KR20110051539A - Transgenic pepper with enhanced tolerance to pepmov and production method thereof - Google Patents

Abstract

PURPOSE: A transformant plant with enhanced resistance to viral diseases due to PepMoV(pepper mottle virus) is provided to reduce virus damage and to reduce agricultural chemical use and labor. CONSTITUTION: A transgenic plant with enhanced PepMoV viral disease resistance is prepared by transforming a plant with a recombinant plant RNAi expression vector containing PepMoV-derived HC-Pro(helper component-proteinase) gene. The HC-Pro gene has a base sequence of sequence number 1. The recombinant RNAi expression vector is pK7GWIWG2(II)::HC-Pro vector. The plant is Capsicum annuum.

Description

Transgenic peppers with enhanced tolerance to PepMoV and production method

The present invention is HC - Pro from Pepper Mottle Virus (PepMoV: Pepper Mottle Virus) The present invention relates to a pepper transformant and a method of producing the same, wherein the plant is transformed with a gene to enhance plant resistance to viral diseases caused by PepMoV.

There are more than 50 kinds of viruses that cause damage to peppers worldwide. In Korea, pepper mild mottle virus belongs to Tobacco mosaic virus (TMV). PMMOV), Tobacco mild green mosaic virus (TMGMV), Cucumber mosaic virus (CMV), Broad bean wilt virus (BWV), Pepper mottle virus (PepMoV) , And six species, including tomato spotted wilt virus (TSWV).

Pepper crops can be damaged by the virus from the seedling stage. Recently, growth inhibition or pepper fruit damage by PepMoV, a kind of potyvirus, is increasing. PepMoV is transmitted by aphids and is the most identified potyvirus in Korea since 1991. However, the virus itself is not more severe than cucumo virus CMV, but in most cases it is more prone to damage caused by multiple infections with CMV, PVY (potyvirus) and PMMoV (tobamo virus).

Since there are no breeding resources resistant to viral diseases caused by PepMoV, it is very difficult to develop PepMoV resistant pepper varieties using conventional breeding methods.

The HC - Pro gene helps the aphid to become a carrier of the virus by helping to bind viral coat proteins to the lining of the aphid's digestive tract. Other features include viral protein processing, long-distance migration of viruses, RNA replication, and gene silencing. Especially HC - Pro The cysteine proteinase domain in the gene is located at the carboxyl terminus of the HC - Pro gene and is highly genetically conserved. When the PepMoV to break into the transgenic peppers that contains Pro HC gene of the virus - - HC not inhibit the role of Pro gene within PepMoV pepper virus does not multiply whereby the pepper is to have resistance to PepMoV virus.

RNA interference (RNAi) phenomenon was first reported in 1998 by the nematode Caenorhabditis elegans (Tavernarakis et al. 2000, Nat Genet. 24: 180-3). Later, RNAi was found to be conserved in a variety of species, including nematodes, insects, plants, and fungi, and was found to be a defense system at the nucleic acid level that is commonly included in living organisms. In these organisms, the introduction of specific dsRNA from outside has been shown to inhibit the expression of target genes, and is also used as a method for producing knocked-down individuals. RNA interference induces degradation of target gene mRNA by introducing double strand RNA (dsRNA) composed of sense RNA having a sequence homologous to mRNA of target gene and antisense RNA having complementary sequence thereto. This is a phenomenon that can suppress the expression of the protein. The gene knock-down method using RNAi is a method that has been in the spotlight recently because of its simplicity and excellent effect of inhibiting gene expression.

The present invention has been devised by the above requirements, and in the present invention, HC - Pro Plants are transformed with a recombinant plant RNAi expression vector that induces gene silencing of the gene, thereby transforming the plant against PepMoV. The present invention has been completed by confirming the improvement of the resistance compared to the wild type plants.

In order to solve the above problems, the present invention is HC - Pro derived from PepMoV It is transformed with a recombinant plant RNAi expression vector comprising a gene to provide a plant and its seeds which have enhanced resistance to viral diseases by PepMoV.

In addition, the present invention is the HC - Pro Plants were transformed with the recombinant plant RNAi expression vector containing the gene, HC - Pro It provides a method for enhancing the plant's resistance to viral diseases caused by PepMoV including inducing gene-silencing of genes.

Red pepper accounts for about 10% of the world's vegetable growing area, and occupies the third largest market. Virus damage from peppers accounts for 30% of the pest damage of all pepper crops. Therefore, developing peppers that are resistant to PepMoV to peppers with a high market value can reduce virus damage and reduce pesticide use and labor in controlling aphids. Therefore, the present invention is a very useful invention in the crop industry.

In order to achieve the object of the present invention, the present invention is HC - Pro derived from PepMoV A recombinant plant RNAi expression vector containing the gene is transformed to provide a plant having enhanced resistance to viral diseases caused by PepMoV.

The HC - Pro gene is HC - Pro Refers to a sequence including a cysteine proteinase domain in a gene. Preferably, the sequence includes the nucleotide sequence of SEQ ID NO. 1, but is not limited thereto. The recombinant plant RNAi expression vector may be, for example, pK7GWIWG2 (II) :: HC-Pro vector described in FIG. 1, but is not limited thereto.

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” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. 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 properties that can be selected by a chemical method, which corresponds to all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol Resistance genes include, but are not limited to.

In the plant expression vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. 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, the constitutive promoter does not limit the selection possibilities.

In the plant expression vectors of the present invention, conventional terminators can be used, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium) terminators of the octopine gene of tumefaciens, but are 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.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 7274; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363373), electroporation of protoplasts (Shillito RD et al., 1985 Bio / Technol. 3, 10991102), Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179185, DNA of various plant elements Or RNA-coated) particle bombardment (Klein TM et al., 1987, Nature 327, 70), incomplete or incomplete in Agrobacterium tumerfaciens mediated gene transfer by plant infiltration or transformation of mature pollen or vesicles. ) Infection by a virus (EP 0 301 316) and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. Plant cells are cultured cells, cultured tissues, cultured organs or whole plants.

"Plant tissue" refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. Plant tissue is planted in planta ) or in organ culture, tissue culture or cell culture.

In the present invention, the transformed plant may include not only the whole plant, but also tissues, cells, or seeds obtainable therefrom.

HC - Pro of the present invention Plants transformed with the fusion gene have excellent resistance to viral diseases caused by PepMoV.

The plant is preferably Arabidopsis, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrots, buttercups, parsley, cabbage, cabbage, gatchi, watermelon, melon, cucumber It may be a dicotyledonous plant, such as pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, pea, more preferably pepper ( Capsicum annuum ).

The present invention also provides seed of the plant.

The present invention also provides a HC - Pro consisting of the nucleotide sequence shown in SEQ ID NO: Plants were transformed with the recombinant plant RNAi expression vector containing the gene, HC - Pro It provides a method for enhancing the plant's resistance to viral diseases caused by PepMoV including inducing gene-silencing of genes.

As a method of inducing gene silencing of the HC - Pro gene, various methods known in the art may be used. The present invention provides a method for a plant to be resistant to a virus by transforming the plant as a gene silencing construct directed towards the plant viral gene.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One: HC - Pro Plant expression expressing genes RNAi  Manufacture of vector

A plant expressing RNAi vector pK7GWIWG2 (II) :: HC-Pro (FIG. 1) was prepared for transformation into pepper.

As a selection marker, kanamycin was used to bind the HC-Pro gene 2 copies of SEQ ID NO: 1 reversely to each other for the RNAi system. PCR was performed with the prepared primers to amplify a region over a portion of the 35S promoter in a portion of the HC - Pro gene to confirm normal cloning. At this time, PCR was denatured at 94 ° C. for 5 minutes as a precycling reaction, and then repeated 35 times in the order of 30 seconds at 94 ° C., 30 seconds at 58 ° C., and 30 seconds at 72 ° C., and then The PCR reaction was terminated by extending the reaction at 72 ° C. for 5 minutes.

Plasmid pK7GWIWG2 (II) :: HC-Pro of each vector prepared as described above was replaced with Agrobacterium tumefaciens EHA105 ( Agrobacterium). tumefaciens EHA105) was used to transform plants.

Example  2: HC - Pro  Transformation of pepper using gene

THK and C15 were used for transformation as the pepper strain. Pepper transformation method was performed according to the patent (Registration: 10-0522437), and was modified slightly because different strains were used.

When the cotyledon has opened about 45 °, the growth point is not followed, and both sides of the cotyledon are cut and placed in a cornical tube containing 20 ml of MS liquid medium, and 2 ml of Agrobacterium solution is added. Then, it was shaken for about 15 minutes to allow the bacteria to be applied throughout. The filter paper was placed on a filter paper to remove moisture, slightly dried, and then incubated for 48 hours. To remove Agrobacterium, washing was performed twice with 800 mg / L of cefotaxime and twice with 500 mg / L, using 1/2 MS, sucrose 1.5%, and pH 5.8 as a washing buffer. . After completely drying the sections using filter paper, they were placed on a selection medium and subcultured at intervals of 3 to 4 weeks to incubate until callus was formed and shoots emerged. Selection of leaves grown in callus (regrowth of leaves 1 cm or more), kidney medium (MS, sucrose 3%, agar 0.8%, pH 5.7, Zein 1.0mg / L, Km Elongate at 60-100 mg / L, Cef 300 mg / L) and organically root in root organic medium (MS, sucrose 3%, agar 0.8%, pH 5.8-Km 0-20 mg / L, Cef 300 mg / L) I was. Green plants with roots were planted in a mixture of vermiculite, perlite, and peat moss (2: 1: 1), and planted in a jiffy pot that had been previously soaked in water so that the roots were not injured. After 10 to 14 days in the culture room was purified. When the plant's above-ground part grew more than 10 cm and the roots adhered to the jiffy pot, it was transferred to the greenhouse.

2 shows the developmental stages of the transformed pepper plant. As can be seen in Figure 2, the transformed pepper plants appeared to grow into normal plants.

As a result of analyzing the transformation rate of the pepper plants transformed as described above, the shoots showing the PCR positive reaction showed 0.4% (Table 1).

Table 1. Pepper transformation efficiency

Number of explants Number of shoots (%) PCR positive shoots (%) 1600 72 (4.5%) 6 (0.4%)

Example  3: transgenic pepper PCR  analysis

HC - Pro after transformation In order to check whether the gene was introduced into the plant, a polymerase chain reaction (PCR) was performed. After separating DNA from leaf fragments of the selected news, primer pairs (35S: F 5'-atgacgcacaatcccactat-3 '<SEQ ID NO: 2>; HC-Pro: R 5'-agaaagctgggtaccaactctatagtgcttta-3'<SEQ ID NO: 3>) PCR was performed using. At this time, PCR was denatured at 94 ° C. for 5 minutes as a precycling reaction, and then repeated 35 times in the order of 30 seconds at 94 ° C., 30 seconds at 58 ° C., and 30 seconds at 72 ° C., and then The PCR reaction was terminated by extending the reaction at 72 ° C. for 5 minutes. Figure 3 shows the PCR analysis of the transformed pepper plants. As can be seen in Figure 3, the transformed pepper plant was able to observe the 300bp PCR product.

Example  4: Southern of transformed pepper plants Blot  analysis

Southern blot analysis was performed to determine whether the HC - Pro gene was introduced into the plant after transformation. Specifically, chromosomal DNA was isolated from the leaf sections of the selected newsletter, and then digested with EcoRI and XbaI, the cleavage product was subjected to agarose gel electrophoresis, and the conventional Southern blot using HC - Pro gene as a probe. Southern blot analysis was performed according to the analysis procedure.

4 shows Southern blot analysis of the transformed pepper plants. As can be seen in Figure 4, when EcoRI cleavage in transgenic pepper plant T20, 2.8kb bands in 1, 2 and 3 samples, 3.6 kb bands in 4, 5 and 6 samples appeared, transgenic pepper plant M6 In XbaI cleavage, only 1.4 kb and 6.0 kb bands appeared in 1, 2 and 3 samples, and 1.4 kb bands in 4, 5 and 6 samples. Thus, samples 1, 2 and 3 are the same origin and samples 4, 5 and 6 also, such as the origin, each HC - Pro gene was confirmed that insertion of a single copy.

Example  5: transformation of pepper plants PepMoV  Immunity test

Pepper T ₁ for PepMoV resistance test Each 200 points were grown in seedlings in a greenhouse. When two main leaves emerged after seeding (after about 3 to 4 weeks), PepMoV was inoculated onto the leaf surface (inoculation: buffer = 1: 4 ratio), and resistance was observed after 3 to 4 weeks of inoculation. As can be seen in Figure 5, in the case of the synthetic HC - Pro transgenic peppers, pepper leaves are not damaged at all by PepMoV, while the leaves of the non-transformed peppers can be seen that the disease of the mosaic pattern.

As shown in Figure 6a, b when the transformants selected after inoculation for about 3 months to maintain a pepper transformant resistant to normal development, non-transformant peppers did not develop normally due to virus damage.

7 is T20 and the M6 pepper traits parties rivers selected objects in the switching element of PepMoV after the water has secured the T ₃ generation seeds by planting each GM Pepper T ₃ seedlings of per generation 100 points were inoculated test each . 98 points in each transformant line were found to have resistance to PepMoV, confirming that GM pepper was highly resistant.

1 is a schematic diagram of a pK7GWIWG2 (II) :: HC-Pro vector.

2 is a step-by-step illustration of the transformation of the pepper plant. A: selection medium, B: shoot induction, C: root induction, D: purified, E: soil purified.

Figure 3 is a diagram showing the results of PCR analysis of the transformed pepper plants. N1: nontransformer; 1-30: transformants; P1: pK7GWIWG2 (II) :: HC-Pro vector; P2, P3: Agrobacterium cell culture containing pK7GWIWG2 (II) :: HC-Pro vector as positive control.

4 shows Southern blot analysis of the transformed pepper plants. Transformants: 1-3 (T20, THK strain); 4-6 (M6, C15 strains); Nontransformers: N1, N2.

FIG. 5 shows a phenomenon in which a mosaic-type disease caused by a virus on the leaves is observed in the transgenic pepper (1 month after purification) after about 2 to 3 weeks of inoculation with PepMoV. Tolerant: resistant; Susceptible: Lee Byung Sung. M6 and T20: transformants; Control: nontransformer.

6 shows the results of resistance to PepMoV of transgenic peppers (3 months post-purification) and untransformed peppers. 6A: M6; 6B: T20.

Figure 7 shows the results of examining the degree of resistance after transgenic pepper was selected for each three generations. C15 is the control line of M6 and THK is the control line of T20.

<110> NONG WOO BIO CO., LTD <120> Transgenic pepper with enhanced tolerance to PepMoV and          production method <130> PN09233 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1308 <212> DNA <213> HC-Pro of PepMoV <400> 1 tcaacatctg aagcattttg gagcggtcta gagaagaagt ggagcgtgat gcgcaagcca 60 accgcgcata cttgtaaacc gacgtattcg gtttcgaatt gtggggaagt agccgctatt 120 atagcgcaag ccttatttcc gtgccacaag ttgacgtgtg gtgaatgctc gaaagagatt 180 tgcgatctca cttcgagtga atgcgtgcaa gagttataca agaatatctc tttggcactg 240 gaaaggatga acaatctaca tcccgaattt caacacattg ttaaggtgtt gagcgttgtt 300 aggcagctca ctgaagcatc caatcatggg atggaagtat tcgatgaaat cttcaaaatg 360 attggatcca aaacacagag tcctttcact catttaaata agctcaatga atttatgttg 420 aaagggaacg agaatacaag tgaggaatgg ttgactgctc gacaacgctt aaaggagctg 480 gtgagatttc agaagaatag aactgataat ataaagaaag gtgacttggc atcattcaga 540 aataagcttt ctgctcgtgc acagtacaat ttgtatttat catgcgataa tcagcttgac 600 aagaatgcta gttttctatg gggtcagcga gaataccatg cacgtcggtt tttcctaaac 660 ttctttcaac aaatagaccc atcaaaaggt tatttgtcgt atgaagatcg gaccatacca 720 aatggttctc gaaagttagc tataggcaac ttaattgttc cactcgattt agctgaattc 780 cgaaaacgca tgaaaggcat cgacactcag caaccaccaa ttggcaagta ctgtacaagc 840 caattggatg ggaattttgt gtatccgtgc tgctgcacga cgcttgatga tggccaacca 900 attcgatcag ctgtttacgc accgactaag aaacatttag ttgttggtaa cacaggagac 960 acaaagtaca tcaacttgcc taaaggagat acagagatgc tatatattgc actcgatggc 1020 tattgttaca ttaacattta tctggcaatg ttggtcaata taagcgagga agaggccaag 1080 gacttcacaa agaaagttcg ggatattttc atgccaaagc ttgggaagtg gccaacattg 1140 atggatttgg ctacgacatg tgctcaactt cggatattcc accctgatgt acatgacgca 1200 gagctgcctc gtattctagt ggatcacaac acacaaacat gtcatgtggt cgattcatat 1260 ggatcaatta gtactgggta tcacattctg aaagctgcaa ctgtttca 1308 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 35S F <400> 2 atgacgcaca atcccactat 20 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> HC-Pro R <400> 3 agaaagctgg gtaccaactc tatagtgctt ta 32  

Claims (7)

HC - Pro (helper component-proteinase) from pepper mottle virus (PepMoV) A plant that has been transformed with a recombinant plant RNAi expression vector containing a gene, thereby enhancing resistance to viral diseases caused by PepMoV. According to claim 1, wherein the HC - Pro Gene is a plant, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. The plant of claim 1, wherein the recombinant plant RNAi expression vector is a pK7GWIWG2 (II) :: HC-Pro vector described in FIG. 1. According to claim 1, wherein the plant is a plant, characterized in that the pepper. Seeds of plants according to claim 1. HC - Pro from PepMoV Plants were transformed with the recombinant plant RNAi expression vector containing the gene, HC - Pro A method of enhancing plant resistance to viral diseases caused by PepMoV comprising inducing gene-silencing of genes. 7. The method of claim 6, wherein the plant is red pepper.

Images