaacagaagg tggtccatcg cgatctcaag gcgaacaaca tcatgctcga 1620 ctcaaacttc aatgcacgcc ttggggattt tggcctagca cgagcaattg acaatgagaa 1680 gacctcgtat gctgatgagg ccgagggggt gcttggcacg atggggtaca tcgcgccaga 1740 gtgcttccac actggaaaag ctactcaaca ttctgatgtc tatgcatttg gagcagtgtt 1800 gttggaagta gtatgtggcc aaagacctgg aaccaaagtt aatggctttc aactccttgt 1860 tgattgggtt tggttcttgc atcgcgatgg aagaatcctc gaagctgttg acaggaggct 1920 cggggatgat tacgtagctg aagaagcaaa gaggttgcta ctacttactc tagcttgctc 1980 acatccaatc gcgagtgaac gaccgaccac acaaactata gttcaaatta tatcaggatc 2040 agtgccagca ccagaagttc caccattcaa gccatcattt gtgtggcctt ctatggttcc 2100 agttgatata gaatcgagca tcgtcgatac aatatccatc acaacacctc agttcagttc 2160 agagaacaac agtattgagt atctaagcaa gtagagatag ctatagcagc atacatatta 2220 ccaacaggaa aaaaaaatta attaacttga ctttgatttc ccttttttaa tgtttttttc 2280 actctgtaat tatgttatat ggtgaggttg tcaaaagaat ttttctcttc cttcttgttt 2340 tgtttttctt ttgggctgca agtttgtaat tctgtaatga caagaacata aacaatgtat 2400 atttcttaga ccttaaaaaa aaaaaaaaaa aaa 2433 <210> 2 <211> 701 <212> PRT <213> Artificial Sequence <220> <223> Capsicum annuum lectin receptor kinase 1 <400> 2 Met Tyr Leu Pro Leu Ile Ile Leu Thr Ser Leu Tyr Phe Ile Ser Leu   1 5 10 15 His Glu Asn Lys Lys Met Lys Phe Leu Thr Pro Lys Ile Ile Ser Ile              20 25 30 Phe Ile Phe Phe Ser Cys Ile Gln Ser Ile Ser Gln Ala Lys Ile Lys          35 40 45 Lys Phe Asp Lys Gln Tyr Gly Asp Pro Phe Asp His Thr Tyr Thr Pro      50 55 60 Ile Phe Glu Ile Lys His Pro Ala Gln Ile Ser Asn Leu Ala Leu Gln  65 70 75 80 Ile Thr Pro Asp Thr Ala Ser Ser Ala Tyr Gln Met Phe Asn Asn Ser                  85 90 95 Gly Arg Ile Leu Leu Lys Arg Pro Phe Arg Leu Trp Asp Ser Ser His             100 105 110 Asp Asp Gly Val Glu Asp Leu Ser Arg Leu Ala Ser Phe Asn Thr Ser         115 120 125 Phe Leu Val Asn Ile Tyr Arg Pro Lys Asn Asp Thr Pro Ala Glu Gly     130 135 140 Leu Ala Phe Leu Ile Cys Pro Asp Leu Asp Leu Pro Lys Asn Ser Gln 145 150 155 160 Gly Gln Tyr Leu Gly Leu Thr Asn Ser Thr Thr Asp Gly Asp Ala Ser                 165 170 175 Asn Arg Ile Ile Ala Val Glu Leu Asp Thr Phe Lys Gln Asp Phe Asp             180 185 190 Ile Asp Asp Asn His Ile Gly Ile Asp Leu His Ser Ile Asp Ser Ile         195 200 205 Lys Ser Glu Ser Leu Thr Pro His Gly Ile Gln Leu Ala Pro Ile Gly     210 215 220 Ala Arg Phe Tyr Asn Ile Trp Ile Gln Tyr Asp Gly Ile Lys Lys Val 225 230 235 240 Leu Asp Val Tyr Ile Val Glu Gln Met Gly Lys Asn Gly Ser Thr Pro                 245 250 255 Pro Arg Pro Asn Asp Pro Ile Leu Thr His Asn Leu Asp Leu Arg Lys             260 265 270 Phe Val Asn Gln Glu Ser Tyr Phe Gly Phe Ser Ala Ser Thr Gly His         275 280 285 Phe Asn Gln Leu Asn Cys Val Leu Arg Trp Asn Leu Thr Val Glu Tyr     290 295 300 Phe Gln Glu Lys Asn His Gly Leu Ile Ile Gly Leu Gly Val Gly Val 305 310 315 320 Pro Ile Val Val Val Leu Met Ile Leu Phe Gly Tyr Phe Gly Tyr Phe                 325 330 335 Tyr Tyr Lys Lys Lys Arg Gly Asp Arg Ser Gln Ser Asn Ile Leu Gly             340 345 350 Ala Leu Lys Ser Leu Pro Gly Met Pro Arg Asp Phe Glu Phe Lys Glu         355 360 365 Leu Lys Lys Ala Thr Asn Asn Phe Asp Glu Lys Asn Lys Leu Gly Glu     370 375 380 Gly Gly Tyr Gly Val Val Tyr Lys Gly Asn Leu Val Asp Glu Lys Leu 385 390 395 400 Glu Ile Ala Val Lys Trp Phe Ser Arg Glu Ser Ile Lys Gly Glu Asp                 405 410 415 Asp Phe Leu Ala Glu Leu Thr Ile Ile Asn Arg Leu Arg His Lys His             420 425 430 Leu Val Lys Leu Leu Gly Trp Ser His Lys His Gly Lys Leu Leu Leu         435 440 445 Val Tyr Glu Tyr Met Pro Asn Gly Ser Leu Asp Lys His Leu Phe Ser     450 455 460 Ala Pro Asp Lys Glu Pro Leu Ser Trp Cys Val Arg Tyr Asn Ile Val 465 470 475 480 Ser Gly Val Ala Ser Ala Leu His Tyr Leu His Asn Glu Tyr Glu Gln                 485 490 495 Lys Val Val His Arg Asp Leu Lys Ala Asn Asn Ile Met Leu Asp Ser             500 505 510 Asn Phe Asn Ala Arg Leu Gly Asp Phe Gly Leu Ala Arg Ala Ile Asp         515 520 525 Asn Glu Lys Thr Ser Tyr Ala Asp Glu Ala Glu Gly Val Leu Gly Thr     530 535 540 Met Gly Tyr Ile Ala Pro Glu Cys Phe His Thr Gly Lys Ala Thr Gln 545 550 555 560 His Ser Asp Val Tyr Ala Phe Gly Ala Val Leu Leu Glu Val Val Cys                 565 570 575 Gly Gln Arg Pro Gly Thr Lys Val Asn Gly Phe Gln Leu Leu Val Asp             580 585 590 Trp Val Trp Phe Leu His Arg Asp Gly Arg Ile Leu Glu Ala Val Asp         595 600 605 Arg Arg Leu Gly Asp Asp Tyr Val Ala Glu Glu Ala Lys Arg Leu Leu     610 615 620 Leu Leu Thr Leu Ala Cys Ser His Pro Ile Ala Ser Glu Arg Pro Thr 625 630 635 640 Thr Gln Thr Ile Val Gln Ile Ile Ser Gly Ser Val Pro Ala Pro Glu                 645 650 655 Val Pro Pro Phe Lys Pro Ser Phe Val Trp Pro Ser Met Val Pro Val             660 665 670 Asp Ile Glu Ser Ser Ile Val Asp Thr Ile Ser Ile Thr Thr Pro Gln         675 680 685 Phe Ser Ser Glu Asn Asn Ser Ile Glu Tyr Leu Ser Lys     690 695 700

Claims (10)

Caylic lectin receptor phosphorylation 1 ( CaLecRK1 ) gene having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.
According to claim 1, wherein the gene is pepper lectin receptor phosphorylation 1 ( CaLecRK1 ) gene, characterized in that having a nucleotide sequence of SEQ ID NO: 1.
Calicectin receptor 1 ( CaLecRK1 ) protein having the amino acid sequence of SEQ ID NO: 2.
The recombinant vector which introduce | transduced the CaLecRK1 gene of Claim 1 or 2.
Plant transformed with the recombinant vector of claim 4.
A composition for increasing infection resistance of a plant comprising the expression inhibitor of the CaLecRK1 gene of claim 1 or 2 as an active ingredient.
According to claim 6, wherein the expression inhibitor is a composition for increasing infection resistance of plants, characterized in that the viral vector for VIGS introduced CaLecRK1 gene.
8. The composition for increasing infection resistance of a plant according to claim 7, wherein the viral vector for VIGS into which the CaLecRK1 gene is introduced is pTRV2 :: CaLecRK1 disclosed in FIG.
The composition of claim 6, wherein the infection of the plant is caused by PMMoV-P ₀ .
The composition for increasing infection resistance of a plant according to claim 6, wherein the composition controls resistance to plant infection.

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