KR101724370B1 - TRSV recombinant vector and uses thereof - Google Patents

Abstract

The present invention relates to a tobacco ringspot virus (TRSV) recombinant vector, and uses thereof. The recombinant vector using TRSV manufactured in the present invention efficiently completes silencing of necessary genes from a wide variety of host plants, such as tobacco, Arabidopsis thaliana, Cucurbitaceae, and Fabaceae, which is considered to facilitate studying intrinsic functions of genes, is considered to be very useful for searching economically valuable and useful genes. Moreover, promotion of new species of plants using the useful genes can be expected, and TRSV recombinant vector can be useful for securing an original technology through a patent.

Description

TRSV recombinant vectors and their uses {TRSV recombinant vector and uses thereof}

The present invention relates to TRSV recombinant vectors and uses thereof.

Plant virus-based vectors are useful tools for efficient expression of target foreign proteins in plants. The plant expression system has significant advantages over other methods of recombinant protein production because the plants are much cheaper and easier to grow than the cell culture methods. This system provides fast and transient expression of heterogeneous genes in plants. These virus-based vectors are used to express genes that have medicinal genes, agricultural management values, and are also used to induce genetically dominant or gene silencing traits to determine the function of an unknown gene . The system can also be used to produce vaccine components that can be used as cancer vaccine therapeutics, or to produce proteins that can be applied as a variety of therapeutic agents. Sequence expression in plants by viral expression vectors results in reprogramming of specific metabolic pathways in plants through virus-induced gene-silencing (VIGS) effects or protein expression. Therefore, viral vectors are being developed to realize gene silencing and / or stable foreign protein expression. The development of virus vectors that can be applied to various crops is still insufficient.

Tobacco ringspot virus (TRSV) is taxonomically classified in the genus Nepovirus and classified as a virus IV (+) sense single strand RNA (sense ssRNA) virus. The TRSV particle is a square with a diameter of about 28 nm, and the nucleic acid is single stranded RNA (RNA) of about 7,500 bp and 3,900 bp. TRSV is a phytopathogenic virus that causes deformations and malformations of fruits and pods as a whole in infected plants, and rotting of roots. In the stem, blight of leaf blight and congenital disease occur, and the infected plants become diminished as a whole, which leads to quantitative and qualitative loss of crops. TRSV is at risk of becoming infected throughout the plant's growing season, such as when it is flowering, when the fruit is opened, and when it is just before germination.

TRSV can be a seed infection, which can be transmitted from plants to plants by grafting, mechanical inoculation, and vectors, and various viral infections have been reported. It can be used as a main host for pepper, watermelon, melon, cucumber, yellow squash, gladiolus, beans, tomatoes, tobacco and blueberries and can also infect geraniums, Danyang cherries, eggplants, potatoes and grapes. For example, it causes bud blight disease in soybean, and mottled leaves in leaves.

The present inventors isolated and identified TRSV and registered a full-length nucleotide sequence in the NCBI genebank. A recombinant infectious clone derived from TRSV was prepared and used as a viral-based vector in various host plants for gene function study through gene silencing, To obtain a plant virus vector capable of simultaneously producing the plant virus vector.

Korean Patent No. 0795367 discloses an expression vector constructed by inserting a transcriptional regulatory gene of a red pepper having resistance to pathogens into a tobacco rat virus vector, and Korean Patent No. 1590404 discloses a novel cucumber fruit mosaic mosaic A virus-derived subgenomic promoter and an expression vector, and their use ". However, as in the present invention, the TRSV recombinant vector and its use are not described at all.

The present invention has been made in view of the above needs, and the present inventors have constructed a recombinant vector using TRSV (tobacco ringspot virus) as a plant viral vector capable of simultaneously studying gene function through gene silencing and expressing foreign proteins in plants , The present inventors have completed the present invention by confirming that the expression vector effectively expresses gene silencing phenomenon and foreign protein expression in various plants such as tobacco, Arabidopsis thaliana, Bacillus subtilis, and soya bean.

In order to solve the above problems, the present invention provides a P2A (protein 2A) coding gene of TRSV (tobacco ringspot virus) RNA2; A movement protein (MP) coding gene; A cleavage site between a movement protein (MP) coding gene and a CP (coat protein) coding gene; MCS1 (multiple cloning site1), which is located at the lower part of the cleavage site and can insert a foreign gene to be expressed; A sequence encoding a self-cleaving peptide located under the MCS1; A coat protein (CP) coding gene; A multiple cloning site 2 (MCS2) capable of inserting a target gene to be silenced, which is located at the bottom of the CP, and a cassette comprising the cassette.

Also, the present invention provides a method of overexpressing a foreign gene in a plant, comprising the step of inserting a foreign gene to be expressed in the TRSV RNA2 recombination vector into MCS1 (multiple cloning site 1) to prepare a TRSV RNA2 recombinant vector do.

The present invention further provides a method for silencing a target gene in a plant, comprising the step of inserting a target gene to be silenced in the TRSV RNA2 recombination vector into MCS2 (multiple cloning site 2) to prepare a TRSV RNA2 recombinant vector do.

The present invention also relates to a method for producing a TRSV RNA2 recombinant vector by inserting a foreign gene to be expressed in the TRSV RNA2 recombinant vector into MCS1 (multiple cloning site 1) and inserting a desired gene to be silenced into MCS2 (multiple cloning site 2) And overexpressing the foreign gene in the plant and silencing the target gene.

Also, the present invention provides a method for producing a plant overexpressed with a foreign gene, comprising the step of inserting a foreign gene to be expressed in the TRSV RNA2 recombinant vector into MCS1 (multiple cloning site 1) to prepare a TRSV RNA2 recombinant vector .

The present invention also provides a plant overexpressed with a foreign gene produced by the above method.

In addition, the present invention provides a method for producing a plant in which a target gene is silenced, comprising the step of inserting a target gene to be silenced in the TRSV RNA2 recombination vector into MCS2 (multiple cloning site 2) to prepare a TRSV RNA2 recombinant vector .

Further, the present invention provides a plant in which the target gene produced by the above method is silenced.

The present invention also relates to a method for producing a TRSV RNA2 recombinant vector by inserting a foreign gene to be expressed in the TRSV RNA2 recombinant vector into MCS1 (multiple cloning site 1) and inserting a desired gene to be silenced into MCS2 (multiple cloning site 2) Wherein the foreign gene is overexpressed and the target gene is silenced.

In addition, the present invention provides a plant in which the foreign gene produced by the above method is expressed and the target gene is silenced.

The TRSV recombinant vector according to the present invention can effectively induce gene silencing and / or gene expression in plants. This can be applied to gene silencing and in vivo expression studies of foreign proteins, which can be used effectively in a wide range of host plants, such as tobacco, Arabidopsis thaliana, and beans. By using the recombinant vector of the present invention, it is believed that it is very useful for searching useful genes that are economically valuable as well as enabling intrinsic function studies of genes by efficiently silencing desired genes. In addition, it can be expected to cultivate new varieties of plants using useful genes and it is expected to be useful for securing original technology through patent.

1 shows a schematic diagram of TRSV viral vectors prepared using TRSV RNA1 and TRSV RNA2. A. cDNA infectious clones (pTRSR1 and pTRSR2) containing respective TRSV RNA1 and TRSV RNA2; B. pT2C2AGFP vector for TRSV-based GFP expression vector; C. pT2S vector for TRSV-based expression vector; D. pT2C2AS vector for TRSV-based gene expression or silencing.
Figure 2 shows the results of tobacco analysis of GFP expression using a TRSV-based expression vector. A. Photograph taken in natural light (top) and UV (bottom) of inoculated tobacco leaves (Mock: buffer-inoculated tobacco leaf, TC2A: TC2A-inoculated tobacco leaf, TC2AGFP: TC2AGFP-inoculated tobacco leaves). B. Expression of GFP by western blotting from the total protein extracted from tobacco leaves inoculated with the anti-GFP antibody. Lane 1, mock; Lane 2, TC2A-inoculation; Lanes 3-6, 8th to 10th leaves of TC2AGFP-inoculated tobacco (lane 3-5) or axillary bud (lane 6).
FIG. 3 shows the result of confirming the silencing phenomenon of NbPDS gene using TRSV expression vector in tobacco. A shows a photograph of tobacco that passed 23 days after TSNbPDS inoculation and the result of confirming NbPDS transcript expression using semi-q RT-PCR. B shows the cigarette photograph 30 days after TC2ASNbPDS inoculation and the result of confirming NbPDS transcript expression using semi-q RT-PCR.
FIG. 4 shows the results of confirming GFP expression and silencing of NbPDS gene using a single vector in tobacco. TC2ASGFPNbPDS215 or TC2ASGFPNbPDS215a This is a picture of a cigarette taken 8 days and 30 days after the vector vaccination in natural light or UV.
FIG. 5 shows the result of confirming the AtPDS gene silencing phenomenon after inoculation of the TRSV expression vector in Arabidopsis thaliana. A is TSAtPDS250 (left) and TSAtPDS250a (right) after 14 days of inoculation. B. Photographs of TSAtPDS250 (right) and TSAtPDS250 (right), TSAtPDS250 (right) and TSAtPDS250 (right) and sepal photographs. C. TS (left), TSAtPDS250 (middle), and TSAtPDS250a (right) Photograph of a baby rod after 37 days of inoculation. D. Photographs of TS (left) and TSAtPDS250a (right) after inoculation of Arabidopsis siliques. E. Expression of AtPDS transcripts by semi-q RT-PCR.
Figure 6 shows the results of confirming the silencing of the CuPDS gene after TSCuPDS vector inoculation in various foliage and plants. A is a photograph of TSCuPDS vector inoculation of melon (top) (21 dpi), melon (middle) (23 dpi) and cucumber (bottom) (34 dpi). B. Expression of CuPDS transcripts by semi-q RT-PCR.
7 shows the result of confirming the virus-induced gene silencing phenomenon of the PDS gene in each of the tobacco (A), melon (B phase) and melon (B bottom) flowers and cucumber fruit (C).
Fig. 8 shows the result of confirming the silencing of the virus-inducible gene of the GmPDS gene of the legume plant. A is a photograph of the inoculation of the soybean 'williams 82' varieties (upper) and wild type (lower) TS (left), TGmPDS209 (middle) and TGmPDS209a (right) vectors. B. Expression of GmPDS transcript by semi-q RT-PCR.

In order to accomplish the object of the present invention, the present invention provides a P2A (protein 2A) coding gene of TRSV (tobacco ringspot virus) RNA2; A movement protein (MP) coding gene; A cleavage site between a movement protein (MP) coding gene and a CP (coat protein) coding gene; MCS1 (multiple cloning site1), which is located at the lower part of the cleavage site and can insert a foreign gene to be expressed; A sequence encoding a self-cleaving peptide located under the MCS1; A coat protein (CP) coding gene; A multiple cloning site 2 (MCS2) capable of inserting a target gene to be silenced located at the bottom of the CP.

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. Recombinant cells can express a gene or a gene fragment that is not found in the natural form of the cell, either in a sense or in an 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 cassette used in the present invention is a P2A (protein 2A) coding gene of tobacco ringspot virus (TRSV) RNA2; A movement protein (MP) coding gene; A cleavage site between a movement protein (MP) coding gene and a CP (coat protein) coding gene; MCS1 (multiple cloning site1), which is located at the lower part of the cleavage site and can insert a foreign gene to be expressed; A sequence encoding a self-cleaving peptide located under the MCS1; A coat protein (CP) coding gene; And multiple cloning site2 (MCS2) which is located at the bottom of the CP and into which a target gene to be silenced can be inserted.

The cassette may preferably consist of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. 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 one embodiment of the invention, the cassette may be the cassette described in Figure 1 D, but is not limited thereto.

The TRSV RNA2 recombinant vector containing the cassette may also be referred to as pT2C2AS.

Preferred self-cleaving peptides (so-called "cis-acting hydrolyzable elements ", CHYSEL, see deFelipe (2002) Curr. Gene Ther. 2, 355-378) are derived from potivirus and cardiovirus 2A peptides. More preferred self-cleaving peptides are selected from 2A peptides derived from FMDV (foot and mouth disease virus), horse rhinitis A virus, Thosea asigna virus and porcine teschovirus. Even more preferred self-cleaving peptides can be, but are not limited to, 2A peptides derived from FMDA.

The term " MCS (multiple cloning site) " of the present invention means a DNA fragment having a restriction enzyme recognition site, which is a site recognized and cleaved by various restriction enzymes. The MCS is recognized by a restriction enzyme, So that insertion of the target gene into the site of the cleaved MCS is enabled. The MCS can be used without limitation as long as it is known to those skilled in the art, and when it is desired to insert the target gene into the MCS of the cassette of the present invention, Enzyme sites can be used.

The MCS1 of the present invention is a site capable of inserting a foreign gene to be expressed, and is located under cleavage site between a movement protein (MP) coding gene and a CP (coat protein) coding gene. Preferably, the MCS1 can include but is not limited to BamHI, MluI and SpeI restriction enzyme recognition sites.

There are no restrictions on the foreign gene that can be introduced into the vector of the present invention. The foreign gene may preferably be a gene encoding a useful protein. The useful protein is all proteins such as pharmaceuticals and industrial products which are currently being produced commercially using plant biotechnology. It is useful as a high value-added protein such as enzymes, hormones, antibodies, cytokines, etc., But are not limited to, proteins capable of accumulating large amounts of nutrients capable of promoting the health of animals, including humans. Also included may be herbicide resistance genes, disease resistance genes, insecticide resistance genes, genes that affect the quality of grain or fruit, screenable marker genes, environmental or stress resistance genes, and the like. More particularly, the present invention relates to a gene encoding a resistance gene of 5-EPSP synthase, a herbicide glyphosate resistance gene, and a herbicide bialaphos (Basta) Phosphinothricin acetyltransferase, a gene encoding a coat protein that is a virus-resistant protein, a Bt toxin-producing gene isolated from an insect resistance gene, Bacillus thuringiensis, (1-aminocyclopropane-1-carboxylic acid deaminase) gene for prevention of infection, INF1 (Phytophthora infestans elicitin gene) which is a necrosis-inducing gene of plant cells capable of easily selecting a virus-resistant plant, But is not limited thereto.

In an embodiment of the present invention, a GFP (green fluorescent protein) gene, which is a reporter gene, may be used in place of a foreign gene. GFP is very useful for identifying transformed plants because it can observe green fluorescence by irradiation with a long wavelength of ultraviolet light.

In the recombinant vector of the present invention, the MCS2 is a region capable of inserting a target gene to be silenced, preferably, but not limited to, under the CP (coat protein) coding gene.

According to an embodiment of the present invention, the MCS2 includes a SnaBI restriction enzyme recognition site, and a target gene to be silenced between the SnaBI restriction enzyme recognition sites can be inserted.

There is no restriction on the gene of interest to be silenced which can be introduced into the vector of the present invention. The target gene to be silenced is preferably a nucleic acid sequence complementary to a sense strand or antisense strand for inducing silencing of the target gene. The target gene to be silenced can be inserted in the sense or antisense direction.

The term "virus-induced gene silencing " (VIGS) refers to a phenomenon in which expression of an endogenous plant gene of an introduced gene is inhibited by introducing a plant gene into a viral vector and infecting the plant. It is a type of post-transcriptional gene silencing (PTGS), characterized by post-transcriptional, RNA turnover and nucleotide sequence-specific. Therefore, using VIGS, the function of transgene can be rapidly and easily mass-analyzed.

In an embodiment of the present invention, a recombinant vector in which a fragment of a PDS (phytoene desaturase) gene having a length of 200 to 350 bp is inserted in antisense or sense direction may be used in place of the target gene to be silenced. Since the PDS gene encodes an enzyme involved in carotenoid biosynthesis, it is very useful in confirming the success of VIGS because it can observe the whitening of the plant leaf when the gene is inhibited.

The recombinant vector of the present invention may contain a promoter. The promoter may be a promoter suitable for in vitro transcription or transformation and may preferably be a T7 promoter, SP6 promoter, CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter, more preferably T7 A promoter, an SP6 promoter, or a CaMV 35S 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.

The recombination vector of the present invention may include a terminator under the MCS2. The terminator can be a conventional terminator, such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens octopine And the terminator of the Octopine gene, but the present invention is not limited thereto.

The first combination vector may preferably comprise one or more selectable markers, but is not limited thereto. 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 phosphinotricin, antibiotic resistance genes such as kanamycin, G418, Bleomycin, hygromycin or chloramphenicol, It is not.

Also,

(1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;

(2) preparing a TRSV RNA2 recombinant vector by inserting a foreign gene to be expressed in the TRSV RNA2 recombination vector of the present invention into MCS1 (multiple cloning site 1);

(3) transforming Agrobacterium with the respective recombinant vectors prepared in the above steps (1) and (2) to obtain respective transformants; And

(4) infiltrating or sapinoculating a mixture of each of the above Agrobacterium transformants to a plant, and (4) overexpressing a foreign gene in a plant.

The polyprotein coding gene of the TRSV (tobacco ringspot virus) RNA1 may preferably be a nucleotide sequence of SEQ ID NO: 2, but is not limited thereto. In addition, homologues of the nucleotide sequences may be included within the scope of the present invention, and details of the homologues are as described above.

In one embodiment of the present invention, the TRSV RNA1 recombinant vector is also referred to as pTRSR1.

The foreign gene of the present invention may be a gene encoding a useful protein, and the gene encoding the useful protein is as described above.

A method of overexpressing a foreign gene in a plant according to an embodiment of the present invention may include an infiltration method or an inoculation method using Agrobacterium tumefaciens transformed with a recombinant expression vector into which a foreign gene is inserted (SAP inoculation) method can be used, but it is not limited thereto.

The method of delivering the vector of the present invention into a host cell can be carried out by various methods such as microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, Agrobacterium- The vector may be injected into host cells, preferably Agrobacterium-mediated transformation or gene bombardment, but is not limited thereto.

As used herein, the term " infiltration "refers to a method for introducing a gene into a plant and expressing it, or for producing a desired protein in a plant. A suspension of Agrobacterium having the gene to be transferred is injected into the leaves of a plant by using a needle-free syringe to transfer the desired gene to the plant cell. This has the advantage of being quick and convenient compared to traditional plant transformation.

The term " sap inoculation "used in the present invention is also referred to as rub inoculation, smearing, or mechanical inoculation as one of the methods of introducing genes into plants. This method generally involves scarring the plant with a friction agent such as carborundum and then introducing the gene into the plant by contacting the inoculum (vector) having the gene to be introduced with the damaged plant. In addition, a needle is made into a bundle, and a sick leaf is inoculated. A needle is inoculated to the leaf surface. The sickle leaf is overlapped and cut with a sharp blade. And a method of spraying. Self-immobilization or expeditious methods can be used to inactivate viral inoculations that are difficult to inoculate.

In one embodiment of the present invention, the plant to be infected by TRSV is not particularly limited, but preferably it can be tobacco, Arabidopsis, bark and plant and leguminous plants.

In addition,

(1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;

(2) preparing a TRSV RNA2 recombinant vector by inserting a target gene to be silenced in the TRSV RNA2 recombination vector of the present invention into MCS2 (multiple cloning site 2);

(3) transforming Agrobacterium with the respective recombinant vectors prepared in the above steps (1) and (2) to obtain respective transformants; And

(4) infiltration or sap inoculation of the mixed solution of each of the above Agrobacterium transformants into a plant; and (4) silencing the target gene in the plant.

The method of silencing a target gene in a plant of the present invention includes a step of inserting a target gene to be silenced in the TRSV RNA2 recombination vector into MCS2 (multiple cloning site 2) to prepare a TRSV RNA2 recombinant vector. Here, the target gene to be silenced is inserted into MCS2, and preferably inserted between SnaBI restriction enzyme recognition sites located under the CP coding gene. Also, the inserted direction may be inserted in the sense or antisense direction, and most preferably inserted in the antisense direction, but is not limited thereto.

The term "silence" refers generally to a phenomenon in which the expression of a gene is downregulated or completely suppressed.

In the method of silencing an endogenous target gene in the plant of the present invention, each step except step (2) is as described above in a method of overexpressing a foreign gene in a plant.

Also,

(1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;

(2) A foreign gene to be expressed in the TRSV RNA2 recombinant vector of the present invention is inserted into MCS1 (multiple cloning site 1), and the desired gene to be silenced is inserted into MCS2 (multiple cloning site 2) to prepare a TRSV RNA2 recombinant vector ;

(3) transforming Agrobacterium with the respective recombinant vectors prepared in the above steps (1) and (2) to obtain respective transformants; And

(4) infiltrating or sapinoculating a mixture of each of the above Agrobacterium transformants to a plant, and overexpressing the foreign gene in the plant and silencing the target gene ≪ / RTI >

In a method of overexpressing a foreign gene in a plant and silencing a target gene, a foreign gene to be expressed in the TRSV RNA2 recombinant vector is inserted into MCS1 (multiple cloning site 1), and a desired gene to be silenced MCS2 (multiple cloning site 2) to prepare a TRSV RNA2 recombinant vector.

In one embodiment of the present invention, the foreign gene to be inserted may be a gene encoding a useful protein, and the target gene to be silenced may be, but not limited to, a nucleic acid sequence capable of silencing a plant endogenous target gene .

In addition,

(1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;

(2) preparing a TRSV RNA2 recombinant vector by inserting a foreign gene to be expressed in the TRSV RNA2 recombination vector of the present invention into MCS1 (multiple cloning site 1);

(3) transforming Agrobacterium with each of the recombinant vectors prepared in the above steps (1) and (2) to obtain transformants; And

(4) infiltration or sap inoculation of a mixed solution of each Agrobacterium transformant into a plant; and (4) providing a plant having an overexpressed foreign gene comprising the step of infiltrating or sapinoculating a mixture of Agrobacterium transformants do.

In the method for producing a plant in which the foreign gene of the present invention is over-expressed, each step is as described in a method of overexpressing a foreign gene in a plant.

The present invention also provides a plant overexpressed with a foreign gene produced by the above method.

Also,

(1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;

(2) preparing a TRSV RNA2 recombinant vector by inserting a target gene to be silenced in the TRSV RNA2 recombination vector of the present invention into MCS2 (multiple cloning site 2);

(3) transforming Agrobacterium with each of the recombinant vectors prepared in the above steps (1) and (2) to obtain transformants; And

(4) infiltrating or sapinoculating a mixture of each of the above Agrobacterium transformants to a plant, and (4) do.

In the method for producing a plant in which the objective gene of the present invention is silenced, each step is as described in a method of silencing a target gene in a plant.

The present invention also provides a plant in which the objective gene produced by the above method is silenced.

Also,

(1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;

(2) A foreign gene to be expressed in the TRSV RNA2 recombinant vector of the present invention is inserted into MCS1 (multiple cloning site 1), and the desired gene to be silenced is inserted into MCS2 (multiple cloning site 2) to prepare a TRSV RNA2 recombinant vector ;

(3) transforming Agrobacterium with the respective recombinant vectors prepared in the above steps (1) and (2) to obtain respective transformants; And

(4) infiltration or sap inoculation of the mixed solution of each of the above Agrobacterium transformants into plants, and (4) overexpressing the foreign gene comprising the step of infiltrating or sapinoculating the plant, ≪ / RTI >

In the method for producing a plant in which the foreign gene of the present invention is overexpressed and the target gene is silenced, each step is as described in a method of overexpressing a foreign gene in a plant and silencing a target gene.

The present invention also provides a plant in which the foreign gene produced by the above method is over-expressed and the objective gene is silenced.

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

Materials and methods

1. Preparation of TRSV-based recombinant vectors for GFP expression

The present inventors have produced pTRSR1 and pTRSR2 vectors, two types of TRSV infectious cDNA clones (Zhao et al., 2015, Virus Genes 51; 163-166) (FIG. 1A). In the present invention, constructs for foreign gene expression or VIGS were constructed by transforming the pTRSV2 vector using PCR.

In order to obtain efficient processing of cysteine / alanine (Cys / Ala, C / A) cleavage site, 19 amino acids at the N terminus of CP are selected and duplicated for several degenerate codons. The nucleotides of the dogs were replaced. Sequences encoding the foot-and-mouth disease virus (FMDV) 2A peptides were inserted from the primers in Table 1. The sequence encoding the additional 19 amino acids was linked to MCS (multicloning site, BamH I- Mlu I- Spe I) and inserted into pTRSR2Part2. PCR I fragments were amplified using T2-1167F and 2 AMCS- R primer sets using pTRSR2 as template and PCR II fragments were amplified using 2 AMCS-F and RZNOS-75R primers set as template pTRSR2. The two PCR fragments (PCR I and PCR II fragments) were mixed in the same amount and subjected to overlapping PCR using T2-1167F and RZNOS-15R primer sets, and then Xba I and Sal I restriction enzymes were cut to obtain Xba I / Sal I PT2C2APart2 was prepared. Fragment I was inserted into the Sac I / Xba I site of pT2C2APart2 to prepare pT2C2A (Fig. 1B). Sequence encoding the GFP is pGFP (Clontech, USA) was obtained by PCR using a GFPBamH-F and GFPSpe-R primer set the template, the PCR fragment was cut with Bam HI and Spe I treated with Bam HI / Spe I PT2C2A vector to construct pT2C2AGFP (Fig. 1B).

2. Preparation of TRSV-based recombinant vectors for VIGS

Two PCR fragments were amplified using the T2-1167F / TRSCP-R or TRSCP3UTR-F / RZNOS-75R primer sets to produce the Sna BI site under the termination codon of the CP coding region, The final PCR products obtained by performing overlapping PCR using -1167F and RZNOS-15R primer sets were cut into Xba I / Sal I and subcloned into the pPZP211NOS vector to prepare pT2SPart2 (Fig. 1C).

Two kinds of cDNA fragments (215 bp and 345 bp) of the tobacco-derived PDS gene ( N. benthamiana PDS, NbPDS ) (GenBank accession no .: EU165355) were amplified using the NbPDSSnaB-743F / 957R and NbPDSSnaB-743F / 1087R primer sets PCR (Table 1). The 250 bp fragment of Arabidopsis Col-0 derived PDS gene ( AtPDS ) (GenBank accession no .: NM_117498) was amplified by PCR using the AtPDSSNAB-1151F / 1400R primer set (Table 1). The 300 bp fragment of cucumber-derived PDS gene ( CuPDS ) (GenBank accession no .: XM_011654729) was amplified by PCR using the CuPDSSnaB-952F / 1251R primer set (Table 1). The 209 bp fragment of soybean-derived PDS gene ( Glycine max PDS, GmPDS ) (GenBank accession no. M64704) was amplified by PCR using the GmPDSSnaB-1353F / 1561R primer set (Table 1). By cutting the PCR products of each of the PDS gene with Sna BI and inserted into the Sna BI- treatment and sense or anti-cent direction to the non-phosphorylated pT2S vector it was produced pT2SNbPDS, pT2SAtPDS, pT2SCuPDS and pT2SGmPDS. These vectors were used to silence the corresponding PDS genes in tobacco, Arabidopsis, cucurbits, and legumes. The pT2SNbPDS vector contained pT2SNbPDS215, pT2SNbPDS215a, pT2SNbPDS345 and pT2SNbPDS345a. The pT2SAtPDS vector contained pT2SAtPDS250 and pT2SAtPDS250a. The pT2SCuPDS vector contained pT2SCuPDS300 and pT2SCuPDS300a. The pT2SGmPDS vector contained pT2SGmPDS209 and pT2SGmPDS209a. Another pT2C2AS vector used for VIGS has been prepared by the Sna BI site is inserted into the Sna BI- pT2S vector processing and non-phosphorylated. The pT2C2ASNbPDS215, pT2C2ASNbPDS215a, pT2C2ASNbPDS345 and pT2C2ASNbPDS345a containing pT2C2ASNbPDS vectors were constructed to silence the NbPDS gene of tobacco, confirming gene silencing ability. pT2C2ASGFPNbPDS215 and pT2C2ASGFPNbPDS215a vectors were constructed by inserting GFP into Bam HI / Spe I in pT2C2ASNbPDS215 and pT2C2ASNbPDS215a, respectively.

3. Plant growth conditions, agroinfiltration and inoculation

All plants used in the present invention were grown in a greenhouse at 23 < 0 > C, 16/8 hour photoperiod condition.

Agrobacterium tumefaciens strain GV3101 was used to deliver vectors in plants used in the experiment. For agroinfiltration, the transformed Agrobacterium tumefaciens was transformed into LB (Luria) containing 28 μg / ml of rifampicin and 100 μg / ml of spectomycin -Bertani) medium. After overnight incubation, the cells were harvested by centrifugation and diluted with infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl ₂ , 200 μM acetosyringone) and incubated at room temperature for 3-4 hours prior to agroin filtration. Agroin filtration was performed using a 1 ml syringe on the 4th and 5th leaves of tobacco seedlings at 6 weeks old (approximately 7 leaves). Agroin filtration was carried out at cotyledon of seedlings of 6-15 days old seedlings. TS-, TSAtPDS-, and TSGmPDS-agrofilated tobacco leaves (6 dpi) were harvested and sap inoculation was performed on soybean leaves.

4. Western blot analysis of GFP expression

For Western blot analysis, protein extracts were prepared on tobacco leaves 10 days after inoculation (10 dpi). Protein extraction samples (20 μg protein / sample) were separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane. The membranes were probed by dilution 1: 800 with mouse anti-GFP antiserum (Santa Cruz Biotechnology, USA). The membranes were diluted 1: 5,000 with goat anti-mouse IgG-HRP secondary antibody. Final results were analyzed using myECL Imager (Thermo Scientific, USA).

5. RT-PCR and semi-quantitative RT-PCR analysis

For RT-PCR and semi-q RT-PCR, total RNA was extracted from inoculated plant samples using TRI samples. RT-PCR was performed using a set of TRS-N30 / C30 primers to demonstrate the infectivity of TRSV cDNA clones in cucumber (Table 1). Semi-q RT-PCR method PDS in each NbPDS-295F / 556R, AtPDS- 544F / 981R, CuPDS-679F / 906R and GmPDS-1627F / 1857R by using the primer set, tobacco, Arabidopsis thaliana, cucumber acids, soybean Gene expression levels (Table 1). The amplified tobacco actin genes (GenBank accession no. EU165355), Arabidopsis actin 2 (NM112764), cucumber actin gene (GenBank accession no. XM_004136807) and soybean tubulin gene (GenBank accession no .: M64704 ) Were used as internal controls.

Example  1. Identification of GFP expression of TRSV-based recombinant vectors

GFP was inserted between the MP and CP genes as RNA2 components and used as a pT2C2AGFP vector for use in TRSV-based foreign gene expression systems (FIG. 1B). GFP was expressed from the polyprotein through the processing of the MP-CP cleavage site and the FMDV 2A catalytic peptide.

Agroinfiltration results on 4th and 5th leaves of tobacco after 4 or 5 days of infiltration (4 to 5 dpi) of tobacco infiltration leaves (pTRSR1 + pT2C2A) or TC2AGFP (pTRSR1 + pT2C2AGFP) (4th, 5th leaf) and 9th leaf (9th), and it was confirmed that the virus construct was infected. On the other hand, the tobacco infiltrated with the buffer (Mock) was not observed to be induced by TRSV. GFP fluorescence was first detected at 9 days (9 dpi) at 7 days of invasion (7 dpi). Strong GFP fluorescence was observed in leaves and axillary buds in which symptoms were observed. After 15 days of infiltration (15 dpi), GFP fluorescence was scarcely visible in newly growing leaves, with little or no symptoms. FIG. 2A is a photograph of the GFP expression of 10-day infiltration (10 dpi) tobacco photographed in UV. In this experiment, GFP fluorescence was observed in about 80% of TC2AGFP-infected tobacco.

Protein was extracted from 10 lobes of TC2AGFP-infected tobacco, 8-10 leaves, compound eyes or buffer-infiltrated, TC2A-infiltrating tobacco, and GFP protein expression was confirmed by Western blotting. As a result, protein bands of ~ 30, 70 and 87 kDa corresponding to GFP-2A, MP-GFP-2A and GFP-2A-CP were confirmed in 9 leaves, 10 leaves and overlapped eyes, respectively (Figure 2B). There was a larger (~ 127 kDa) protein band in 10 lobes and folds, indicating that the protein cleavage was insufficient (MP-GFP-2A-CP fusion protein) (Fig. 2B). In the compound eyes, a 27 kDa protein band corresponding to the GFP protein size was identified. No protein bands were found in the buffer-infiltrated, TC2A-infiltrating tobacco leaves. Western blot results were consistent with the results of GFP fluorescence expression in invading plants.

Example  2. Into the TRSV-based recombinant vector PDS Confirm gene silencing

To carry out the VIGS in the tobacco, pT2S and pT2C2AS with Sna BI inserted under the termination codon of CP in each pTRSR2 and pT2C2A vector were prepared (Fig. 1C, D). Two cDNA fragments (215 and 345 bp) from the tobacco NbPDS gene were inserted in the sense and antisense orientation into pT2SNbPDS (pT2SNbPDS) inserted in the Sna BI site of pT2S and pT2C2AS, respectively, in order to confirm whether the size and orientation of the insertion site affects VIGS efficiency And pT2C2ASNbPDS were prepared and infiltrated into tobacco. Buffer-infiltration, TS- (pTRSR1 + pT2S) and TC2AS- (pTRSR1 + pT2C2AS) -invasive tobacco were used as controls.

In the infiltrated leaves, 5 days (5 dpi) of infiltration started, the symptoms of TRSV deciduous (microcircular spot) appeared but not in buffer - infiltrated lobe. A photo-bleaching phenotype, a silent effect of NbPDS, began to appear in all VIGS constructs, but showed a difference in uniformity (Fig. 3A, B). Partial whitening was observed at 10 dpi and 13 dpi, respectively, for 10 leaves and mixed eyes of TSNbPDS (pTRSR1 + pT2SNbPDS) - inoculated tobacco and TC2ASNbPDS (pTRSR1 + pT2C2ASNbPDS) - inoculated tobacco. TSNbPDS (pTRSR1 + pT2SNbPDS) -inoculated cigarettes were completely whitened due to strong silence in the whitened leaves, stems, and flowers within 13 days (Fig. 3A, 7A, Panel 1). In TC2ASNbPDS-inoculated tobacco, an incomplete whitening phenomenon was observed throughout the plant until the development of new green leaves on the 70th day of inoculation (Fig. 3B, 7A, panel 2). Semi-quantitative RT-PCR analysis showed a marked decrease in NbPDS mRNA expression levels in white-leaf of TSNbPDS-inoculated plants (23 dpi) or TC2ASNbPDS-inoculated plants (30 dpi) as compared to the control (Fig. VIGS efficiency in TSNbPDS was higher than TC2ASNbPDS in whitening results of TSNbPDS and TC2ASNbPDS, but TC2ASNbPDS persisted more than 1 month in terms of persistence of VIGS.

As a result of examining the effect of size and direction of the inserted fragment in gene silencing efficiency, a similar whitening phenomenon was observed in both the NbPDS 215bp fragment and the 345bp fragment irrespective of the size and direction (FIG. 3A, B). Semi-quantitative RT-PCR analysis also confirmed that the amount of NbPDS transcript reduced by both fragments was similar (FIG. 3A, B). The above results confirm that the VIGS efficiency in the tobacco is not affected by the insert length and direction of ~ 200-350 bp.

To determine the exact location of gene silencing, a marker gene-assisted silencing method previously described by Zhang et al. (2010, Plant physiol. 153: 52-65) was performed. For this purpose, pT2C2ASGFPNbPDS215 and pT2C2ASGFPNbPDS215a prepared by inserting GFP into each of pT2C2ASNbPDS215 and pT2C2ASNbPDS215a were used. GFP fluorescence was observed from day 7 to 14 after inoculation of the tobacco inoculated with TC2ASGFPNbPDS (pTRSR1 + pT2C2ASNbPDS) and weak bleaching occurred after 17 days of inoculation (FIG. 4). Based on the above results, it is expected that marker-gene assisted silencing will not be feasible in this system, since the expression of the marker gene and the silencing of the endogenous gene do not occur simultaneously by TRSV-based vectors.

Example 3. In vitro transcription of TRSV-based recombinant vectors PDS Confirm gene silencing

TRSV showed variation depending on the Arabidopsis ecology. For example, Arabidopsis Col-0 did not show visible signs of disease, while Estland showed lethal systemic necrosis (LSN).

In this experiment, a 250 bp fragment of the PDS gene derived from Arabidopsis Col-0 was inserted into the Sna BI site of pT2S in the sense and antisense direction to prepare pT2SAtPDS. TSAtPDS (pTRSR1 + pT2AtPDS) - Inoculated with Arabidopsis Col-0 with the juice from tobacco leaves 5 or 6 days after inoculation, and the inoculated Arabidopsis showed little symptoms.

On the 7th or 8th day of inoculation, whitening was observed in stems and newly developed leaves. After 2 weeks of juice inoculation TSAtPDS (pTRSR1 + pT2AtPDS) - inoculation was observed in most stems and calyxes of Arabidopsis thaliana (Fig. 5A, B). The PDS silent phenotype lasted up to 6 weeks, confirming the formation of white silica (Fig. 5C, D). Thereafter, as the gene silencing recovery progressed, a green silica was formed at the top of the stem. It was confirmed that gene silencing phenomenon is similar in both sense and antisense DNA fragments. In addition, the reduction of AtPDS gene expression in TSAtPDS (pTRSR1 + pT2AtPDS) -involved Arabidopsis thaliana was confirmed by semi-q RT-PCR (Fig. 5E).

Example  4. Agroinfiltration confirmation of TRSV cDNA infectious clone in cucurbits

Oysters TRSV The biological activity of the cDNA clone has not been previously reported and has been confirmed in this experiment. Five kinds of cucumber (melon, melon, cucumber, watermelon, pumpkin) cDNA clone (pTRSR1 + pTRSR2) was inoculated. In the case of TRSV-inoculated melon and melon, a micro-circular spot appeared on 2 ^nd true leaf from 7th day after inoculation, and a weak symptom appeared on 4th leaf after 14th day. The TRSV-inoculated cucumber showed little symptom except for fine mottling after 10 days of inoculation in the two leaflets.

Systemic infections of TRSV in melon, melon and cucumber were identified by RT-PCR. On the other hand, as a result of the RT-PCR method, the method of agroinfiltration or leaf-juice in watermelon and amber did not succeed. It was confirmed that melon, melon and cucumber could be inoculated through the agroinfiltration method.

Example 5. Construction of TRSV-based recombinant vectors in cucurbits PDS Confirm gene silencing

To identify VIGS in the oocyte , the pT2S vector was loaded with CuPDS The pT2SCuPDS vector was prepared by inserting the gene in sense or antisense orientation. In order to confirm the gene silencing phenomenon, TSCuPDS (pTRSR1 + pT2SCuPDS) was inoculated with melon, melon and cucumber as confirmed in the results of Example 4. In the case of melon and melon, partial whitening occurred on 3 ^rd true leaf on 10th day after inoculation on cotyledon. In case of cucumber, 4th or 5th leaf showed similar phenomenon on 20th day. The silent plants then formed completely whitened new leaves, petioles, petals, calyxes, and tendrils (Figs. 6A and 7B). Melon and melon persists the phenotype of CuPDS gene silencing for a month, but recovered from gene silencing before fruit development. In the case of cucumber, TRSV-mediated VIGS persisted in the plant. In the TS- and TSCuPDS-inoculated cucumber plants, the fruit of the sub-node did not grow due to TRSV infection. TSCuPDS-inoculated cucumber died faster than TS-inoculated plants, and one or no fruit developed in the upper node. In the case of the developed fruit, whitening occurred partially and the shape was twisted (FIG. 7C). Semi-q RT-PCR analysis showed that CuPDS mRNA was significantly reduced in silenced leaves of TSCuPDS-inoculated plants (Fig. 6B).

Example  6. In the legume species of TRSV-based recombinant vectors PDS Confirm gene silencing

In previous studies, the method of agroinophilization of Arabidopsis and soybeans directly with TRSV cDNA infectious clones was unsuccessful and a method of mechanically inoculating the juice of infectious clone-inoculated tobacco was performed.

To identify VIGS in the legumes, a pT2SGmPDS vector was prepared by inserting a 209-bp fragment of the GmPDS gene in the sense or antisense orientation into the pT2S vector Sna BI site. The prepared vector was inoculated on tobacco and the juice was prepared on the sixth day and mechanically inoculated into the soybean variety 'Williams 82' (Williams 82 ') and wild type unifoliolate leaves. TSGmPDS (pTRSR1 pT2SGmPDS +) - inoculated soybean plants in the bleaching phenotypes appeared in 14 days after inoculation in inoculated gradually leaves the upper lobe of the second effusion (2 ^nd trifoliolate leaf) or 3 effusion lobe (3 ^th trifoliolate leaf). Afterwards, the white pigment phenotype became complete and expanded to the monocyte and compound eyes (Fig. 8). The TSGmPDS-inoculated wild type could not observe the pod. Semi-q RT-PCR analysis showed that the GmPDS mRNA was significantly reduced in the wild type on the 24th day of TSGmPDS-inoculation and in the silent leaves of the 'Williamsa 82' plant on the 45th day (FIG. 8B).

<110> Korea Research Institute of Bioscience and Biotechnology <120> TRSV recombinant vector and uses thereof <130> PN15397 <160> 32 <170> Kopatentin 2.0 <210> 1 <211> 4105 <212> DNA <213> Artificial Sequence <220> <223> TRSV RNA2 cassette <400> 1 ttgaaaattc tctcacaggg ttccggttac gttgttcttt tactctcttt ttattttaat 60 tgttcaaaat tgcgattctg aagcttccta ctttctggtg cccgcatttt ctctctgctc 120 cgatggagcc cctcttatgg cacgttgatg ccaccactcc ttcccacatc caggctctcc 180 agtcagggtc tctccctcct gcttctccgg ccgcggctct cacccgcgtc caacgcgccc 240 tttccttctt ccggactgct gcccggaagt actgcaagca ggccgatgcg cctgacttgt 300 ttgccctggc aatgaccagg gttgctgagc acaacgacat tgtcgttgat gcaagaaatg 360 tggaacagct gttccacttt gttggccaac atgtggccaa cactgctgag cgcaaggcgc 420 tccgggctgc tcttcgtgag cagcgggctt tatttaaggc atccctgccc ggtgcctgtt 480 ttcctgctcc agctcctcca ggctggggta ttccaaaacc cccccctctt cctccacctt 540 ttgtgtggaa aggatgccgc tacaatgtgg tggcccctcc tccacgcatc ccgcaaccac 600 ctccactgcc taaatttgcg ccttttgtgc gcaacaactt tagagtggtt gctccccctc 660 cccttggtga ggtgtaccaa cccgttggtg cacctttccc acagacccgg gcctccgcgg 720 ccctctcctt ctttcgcact gcctccacct gcaggcaggt cttggttgag agctgcattc 780 agcagcccgc cttcatgact tgctgtgctt ctaccgggga agtgcaagaa atgacgagca 840 tgcttactga agctcgtcag actggaaaaa tcctgactcc caaagaggtg agtcaggcct 900 tggcccaaaa acgcaaagaa atcaaagggg ctgaggaaaa tcgcatctcc tttgatgaag 960 gggtgcattt gacagaggcg gatgtcttcc atcgtctcag tcttgcgaag cgcttcatgg 1020 cgcataagcg agaccggact ttggtggacg ttttaatgcc tactgaacat gaagttgttc 1080 ggtatccagg cacccgccct gacgggacat tgcagatgtg cgtgtctgcc cttccacgca 1140 tgtctgagga agcagctagg aagctgcttg agaaggggtg gaagaactcc aaaaacgtct 1200 ctctagacat tggggttact tcctatatgc catatggtgc gcccatagtt gcattcatga 1260 ctattatgga tgggcgtacc gatgatccac aagaggcagc actttgtgcg aattacatgg 1320 accttggtcg agaaaagtcc aaggtgttgt ctcttccact tgttaccatt cccctctctg 1380 agattgaaca tgatcaaggc attttggatt gcctttatat tgttacatat tttcatggtg 1440 ttcaatctta tcaacccgga actttaatga tgagttatgg aactcttgag tttcaagaat 1500 attccaataa ctcttttaca actgctactc gggttagaga gagttgggac cagattctca 1560 aacgcaatga gaatcttggg aaaagagtcc atgccggcat cggggtgctt ggcactattg 1620 aaaaagaaat ggaccagcaa cttgaggact tccccgccat aaatctggaa actaggccac 1680 gccctgttgt gcgaactttt cagaatgcac atcagccgtt gcataagacc agatctatgc 1740 gaattggcac tacctctttt actgggaaca ctggtaggac tgtactccca ccagtggtta 1800 aaacctatga agagggaaat gctaattttg attccctaca atccaaacca cgtcacagct 1860 ctgccagcac tgctcatttg atgtgtgctg tcacagtagt acctgaccct acatgctgcg 1920 gtactcttag tttcaaagtg cctggatcca cgcgtactag tgcgcctgca aaacagctct 1980 taaactttga cctacttaag ttagcaggtg acgttgagtc caaccctggg cccgctgtga 2040 cggttgttcc cgatcccact tgttgtggga cattgtcctt taaggttccc aaagatgcga 2100 agaaaggaaa gcatcttgga acttttgaca ttcggcaagc cattatggaa tatggtggtt 2160 tgcattccca agaatggtgt gcaaagggca ttgttaatcc cacttttaca gtgaggatgc 2220 atgccccacg caacgccttt gcaggtttgt ctatagcgtg cacctttgat gattacaaac 2280 gcatagactt accagcgctt gggaatgaat gtcctccctc cgagatgttt gaactgccta 2340 ccaaggtttt catgcttaaa gatgcagatg tgcatgaatg gcagttcaac tatggggaac 2400 ttacaggaca tgggttgtgc aattgggcaa atgtagttac ccagcccaca ttgtactttt 2460 ttgttgcgtc cacaaatcaa gtgacgatgg ctgctgattg gcagtgtatt gttactatgc 2520 atgtggacat ggggcccgtc attgatcgtt ttgagttagt tccaactatg acgtggccta 2580 ttcaattggg tgacactttc gccattgata gatattatga ggcgaaagaa attaaacttg 2640 acgggtcaac ctccatgttg tctatatctt ataattttgg aggtcccgtc aagcattcta 2700 agaaacatgc catttcatat tcccgggcag ttatgtctag gaatcttggg tggtctggca 2760 ctataagcgg aagtgtcaag agtgtttctt ctttattttg taccgcttct tttgttattt 2820 ttccatggga acatgaagca cctccaacct tacgtcaggt gttatggggc ccacatcaga 2880 taatgcacgg agatggccaa tttgaaattg ctatcaaaac tcgtcttcat tcagctgcta 2940 caactgaaga agggtttggt agacttggta tactcccgct ttctgggcct atagctcctg 3000 atgcacatgt tggatcgtac gagtttattg tacatataga cacttggcga cccgactctc 3060 aggtgcatcc tcccatgttt tctagtgcgg agctttataa ttggttcact ttaaccaatt 3120 tgaaaccaga tgcgaacact ggcgtagtca actttgatat tcccggatac atccatgact 3180 tcgcctctaa ggacgcaact gtgacgctcg catcaaatcc cctctcttgg cttgtcgcag 3240 ctactggctg gcattatggt gaggtggatc tctgcatctc ctggtcaagg tccaaacagg 3300 cccaggctca ggagggtagt gtttccatta ccactaatta tagagattgg ggtgcttact 3360 ggcaaggcca ggcccggatt tatgatttgc ggcgtactga agcggaaatt cccatcttct 3420 tgggttctta cgctggtgcg acgccatctg gtgccttggg taagcaaaac tatgtccgga 3480 tttcaattgt caatgctaag gacatagttg cactgcgagt gtgtttgcga cccaaatcta 3540 taaagttctg gggtcgctcc gccactttgt tttaaatgcc tttacgtagg tgttttgtct 3600 tttgattctt ctaaccagga ttggtagccc ttctggcatt catttgctgg aaaccatgct 3660 gtacatggtt ggttagatag aagcgtcaat aaaccgggac tttaattagt tccgttttag 3720 gttcctgccc ttactagggt ggtccggtcc ttaggtggat cgggaagctg tataaactca 3780 gcttcttggg ggtgagagcc caagaatgtc tgtctgtcag ctgcttgtgt aaatgagctt 3840 tctcccagga tagctctcct gggcacaagt gaaaactact gtcgtggcga gttagtagcc 3900 agacgaccgg agtaaaatct ctagttaaca aaatgtgtta gaccgttgtt tctggcagct 3960 ttgttgggtc tgttgagttt tctaaagctg ctttgctatt tgctgttgtt tgagcccact 4020 gttcatattt ggtttggggc cttttatgtt tcctagtgtc gtgtttgtcc aacacaaaaa 4080 aaaaaaaaaa aaaaaaaaaa aaaaa 4105 <210> 2 <211> 7543 <212> DNA <213> tobacco ringspot virus <400> 2 ttgaaaattc tctcacaagg ttcccgttac tttcaaaact ctcttttctt cttataattg 60 tctactgaat gtgattttgt tttgtttcaa cttgtgtaaa gatgggcttt acttgcccaa 120 actccgattg tctctactcg cgtagcgagt ggagcaatcg tgccctccgc gaggagggtc 180 ttacttttag catgcgttgt cctggcgtat gctgcggagc actgttggtc cgcaagcaac 240 aggaacctga agctgtggat agcgccacag caccccgaaa gagggtggat agcgccaccc 300 caaaatgttt gtgctggctt gccagcgtgg ggccttccaa gtgccccaaa cactcccagg 360 tgcctctggc accaaaatcc aaacctacgc ccacggcggt tgtagtttct gcttctcctc 420 tcaagaagca gagctgcgac gttgtggtcg ctgttggccc accggccaac ctagagctgg 480 tttatccggc tctagtctct cttggtgctg ctgccctacc taaggcggag aaaaagagct 540 tcaatgaggc tcttttggag aagcgtgcgg cctaccaggc gcgcaccgct gtaccaccac 600 ccgggcctat tcgggtggtg aagaccgttg ctgccccggt taaggcagaa aaggtgaagt 660 tcccaaaagg ggctgtcgcc ttcaatggca tcaattttgt tgatgctaag ggccatgtgg 720 tcctgagcgc tggagcgctt aaaattctgc gcggagctaa aaaactccgg caacagcagg 780 cgcgatctat gcgccgcatg gctgcctgcc gccgtgtgcg gttggcagct tttgctgcta 840 gagtgccttc ccttttgagg aaggcagatg aagccacatc tggtggcttt aaatatgttg 900 atttaaatgc accaagggcg gtgcgtgagg ccgagaagcg gccaaaaaag aagcccgcca 960 agaaggcggc caggtctgcc tcccctgttg aggaggaaat taactgggat gattttatca 1020 ttccagattc tgagaggact gcctccccga tgaaggagga aaaaccaaag cgccctttgg 1080 ttcccaattc tttgggtttc ggctggtggc gtccagcttc tggaaatctt tgggacgctg 1140 tgtcccagtg tcagcgtgcc tgtaagggca cctttcttga agtctctgct gaggctaagc 1200 tggtgtgtgc tggcaccgat gatggagccc tatctgtttg ggccagaatt tcaaagagtg 1260 ttgtgcaact ttctgcttat tatgatgcca acactcttct ggataattat aatgcactgt 1320 ctgagtgtac catggatgaa ttacaatctg tggcagtgca gttggattct gagtaccagg 1380 aacttggacc acctacgcat tttacgtgtg ggttgtccaa ctgggctcgt ggggctggca 1440 aaattctcta taattttgta gctcctactg tggaagggat agcaggcgca ggatgtcgta 1500 ttgttgagcg cgcctatgag ttgtctaaag tcgttattga cgagatcttc agtaaaatga 1560 agtccctttt ttatgactgc tttggcaatt tatttggcca tttgaatgtg ctgctatcca 1620 caattgatag tttttgggcc cgtgcgtcta catggataat gaatatctta gagaaaacgc 1680 atgattgcct taaggtgctt agggacagtg ctgtttggtc cctattgttg attctggttg 1740 gaggtttaat acttctttct gaaaggttcc tgcaatccat tggtataatt tcaaagccag 1800 ggaccatttt gggtattttc cttgcaacct ttcttggcat atttgggtac acctttttcc 1860 gtaaggacga cactttagtg tcagatcttt tgtgtgcctt taaaattgct attacaaacc 1920 ttttccggac aaagcctggt cctccaggat ctcccatcat tgtggacggt gatgtggtga 1980 taccggaaag tgctgtcgaa atgtccacat gtagtttcat gggcggtctt gatattgcaa 2040 ttgctgctat tggaaatgtt ggagcttcta tccttggttt taaggttgga gctttacaat 2100 atgcagccaa gatcgctaca tgtctagacc agttgcgcaa gggtaaagac gtgcttaaag 2160 agatgacctg ctggatcatt gaaacccttg gcgccctgtg gaataaaatc actggtcgtg 2220 aagccacttt cttcgacgaa gtttctgcta ttgtagcagt tgatattagg gagtggcttg 2280 aagaatccca aaacttatgt ctcgctgcgc aaactttttc catcggagat aagattgttc 2340 ttgaacaatg tgaacgtctt attgctgatg gccacaaact tttgcggggc atgggtgatg 2400 ccgatcggaa gctttctagt tctttccttt caactgtaca aaggaaagtt tctgatcttg 2460 agaaaataca tactcagtct gtccgtgctg ggtattttga aggaaggagg atggaaccct 2520 tttgggtgta catccacgga ccctcccatt gtggcaagtc tctcttaatg gaacccatgt 2580 ctagggaact attgagagcg ggaggttttt ctgaatcgtc tatctataca aagaattctt 2640 gtgataaata ctggtcaagg tatagaagac aagcttgtgt acagattgat gacttgtcag 2700 ctggcaaaac ggatccatct ttagagagtc agctcatcaa tcttgttgct tcaaaggagg 2760 tgccacttga tatggctgaa gttgaagaca agggcatatt attcgattca gccattcttg 2820 tcacctcctc taatacagcc cacgtgccga cgaatgctaa cgtcaaccat gcagaagctt 2880 acaagaaccg catgaatgtt gttatccaat gtaggaggaa gcctgagtac tccaacatag 2940 gtgtcgagct tgaaggcacg tttcaaccat ttgacccgcg gaatccacaa gcttcaattg 3000 agtgcatgtt acaacaccgt gaaacgcatg cgcccattac cggatggatt tctgctgggg 3060 ctgccatggc tgaggcagtg aaccagttcc gcttgcacag agagaaggag atgatcttac 3120 aaagtaatca tctttcctcc ttccgccctg cgcaccccat ttacactgag tgtgccactt 3180 ttttaagtat gtacgcgcga gacgcaagtt ttgtgccacc tgtggacctt ggttgtaaat 3240 gggaaattcc aagtgggtac atgaccattg ctgctgtgga tggtcgcgtt tttggtttca 3300 cacagcttgg agtgtgtact gagataacta aacagttaaa gttcactgac gaaatggaac 3360 agtacacact cgataaattt gccccagata ttacgaagac aatggcttcc caaagtcgtt 3420 ttaagcttgt tggggcattt ttgaaaggaa tgattaggga ggaagacaat gttatttcac 3480 tcacctccct agggcccaag agtactgcaa ctcaaagaga attttatgaa actcttgggc 3540 ttgctgagcg ggtttacctc cgtgctgtgc aaaagaaagt caataaaatt cgcacggacc 3600 ccgcttttga tgttgaggcg ttgcatgcta gactactgag caacattgca acttcctatg 3660 agtatgttag gacacatgga cctaaaattt tccctttact catgggattt gtttgtgtag 3720 ttctttgcatg ctatggtttt attatgcccc tgctttcttt tgcctcaggg ggctctgctg 3780 taggtgggat ggttgcaatg gaacaaatgt ctgcagcctc tgttgtttct tctggatcaa 3840 gtccagttgc ccaccgcaac cgtgcaccac cagtgcaacc aagatatgct aggcatcgtt 3900 tggctggagc ttcagccgat gatgcctatg cttatgaaga aatgatggtg gtgttgtacg 3960 ttgactcgac cgttgctccg gtagtcaatg ctgttagagg gcctggccgt tcaattttca 4020 taacccggca ccaggccctc atgattccca acaatagtac cgttgtggct catttttcca 4080 cacgtgatgt tgttgagatc cactgggagc acgacgtcgt caggaaaggc gagaagaagg 4140 ataccgaaat tgttcaatat cgctgtcctt ctattcctga acttccctct cgctgtaaga 4200 aatattttga atatgattta gagagggatt tccctggacc atttacttta gatgccagct 4260 gttatagaat gcaaagtcca gggaaaatcg atatagagct ggtgagctgg accgaccatg 4320 atgcggaact ccgcactcgt cctcttgtta tcgctgatcc atttggagag gacagatata 4380 gaagggaaat tccccgatat atccagtatg gtaggccagc gcaactccat gattgtggtg 4440 ctatctgtgt tgccaaaatt ggaggccaac atagaattgt gggcctagtt atttccacag 4500 ataagcacaa cactggagtt ggtctgctgc cgtcggcgct tcatatgata acttgttccc 4560 ttacctatgt gcctgaggaa tgggaagatg cgccacgggg tttgaagaaa ttgggttgga 4620 agcacgcttc tgaacttcca catatgccgc ggaaaaccca atacgttgct gttaatgagg 4680 atcttgcaat tccatttgat aaccccaaga taccaagtgt cttggtccca gatgatcctc 4740 gtactgtagg cacgcccgtt gaaggtaaag atcctgtctt ggttgcaatg gaaaaatttt 4800 atgaaccaat gacagacttt acagaagagg aggtccgccc aggtcagacc gaggttagct 4860 tgtttgagca agtgtgtgat gatattgtgc aaacctggtt tgatgctggt gcggaatttg 4920 aagatgttga agatgatgtc gtgatcaatg gtgatgatga ctttgataaa ttgatcatgg 4980 acacatctga aggttatccc tatgtgcttg aaaggacgca tggggaaaaa ggaaaaactc 5040 gatatttcga gggtggacct ggggcctaca ctctaaagcc cggtacttcc gtgtataatg 5100 attatcatag actacaagag gaggtacagg ttgaaggggg tatccctgag atggtctgta 5160 ttgagtgccc aaaagacgaa ttgcttgtgg agcgtaaggt tctgcaaaaa cttggcactc 5220 gaaacttcga gatattggaa ttgcccaaaa acatgctttt cagaaagaaa tttttgcatt 5280 gggctttatt cctttcagat atgcgatggt gcctgccgtg tcaagtgggc atcgttgttc 5340 aggggcgtga atgggggctc ctgatggatc gtcttgctgc gaagaattcg gttgcttaca 5400 actgtgatta ttctaagttt gatggtctca tgtcttgtca ggtgttggat gctataggga 5460 aaatggtaaa taaatgctat tccaacgcca accccaattt aaagaagaag gggaagggtg 5520 agctgcccgg tagtcctccc caattagcta ggcataattt gttaatgtcc atttttggta 5580 gaaagtgctt agctagatcc caagtttttg aagttcgggg tgggatcccg tcggggtgtg 5640 cacttaccgt attgttaaac tctgtattta atgaaattct catacggtat gtgtataaaa 5700 cagtaattcc atcaccagag tttaatcgct ttgaaacttt tgtgactctg gtggtatatg 5760 gtgatgataa cctcattgca gttgatcctt caatgcaaaa aatttttact ggtgaggtta 5820 ttaagaagac attggcgagg aagaaaatca ctattactga tggtagcgat aagttatctc 5880 ctgtgctaga ggcaaaacca ctcgcgcagt tagattttct taagcgttcc ttcctcattt 5940 ctgattcggg gcaagtcatg ccagcgttag atagaacttg catatattca tctttgctat 6000 atctacgctc ggctgattgt gatccaatac ctctcttgca ccaaaatgtg caaaatgctt 6060 tgcaagagct gtattaccgc caagatcggg acgagtttga taatctccgt attttttatc 6120 tcgagagatt accaatgtgg cggaatggtc aacaccggct cctcgattgg aaccaatgtg 6180 gtgaacactg gcgggcgcgt tatactgggt gtccttccga taatcctgct ggcgttctcg 6240 atatgttgat tgatccaagg tgtaagagct ttattcttcc tgctggtccg gctaattggt 6300 ccatgccaat agcagaccgt atctttgttt gtgggcctaa attttgtcca agtgggtcct 6360 catacacctt gtgtttcaat cgtcttgctg caggtgagac aggtgtacaa ataaaacctg 6420 ttcatgctgc aactcaggga gctatgccta ctgctaagtt tgtcgagagc ttccgctcca 6480 ttaagaagag acctgagctt gaattagcta tctctgctta tgagagtgga agtaatcttt 6540 atttcaaagg ctgtgctcct tacaatgaca tctgggcttg cgctatatct ttttgctctg 6600 cttttggata tgcccagaaa caagtgctcc tccacatgta tgataattgc aagcctttag 6660 gggctagttc tttgaggagt tactttaata aaaacctggt tggtgatggt tgtgcgcgac 6720 gttgtgaaat tcatgcaacc tcagctattg ccaggcaggt cgaacgcctt cttccacagg 6780 ttcaatgcaa acattgtgaa tatgatcctg agtttgcatc aaaacctact acccaattgc 6840 gtaaatgcac ggatcctggg gtagatggag gtaaggcaat gtatatagtt cgtggcctgg 6900 gcagaactgc agccaagttg gtttgttcag acatgtgtga tggacatcta atgtcctgta 6960 atacaacttt tgacaaaatg gttgtggatc tgttcaggca atcttgtttt taaatgcctt 7020 tagttaggtg ttttgtcttt tgattcttct aaccaggatt ggtagccctt ctggcattca 7080 tttgctggaa accatgctgt acatggttgg ttagatagaa gcgtcaataa accgggactt 7140 taattagttc cgttttaggt tcctgccctt actagggtgg tccggtcctt aggtggatcg 7200 ggaagctgta taaactcagc ttcttggggg tgagagccca agaatgtctg tctgtcagct 7260 gcttgtgtaa atgagctttc tcccaggata gctctcctgg gcacaagtga aaactactgt 7320 cgtggcgagt tagtagccag acgaccggag taaaatctct agttaacaaa atgtgttaga 7380 ccgttgtttc tggcagcttt gttgggtctg ttgagttttc taaagctgct ttgctatttg 7440 ctgttgtttg agcccactgt tcatatttgg tttggggcct tttatgtttc ctagtgtcgt 7500 gtttgtccaa cacaaaaaaa aaaaaaaaaa aaaaaaaaaaaaa 7543 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tggaggtccc gtcaagcatt cta 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tggccatctc cgtgcattat ctga 24 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cttgagaagg ggtggaagaa ct 22 <210> 6 <211> 92 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gcactagtac gcgtggatcc aggcactttg aaactaagag taccgcagca tgtagggtca 60 ggtactactg tgacagcaca catcaaatga gc 92 <210> 7 <211> 111 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggatccacgc gtactagtgc gcctgcaaaa cagctcttaa actttgacct acttaagtta 60 gcaggtgacg ttgagtccaa ccctgggccc gctgtgacgg ttgttcccga t 111 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tggtcacctg taattcacac gtgg 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ctttagggac tcgtcagtgt actg 24 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ccaaacacct acgtaaaggc at 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 atgcctttac gtaggtgttt tg 22 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aaaggatcca tgagtaaagg agaag 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ccgactagtt ttgtatagtt catcc 25 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aaatacgtaa tgcagaacct gtttgg 26 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 aaatacgtag gcaagagtcc aatag 25 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 aaatacgtag ttaagtgcct ttgac 25 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 tggactctgg tgatggtgtc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 cctccaatcc aaacactgta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gaggtcttcg ttgggaactg 20 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 caatggttta gttgggcgtg ag 22 <210> 21 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 aaatacgtat ttggggctta tcccaa 26 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 aaatacgtat ctcatccact cttgc 25 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 ttggtgatga agctcagtc 19 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 taggatagca tgagggagtg 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 tgtgtggatt accctagacc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 ccaagctgct acctttccac 20 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 ccgtacgtaa taactggaaa gg 22 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 ccgtacgtaa accaactcta ac 22 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 aacctcctcc tcatcgtact 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 gacagcatca gccatgttca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 gcttgccaaa ctctttcctg 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gcacaaagct tcccagaaag 20

Claims (15)

P2A (protein 2A) coding gene of TRSV (tobacco ringspot virus) RNA2; A movement protein (MP) coding gene; A cleavage site between a movement protein (MP) coding gene and a CP (coat protein) coding gene; MCS1 (multiple cloning site1), which is located at the lower part of the cleavage site and can insert a foreign gene to be expressed; A sequence encoding a self-cleaving peptide located under the MCS1; A coat protein (CP) coding gene; A multiple cloning site 2 (MCS2) capable of inserting a target gene to be silenced located at the bottom of the CP. 2. The recombinant vector according to claim 1, wherein the cassette comprises the nucleotide sequence of SEQ ID NO: 1. The recombinant vector according to claim 1, wherein the foreign gene to be expressed is a nucleic acid sequence encoding a useful protein. 2. The recombinant vector according to claim 1, wherein the target gene to be silenced is inserted in a sense or antisense direction. (1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;
(2) preparing a TRSV RNA2 recombinant vector by inserting a foreign gene to be expressed in the TRSV RNA2 recombinant vector of claim 1 into MCS1 (multiple cloning site 1);
(3) transforming Agrobacterium with the respective recombinant vectors prepared in the above steps (1) and (2) to obtain respective transformants; And
(4) infiltrating or sapinoculating a mixture of the respective Agrobacterium-containing transformants to the plant; and (5) overexpressing the foreign gene in the plant. 6. The method according to claim 5, wherein the polyprotein coding gene of the TRSV (tobacco ringspot virus) RNA1 comprises the nucleotide sequence of SEQ ID NO: 2. 6. The method according to claim 5, wherein the plant is selected from the group consisting of tobacco, Arabidopsis, poultry, and leguminous plants. (1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;
(2) preparing a TRSV RNA2 recombinant vector by inserting a target gene to be silenced in the TRSV RNA2 recombinant vector of claim 1 into MCS2 (multiple cloning site 2);
(3) transforming Agrobacterium with the respective recombinant vectors prepared in the above steps (1) and (2) to obtain respective transformants; And
(4) infiltration or sap inoculation of the mixture of the respective Agrobacterium-containing transformants into a plant; and silencing the desired gene in the plant. (1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;
(2) A foreign gene to be expressed in the TRSV RNA2 recombinant vector of claim 1 is inserted into MCS1 (multiple cloning site 1), and the desired gene to be silenced is inserted into MCS2 (multiple cloning site 2) to prepare a TRSV RNA2 recombinant vector ;
(3) transforming Agrobacterium with the respective recombinant vectors prepared in the above steps (1) and (2) to obtain respective transformants; And
(4) infiltrating or sapinoculating a mixture of each of the above Agrobacterium transformants to a plant, and overexpressing the foreign gene in the plant and silencing the target gene Way. (1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;
(2) preparing a TRSV RNA2 recombinant vector by inserting a foreign gene to be expressed in the TRSV RNA2 recombinant vector of claim 1 into MCS1 (multiple cloning site 1);
(3) transforming Agrobacterium with each of the recombinant vectors prepared in the above steps (1) and (2) to obtain transformants; And
(4) infiltrating or sapinoculating a mixture of each of the above Agrobacterium transformants to a plant, wherein the foreign gene is overexpressed. A plant overexpressed with a foreign gene produced by the method of claim 10. (1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;
(2) preparing a TRSV RNA2 recombinant vector by inserting a target gene to be silenced in the TRSV RNA2 recombinant vector of claim 1 into MCS2 (multiple cloning site 2);
(3) transforming Agrobacterium with each of the recombinant vectors prepared in the above steps (1) and (2) to obtain transformants; And
(4) infiltrating or sapinoculating a mixture of each of the above Agrobacterium transformants to a plant, wherein the target gene is silenced. A plant in which the objective gene produced by the method of claim 12 is silenced. (1) preparing a TRSV RNA1 recombinant vector containing a polyprotein coding gene of TRSV (tobacco ringspot virus) RNA1;
(2) A foreign gene to be expressed in the TRSV RNA2 recombinant vector of claim 1 is inserted into MCS1 (multiple cloning site 1), and the desired gene to be silenced is inserted into MCS2 (multiple cloning site 2) to prepare a TRSV RNA2 recombinant vector ;
(3) transforming Agrobacterium with the respective recombinant vectors prepared in the above steps (1) and (2) to obtain respective transformants; And
(4) infiltration or sap inoculation of the mixed solution of each of the above Agrobacterium transformants into plants, and (4) overexpressing the foreign gene comprising the step of infiltrating or sapinoculating the plant, Way. 14. A plant wherein the foreign gene produced by the method of claim 14 is overexpressed and the gene of interest is silenced.

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