KR20150051283A - Multiple plant pathogen-resistant transgenic plant using antimicrobial peptide and uses thereof - Google Patents

Abstract

The present invention relates to a cultivation of a complex disease resistant transformant using antimicrobial peptide, and a usage thereof. More particularly, the present invention relates to a method for regulating complex disease resistance of a plant, which includes a step of transforming a plant gene to a recombinant vector containing human-derived cathelicidin peptide (CAP) protein coding genes so as to regulate the expression of the CAP protein coding genes; a method for preparing a transformed plant having the regulated complex disease resistance, which includes a step of transforming a plant cell to a recombinant vector containing the CAP protein coding genes; and to a compound for regulating complex disease resistance of a plant, wherein the compound contains as an active ingredient, a recombinant vector containing the transformed plant with the regulated complex disease resistance, seeds of the plant, and the human-derived CAP protein coding genes.

Description

[0001] The present invention relates to an antimicrobial peptide,

More particularly, the present invention relates to a method for transforming plant cells with a recombinant vector containing a CAP (cathelicidin peptide) -coding gene derived from a human, thereby producing a CAP protein coding gene A method for controlling the complex tolerance of a plant comprising the step of regulating the expression of the CAP protein coding gene, and a step of transforming the plant cell with a recombinant vector comprising the CAP protein coding gene , A composition for regulating the complex tolerance of a plant, which comprises the recombinant vector comprising the transgenic plants with controlled complex tolerance and the seeds thereof and the human-derived CAP protein coding gene prepared by the above method as an active ingredient.

Antimicrobial peptides produced in response to bacterial infections play a very important role in the forefront of host defense systems against pathogen invasion. These peptides have antimicrobial activity against a very wide range of microorganisms including Gram-positive bacteria, gram-negative bacteria, fungi (fungi) and certain viruses. Antimicrobial peptides are divided into two groups called defensin and cathelicidine. In the case of dipenthine, three types of α, β, and θ are identified. α-Dipenesin is also known as the human neutrophil peptide (HNP) 1-3 because α-diphenesin is located in the neutrophil and large intestine paneth cells. On the other hand, beta -dipensin is expressed in many epithelial tissues.

On the other hand, the damage caused by diseases or pests of plants has been a major obstacle to the stable food supply of mankind along with natural disasters. According to general statistics, plant diseases or insect pests account for about 10% of the total production, respectively, and will be much higher if the pesticides produced by the crops are considered. In addition, large outbreaks of certain pests due to meteorological changes or special environmental changes may cause great social problems. For example, in 1979 and 1980, the outbreak of rice blight in Korea caused serious problems in terms of food security, resulting in the introduction of large quantities of rice in foreign countries and social problems for a while. Meanwhile, mass rainfall in central Chungcheong province in 1995 caused a great loss due to diseases of pepper and other crops. It is estimated that pepper has lost at least 50% of water loss due to damage of anthrax and plague.

As part of efforts to control such pests, artificial synthetic pesticides have been used in agriculture for their development. However, due to the ever-increasing population, it is necessary to secure more food. Of the same variety leads to the mass production of pests and the abuse of synthetic pesticides for their control prevents the destruction of natural ecosystems as well as toxicity to residual toxicity, Research has been carried out on the development of new pesticides (low toxic pesticides, natural pesticides or biological pesticides) in order to replace them, but the speed of development has not satisfied the demand.

In addition, the recent proliferation of environmental protection campaigns and the tendency of mankind to take clean, uncontaminated agricultural products are urgently demanding new methods that are harmless to mankind and free from residual toxicity and destruction of natural ecosystems. Recently, as genetic engineering technology has been developed, disease-resistant transgenic plants have been proposed as an alternative to produce stable food crops while using less pesticides. In addition, research on the disease resistance mechanism of plants has been carried out and a new concept And to develop a plant protection product for the developed countries.

Korean Patent No. 0803393 discloses a method for enhancing disease resistance using OsLRP gene in rice and Korean Patent No. 0607701 discloses a plant disease resistance gene oopjial 10 isolated from wild rice, Although a transformant plant has been disclosed, the cultivation of an antimicrobial peptide-use complex tolerant transformant and its use have not been disclosed as in the present invention.

The present inventors have found that the inventors of the present invention have found that when a transgenic rice plant that overexpresses a CAP protein coding gene derived from a human is treated with rice blast fungus and blast fungus, The present inventors have completed the present invention by confirming that they exhibit superior disease resistance to the genus Bacillus strains as compared with the resistant strains of the genus Bacillus,

In order to solve the above problems, the present invention relates to a method for regulating expression of a CAP protein coding gene by transforming a plant cell with a recombinant plant expression vector containing a CAP (cathelicidin peptide) protein coding gene derived from a human, Thereby providing a method for controlling complex tolerance.

In addition,

Transforming a plant cell with a recombinant vector comprising a human-derived CAP (cathelicidin peptide) protein coding gene; And

And regenerating the plant from the transformed plant cell. The present invention also provides a method for producing a transformed plant having a controlled complex tolerance.

In addition, the present invention provides a transgenic plant having a controlled complex tolerance prepared by the above method and seeds thereof.

In addition, the present invention provides a composition for controlling the complex tolerance of a plant, which contains, as an active ingredient, a recombinant vector comprising a human-derived CAP (cathelicidin peptide) protein coding gene.

In the present invention, when the CAP protein coding gene derived from human and overexpressing transgenic rice plants were treated with the rice blast fungus and the blast fungus, respectively, the resistance to the rice blast fungus and the blast fungus strains was higher than that of the resistant varieties, And it was confirmed that they were resistant to one disease. Accordingly, the present invention can be applied to molecular breeding for development of disease-resistant crops in rice and other crops, and it is possible to reduce the use of pesticides by cultivating disease resistant cultivars, thereby improving productivity and being useful for safe agricultural products supply .

FIG. 1 shows the germination inhibition level of spores of Fusarium culmorum strain treated with antimicrobial peptide material of hCAP18 (same as CAP protein of the present invention) at different concentrations (2 and 10 μM).
FIG. 2 shows the antifungal activity assay of GST-CAP fusion protein by treatment with GST-CAP protein expressed and purified in E. coli to a culture of Botrytis cinerea. A, B, and GST-tags processing; C, D, and GST-CAP processing. D arrows show that cytoplasmic material was spilled around the destroyed spore.
Fig. 3 shows the inoculation process and the complex disease tolerance test procedure of transgenic rice. (A), (E), growth of transgenic plants in pots; (B), inoculation with rice blast fungus; (C), phenotype of the lesion after infection with rice blast fungus (WT; (D) Pathogen proliferation in CAP transformant (TG) after 2 weeks of inoculation with rice blast fungus (Cont 1, Dong 2; (F), inoculation with blast fungus; (G), the phenotype of the lesion after infection with blast fungus (WT; (H), two weeks after the inoculation of the rice blast fungus, the pathogen proliferation in the CAP transformant (Cont 1, Dong 3,
Fig. 4 shows the vector structure for the rice expression of the CAP gene and the production process of the transgenic rice plant of the vector. A, CAP amino acid sequence; B, a CAP base sequence including a signal peptide (underlined), an initiation codon and a stop codon; C, a CAP transformation vector for introduction into rice plants; (D) seedling growth and growth after 6 weeks, (f) seedling growth in the greenhouse, (d) seedling growth, sublimation
FIG. 5 shows a process for producing a rice plant transformant through transformation of an Agrobacterium-mediated vector of a CAP gene-introduced vector. (A), (B), rice seed germination in N6D medium; (C), Agrobacterium inoculation; (D) Co-culture of Agrobacterium strain and rice callus in 2N6-AS medium; (E) callus proliferation in 2N6-C medium; (F), (G) and (H), shoot formation and elongation; (I), (J), root formation and elongation; (K), (L) purification
FIG. 6 shows the result of measuring the size of the lesion 10 days after the inoculation with the strain of rice blast fungus (Xanthomonas oryzae KACC10859) at the time of transformation. Cont 1, Dong Jin - jin; Cont 2; 1 to 5, CAP T0 transgenic plant line

In order to achieve the above object, the present invention provides a method for regulating expression of a CAP protein coding gene by transforming a plant cell with a recombinant plant expression vector containing a CAP (cathelicidin peptide) protein coding gene derived from a human, Thereby providing a method for controlling complex tolerance.

In the method of controlling complex tolerance of a plant according to an embodiment of the present invention, the CAP protein may be composed of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The range of the CAP protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous physiological activity" means an activity of regulating complex tolerance of a plant.

The present invention also provides a gene encoding the CAP protein. The gene of the present invention includes both genomic DNA encoding cPP protein and cDN. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In a method for controlling complex tolerance of a plant according to an embodiment of the present invention, the CAP The gene may be overexpressed to increase the complex tolerance of the plant, but is not limited thereto.

In the method of controlling complex tolerance of a plant according to an embodiment of the present invention, the plant disease may be a disease caused by a phytopathogenic bacterium or a phytopathogenic fungus, and preferably a disease caused by a disease such as a blister, a spot blight, A rice blast, a blight of blight of rice blast, or blast disease, and most preferably, it may be blight of blight of blight of white blight or blast disease, but is not limited thereto.

In addition,

Transforming a plant cell with a recombinant vector comprising a human-derived CAP (cathelicidin peptide) protein coding gene; And

And regenerating the plant from the transformed plant cell. The present invention also provides a method for producing a transformed plant having a controlled complex tolerance.

In the method of the present invention, the CAP protein may be composed of the amino acid sequence of SEQ ID NO: 2, but the present invention is not limited thereto.

In the method for producing a transgenic plant having controlled complex tolerance according to an embodiment of the present invention, the CAP The gene may be overexpressed to increase the complex tolerance of the plant, but is not limited thereto.

In the method for producing a transgenic plant having controlled complex tolerance according to an embodiment of the present invention, the plant disease may be a disease caused by a phytopathogenic bacterium or a phytopathogenic fungus, and preferably a disease caused by a disease such as blight, , Pest disease, ulcer disease, blight of blight of white blight of rice blast or blast disease, and most preferably blight of blight of white blight of blight or blast disease.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

A preferred example of a plant expression vector is a Ti-plasmid vector which is capable of transferring a so-called T-region into a plant cell when present in a suitable host such as Agrobacterium tumefaciens . Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as Kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

In the plant expression vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) Terminator of the Octopine gene, and the rrnB1 / B2 terminator of E. coli, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae), and the like insect cells, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cell may be used. The host cell is preferably a plant cell.

The "plant cell" is any plant cell. Plant cells are cultured cells, cultured tissues, cultivated or whole plants.

The method of delivering the vector of the present invention into a host cell can be carried out by injecting a vector into a host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, can do.

In addition, the present invention provides a transgenic plant having a controlled complex tolerance prepared by the above method and seeds thereof.

Plants to which the method according to the present invention can be applied include monocotyledonous plants or dicotyledonous plants. Examples of the monocotyledonous plants include rice, wheat, barley, bamboo shoots, corn, taro, asparagus, onion, garlic, leek, leek, soya bean, and ginger. Examples of dicotyledonous plants include, but are not limited to, Arabidopsis thaliana, eggplant, cigarette, red pepper, tomato, burdock, ciliaceae, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, parsley, parsley, cabbage, cabbage, Watermelon, melon, cucumber, pumpkin, strawberry, soybean, mung bean, kidney bean, camelina and pea. The plant is preferably rice.

In addition, the present invention provides a composition for controlling the complex tolerance of a plant, which contains, as an active ingredient, a recombinant vector comprising a human-derived CAP (cathelicidin peptide) protein coding gene. The composition comprises, as an active ingredient, a CAP protein coding consisting of the amino acid sequence of SEQ ID NO: 2 Gene, and the complex tolerance can be controlled by transforming the gene into a plant.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

Example  One. hCAP18 Analysis of Antifungal Activity in vitro

37 peptides (NH2'-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES-COOH ': SEQ ID NO: 2) were synthesized with a consulting company of Youngin, Frontia and diluted to low and high concentrations to inhibit spore germination of Fusarium culmorum As a result, spore germination of the strain occurred at a low concentration of 5 μM of the peptide. However, it was confirmed that the spore of the bacteria did not occur at a high concentration of 10 μM and the antifungal activity was very high (FIG. 1).

In order to measure the antifungal activity of the GST-CAP fusion protein, the fungus Botrytis cinerea was cultured with the purified GST-CAP protein in E. coli, The spores did not germinate at all in the treatment with the GST-CAP fusion protein (Fig. 2D). Therefore, it is suggested that CAP peptides are expressed in Escherichia coli and have antibacterial activity (Fig. 2).

Example  2. VB2 :: hCAP18  Antibacterial activity analysis of bacterial pathogens and fungal pathogens of transgenic lines transformed with vectors

In order to verify the antimicrobial activity of the antimicrobial peptide CAP gene in the transformant into which VB2 :: hCAP18 was introduced, the strains of Xanthomonas oryzae KACC10859 and blast were isolated from the transformant T3 generation After culturing, the cells were inoculated at a concentration of 10 ⁶ CFU. In this study, we used Jinbagi rice, which is resistant to resistant cultivars such as Yongkwangbyeo and mutant strains, to Dongjinbyeong, rice blastomycetes and rice blast fungus strains. On the 14th day after inoculation of the pathogens into the transformant and wild type (WT), the CAP-introduced transformants were not infected with the blight-susceptible bacteria (Fig. 3C). In addition, it was confirmed that resistance to the mutant strains was greater than that of the cultivar Jinbagi (Fig. 3D). Transgenic plants inoculated with blast fungus were also significantly smaller than those of Dongjinbya WT in terms of the size of the lesion as in 3G, and pathogen transitions were smaller than those of the previously reported resistant cultivar (Figure 3H).

Example  3. pPZP :: Preparation of CAP vector and pPZP :: Cultivation of transgenic lines transformed with CAP vector

In addition to the existing constructed VB2 :: hCAP18 vector, the vector was reconstructed by inserting the antimicrobial peptide CAP gene into the pPZP vector regulated by the PGD1 promoter distributed from Myongji University in order to easily identify and introduce the single copy of the gene in the transformant Respectively. The pPZP :: CAP vector constructed as shown in Fig. 4C was introduced into a Dongjin-jin rice variety to prepare a transformant (Fig. 4).

A common method commonly used to transform rice plants is to inoculate the callus in rice seeds and then inoculate the callus with Agrobacterium spp. It usually takes about 4 months to obtain the transformant . In this experiment, agar bacterium was inoculated with seeds that had been swollen at the blastocyst stage after washing rice seed matured seeds to shorten the time consumed for transplanting the rice, and soaking them in N6 liquid medium for 24 hours. The surface-sterilized seeds were soaked in N6 liquid medium containing 2 mg / L 2,4-D and germinated for 24 hours at 30 캜 in a dark state (Fig. 5A and Fig. 5B), resulting in swelling of the abdomen, Was inoculated in a tube containing 15 ml of Agrobacterium suspension, and inoculated for about 20 minutes. Then, seeds were placed on the sterilized filter paper to remove excess Agrobacterium. The seeds inoculated with Agrobacterium were placed on filter paper on 2N6-AS medium containing 1 mM DTT (dithiothreitol), 3 mg / L silver nitrite (AgNO ₃ ) and 0.5% gelite, (Fig. 5C). Then, in order to enhance the influence of the medium components, the filter paper was dentated to the medium from which the filter paper had been removed, and co-cultured for 4 days at 25 캜 in dark condition (Fig. 5D).

1 mM DTT, 3 mg / L silver nitrite (AgNO ₃ ) and the like were added to the culture medium to prevent excessive growth of Agrobacterium during the co-cultivation period and to reduce the substances that could be produced by the defense mechanism of plant cells due to the introduction of Agrobacterium . After 7 days of coculture, the callus formation rate by hormone was less effective than the callus formation and growth after infection with bacterium. In addition to the addition of antioxidants that can inhibit excessive growth of Agrobacterium during co-culture, proper regulation of the hormones used may enable efficient callus formation and introduction of target genes by Agrobacterium in co-culture .

Since the seeds used in this experiment are not yet sufficient for callus proliferation, healthy calli are proliferated and transferred to N6D medium containing only 400 mg / L carbenicillin to inhibit the growth of Agrobacterium. And cultured for 1 week in a continuous light condition to proliferate callus (FIG. 5E). After 1 week of callus proliferation, the callus was transferred to N6D medium containing 6 ㎎ / L ppt (phosphinothricin) and 400 ㎎ / L carbenicillin for 2 days Lt; / RTI > After cultivation, the calli propagating proliferately from the blastocyst were transferred to SF (shoot formation) medium to induce plant regeneration. Green spots began to form on the callus after 2 weeks in the medium containing antibiotics (Fig. 5F), and shoot formation began in the callus after approximately 3 weeks of culture (Figs. 5G and 5H) . After transferring from antibiotic medium to SF medium, the efficiency of green spot formation and regeneration was different depending on the gene inoculated. The subse- quently regenerated plant was transferred to root formation medium (Fig. 5I and 5J) to induce root development. The purified rice transgenic plants were transplanted into pots in the greenhouse and cultivated (Figs. 5K and 5L). A total of 400 transgenic plants were obtained from 1200 transgenic calli.

Example  4. pPZP :: CAP transformants PCR  Identification of transgenes through analysis

400 pPZP :: CAP transformants prepared using the Agrobacterium method were transplanted into the pots and cultivated in a greenhouse at 28 ° C. Each phenotype appeared in various forms such as leaf color, leaf vein, kidney and till number, and grew as a healthy plant after one month. Total DNA was extracted for 400 transfected pPZP :: CAP transformants. The leaves were weighed 1 g each from the transformants and control plants, finely pulverized using liquid nitrogen, and the ground tissue was placed in a 1.5 ml centrifuge tube containing 400 μl of the DNA extraction buffer, and the mixture was blended up and down 20 times . Then, 200 μl of 2 × CTAB buffer was added, mixed in the same manner, 10% SDS was added, and the mixture was mixed up and down about 10 times, and then left in a constant temperature water bath at 65 ° C. for 20 minutes. 200 μl of 5M potassium acetic acid (pH 7.5) was added and mixed up and down 50 times. 700 μl of PCI (25: 24: 1) was added and the mixture was centrifuged at 12,000 rpm for 10 minutes at room temperature. About 400 μL of the upper layer was transferred to a new 1.5 mL tube, and the same amount of isopropanol was added thereto. The mixture was stored in a freezer at 20 ° C. for 20 minutes and then centrifuged at 12,000 rpm at 4 ° C. for 15 minutes to precipitate the DNA. The precipitated DNA was washed with 1 ml of 70% ethanol, completely dried at room temperature, sufficiently dissolved in 50 μl of TE buffer to which 2 μl of RNase (1 mg / ml) had been added, and allowed to stand at 37 ° C. for 1 hour. PCR analysis was performed to confirm the gene introduction using the extracted DNA.

In order to investigate the introduction of pPZP :: CAP gene in the rice genome of the regenerated plant obtained from the transfection experiment, genomic DNA was isolated from the regenerated leaf to obtain Bar gene and CAP gene-specific PCR analysis was performed using the primer set for amplification. Bar and CAP genes were detected in 400 sown seedlings. This means that the pPZP :: CAP vector is well introduced into the rice genome, and these transformants are numbered from the T0 generation.

Example  5. TaqMan PCR Single copy by Single copy ) Introduced transformant Selection

In this study, based on the probe information obtained from the myoji region by the protocol provided by Takara for the selection of single copy transfected cells by TaqMan PCR, the specificity of the nos terminator region at the terminal of CAP gene of PGDI-pinII bar vector A probe primer labeled with an array was used. The probe used herein includes a target gene probe; 3'NOS, reference probe; Respectively. Transformants were able to calculate the absolute standard concentration for the quantification of CAP genes in the genome from 400 plant genomes identified by PCR analysis with bar and CAP genes. A total of 400 transformants selected from the regeneration plant were confirmed by TaqMan PCR using T2-homo DNA and T2-heterogeneous DNA from Myongji University as a control.

From the regenerated plants into which the pPZP :: CAP vector was introduced, it was confirmed by PCR analysis that 170 transgenic plants were inserted in a single copy among 400 transgenic T0 individuals.

qRT-PCR was performed to analyze the expression levels of the CAP gene-introduced transformants from the cDNAs obtained after extracting the 170 single-copy plants selected from the regenerated plants into which the pPZP :: CAP vector was introduced. Compared with the control, the expression of CAP gene by PCR method was higher in most transformants (data not shown).

Example  6. pPZP :: In the transgenic line introduced with CAP, rice Blight of white blight  Inoculation and selection

In order to verify the antimicrobial activity of 170 single-copy plants selected from the TO generation of the pPZP :: CAP vector, the plants were inoculated with 10 ⁶ CFU of Xanthomonas oryzae KACC10859 strain. As a result, CAP overexpressing transformants 1 to 5 showed resistance to the pathogenic bacteria infecting the cells as the antimicrobial peptides were expressed, compared with the Jinbongbyeo variety, which is resistant to the wild-type and rice blight-resistant bacteria used in the control. (Fig. 6). The size of the lesion was 11 cm and the size of the transgenic plants was less than 2 cm and the transmission of pathogens was not occurred. These results are analyzed to be obtained through the action of the CAP gene as an antimicrobial peptide in the plant cell tissue.

Example  7. T2  Gene expression analysis from generation Agricultural trait  Analysis, generation upbringing

The expression of genes in the T2 line developed from the homozygous T1 line was examined and the phenotypic and agricultural traits of the individuals raised in the field were analyzed. As a result, it was confirmed that CAP was stably expressed in all individuals, As a result of analyzing the traits, the phenotype may be somewhat lacking in all aspects if it is considered to be a transgenic animal compared to the wild type of Dongjinbang, as shown in Table 1, but the overall agronomic traits were not significantly different.

<110> Hankyong Industry Academic Cooperation Center <120> Multiple plant pathogen-resistant transgenic plant using          anti-microbial peptides and uses thereof <130> PN13366 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 162 <212> DNA <213> Homo sapiens <400> 1 atgttgttgg caattgcttt cctagcctca gtttgtgtct cttctatgct gctgggtgat 60 ttcttccgga aatctaaaga gaagattggc aaagagttta aaagaattgt ccagagaatc 120 aaggattttt tgcggaatct tgtacccagg acagagtcct ag 162 <210> 2 <211> 37 <212> PRT <213> Homo sapiens <400> 2 Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu   1 5 10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val              20 25 30 Pro Arg Thr Glu Ser          35

Claims (14)

A method for regulating the complex tolerance of a plant, comprising the step of transforming a plant cell with a recombinant plant expression vector containing a human-derived CAP (cathelicidin peptide) protein coding gene to regulate expression of a CAP protein coding gene. 2. The method according to claim 1, wherein the CAP protein comprises the amino acid sequence of SEQ ID NO: 2. The method of claim 1, wherein the CAP A method for regulating the complex tolerance of a plant, comprising overexpressing a gene to increase the complex tolerance of the plant. The method according to claim 1, wherein the plant disease is a disease caused by a phytopathogenic bacterium or a phytopathogenic fungus. [Claim 3] The method according to claim 1, wherein the plant disease is rice blight blight disease or blast disease. Transforming a plant cell with a recombinant vector comprising a human-derived CAP (cathelicidin peptide) protein coding gene; And
And regenerating the plant from the transformed plant cell. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; [Claim 6] The method according to claim 6, wherein the CAP protein comprises the amino acid sequence of SEQ ID NO: 2. 7. The method of claim 6, wherein the CAP Wherein the plant is overexpressed to increase the complex tolerance of the plant. [Claim 7] The method according to claim 6, wherein the plant disease is a disease caused by a phytopathogenic bacterium or a phytopathogenic fungus. [Claim 7] The method according to claim 6, wherein the plant disease is rice blight blight disease or blast disease. 10. A transgenic plant having a controlled complex tolerance prepared by the method according to any one of claims 6 to 10. 12. The plant according to claim 11, wherein the plant is a monocotyledon. 12. A plant seed according to claim 11. A composition for controlling the complex tolerance of a plant, which contains, as an active ingredient, a recombinant vector comprising a human-derived CAP (cathelicidin peptide) protein coding gene.

Images