WO2012053710A1

WO2012053710A1 - Recombinant virus-induced gene silencing vector from symmv useful for functional analysis of useful genes in soybean and uses thereof

Info

Publication number: WO2012053710A1
Application number: PCT/KR2011/003161
Authority: WO
Inventors: Su Heon Lee; Moon Nam; Jae Sun Moon; Hong Soo Choi; Jeong Soo Kim; In Youl Baek; Jung Kyung Moon; Kook Hyung Kim
Original assignee: Republic Of Korea (Management: Rural Development Administration)
Priority date: 2010-10-21
Filing date: 2011-04-28
Publication date: 2012-04-26
Also published as: KR20120041608A; KR101352039B1

Abstract

The present invention relates to a recombinant virus-induced gene silencing (VIGS) vector from soybean yellow mottle mosaic virus (SYMMV), comprising a promoter and the full-length nucleic acid sequence of SYMMV, wherein a restriction enzyme site for inserting a foreign gene is included within the gene encoding the coat protein in the full-length nucleic acid sequence of SYMMV, a recombinant VIGS vector constructed by inserting phytoene desaturase (PDS) gene to the recombinant VIGS vector, a method for functional analysis of a foreign gene by using the recombinant VIGS vector, a method for production of a plant having a silenced target gene by using the recombinant VIGS vector, a plant having a silenced target gene that is produced by the method and seeds thereof, and a combination of primers useful for amplification of the recombinant VIGS vector.

Description

RECOMBINANT VIRUS-INDUCED GENE SILENCING VECTOR FROM SYMMV USEFUL FOR FUNCTIONAL ANALYSIS OF USEFUL GENES IN SOYBEAN AND USES THEREOF

The present invention relates to a virus-induced gene silencing (VIGS) vector from soybean yellow mottle mosaic virus (SYMMV) useful for functional analysis of useful genes in soybean. More specifically, it relates to a recombinant VIGS vector from SYMMV which comprises a promoter and the full-length nucleic acid sequence of SYMMV, wherein a restriction enzyme site for inserting a foreign gene is included within the gene encoding the coat protein in the full-length nucleic acid sequence of SYMMV, a recombinant VIGS vector constructed by inserting phytoene desaturase (PDS) gene to the recombinant VIGS vector, a method for functional analysis of a foreign gene by using the recombinant VIGS vector, a method for production of a plant having a silenced target gene by using the recombinant VIGS vector, a plant having a silenced target gene produced by the method and seeds thereof, and a combination of primers useful for amplification of the recombinant VIGS vector.

Soybean is an economically important crop all over the world and widely consumed as everyday food products and animal feeds. Recently, many studies are performed regarding its use as a raw material for biodiesel. Thus, many researchers all over the world now concentrate on development of new varieties based on identification and characterization of many useful genes contained in soybean. Many efforts are also given to have an original technology by obtaining patent right.

In order to study the function of genes contained in a crop, it is essential to have a mutant of each gene. Until now, T-DNA knockout mutant has been mainly used to obtain the mutant in a large amount. For some crops, VIGS method using a plant virus is used. However, to obtain a T-DNA knockout mutant, the transformation technique is essential. At this moment, unfortunately, the large scale transformation technique is not available for soybean. Further, to actually practice the VIGS method, an effective viral vector that can be used for soybean is very much required.

To develop a VIGS vector usable in soybean, a search for a new virus which occurs in soybean is necessary. As every virus has different pathogeneticity, host range, and silencing suppressor efficiency, the continuous search and investigation of efficient viruses are required. Further, it is important not to have any symptom of disease in host due to the virus. This can be achieved by selecting the varieties which show normal viral growth without exhibiting a symptom of disease, based on studies of viruses regarding various symptoms of disease.

In terms of developing a virus which can be used as a VIGS vector in soybean, viruses reported so far has a limit. Because many researchers have already finished most studies relating to the possibility as a vector, a continuous search for a new virus is necessary. SYMMV has been recently found in Korea for the first time and until now very few studies have been made related thereto. Further, most of the VIGS vectors use a double-stranded RNA virus (tobacco rattle virus and bean pod mottle virus, etc.) and use of a single-stranded virus like soybean yellow mottle mosaic virus is extremely rare. Thus, it is desired to have more improved gene silencing effect by using a new virus, i.e., SYMMV, than conventional vectors.

In a viral gene of a plant, there is a suppressor which inhibits the silencing occurring in a plant. It is known that there are many cases in which VIGS effect cannot be efficiently obtained due to the presence of such gene and mechanism and ability of each suppressor are somewhat different for each virus. Further, the important thing in VIGS experiment is that the symptom of a disease caused by the virus used as a vector should not be expressed in the plant. This is due to the necessity of avoiding any confusion between the symptom of a disease caused by virus and the phenotype expressed by VIGS. Thus, it is necessary to develop a vial vector satisfying all of these requirements.

Currently, gene function analysis using VIGS is carried out for many crops. In tobacco, tomato, pepper, and Arabidopsis thaliana, etc., tobacco rattle virus (TRV) vector is mainly used (Ratcliff, Frank. Tobacco rattle virus as a vector for analysis of gene function by silencing. 2001 Vol. 25, 237-245). For soybean, a viral vector like TRV vector from which a potent silencing effect is expected is not available yet. In US, the bean pod mottle virus (BPMV) is developed and currently being studied (US 2007/0214518 A1 (Ghabrial, S. A.) Sep. 13, 2007).

In Korean Patent Registration No. 10-0795367, a VIGS vector constructed by inserting a transcription regulating gene of a pepper having resistance of a pathogen to TRV is disclosed. In Korean Patent Application Laid-Open No. 2009-0114715, a VIGS vector constructed by inserting a pepper lipoxygenase gene to TRV is disclosed.

The present invention is devised in view of the needs described above. The inventors of the present invention constructed a recombinant VIGS vector by using SYMMV isolated from soybean, and as a result of inoculating an RNA transcript prepared with the vector into a plant, it was found that the silencing occurs effectively in plant cell gene. Accordingly, the present invention is completed.

To solve the problems above, the present invention provides a recombinant VIGS vector from SYMMV comprising a promoter and the full-length nucleic acid sequence of SYMMV, wherein a restriction enzyme site for inserting a foreign gene is included within the gene encoding the coat protein in the full-length nucleic acid sequence of SYMMV.

Further, the present invention provides a recombinant VIGS vector constructed by inserting phytoene desaturase (PDS) gene to the recombinant VIGS vector.

Further, the present invention provides a method for functional analysis of a foreign gene by using the recombinant VIGS vector.

Further, the present invention provides a method for production of a plant having a silenced target gene by using the recombinant VIGS vector.

Further, the present invention provides a plant having a silenced target gene produced by the method, and seeds thereof.

Still further, the present invention provides a combination of primers useful for amplification of the recombinant VIGS vector.

Soybean has a nucleotide sequence of about 975 Mb which encodes about 66,000 proteins. As a tool for analyzing characteristics of those many genes, VIGS is presented as the most preferred solution, and it is believed that a vector using SYMMV can be used therefor.

Thus, it is considered that, by having effective silencing of target genes using SYMMV, researchers will be able to study unique functions of a gene and it will be very useful for a search for economically valuable and useful genes.

Further, it is expected to achieve a development of new soybean varieties by using such useful genes, and in particular, an original technology can be attained via obtainment of patent right. Therefore, in terms of a future seed war over new breeds, a basis for the development of new soybean varieties having competitive edge on world market can be established.

FIG. 1 is a schematic diagram of SYMMV, a new virus isolated from soybean.

FIG. 2 is a schematic diagram of an infectious clone which has been constructed by using SYMMV.

FIG. 3 shows the result of examining pathogenicity of SYMMV after inoculating standard SYMMV strain or SYMMV infectious clone to soybean.

FIG. 4 is a schematic diagram illustrating the insertion of gmPDS fragment to SYMMV infectious clone.

FIG. 5 is a schematic diagram illustrating the recombinant VIGS vector which has been constructed by using SYMMV infectious clone.

FIG. 6 shows the result of RNA synthesis of SYMMV infectious clone with inserted PDS.

FIG. 7 shows the photo-bleaching induced by pSYMMT7-PDS3.

FIG. 8 shows determination of a silencing effect of the recombinant VIGS vector based on RT-PCR.

FIG. 9 is a schematic diagram showing the construction of USV3 VIGS based on single-joint PCR.

FIG. 10 shows the photo-bleaching effect induced by pSYMMT7-PDS3.

In order to achieve the object of the invention, the present invention provides a recombinant VIGS vector from SYMMV comprising a promoter and the full-length nucleic acid sequence of SYMMV, which is characterized in that it contains a restriction enzyme site for inserting a foreign gene within the gene encoding the coat protein in the full-length nucleic acid sequence of SYMMV.

VIGS is a phenomenon of inhibited expression of endogenous plant gene that is induced by infection of a plant with a viral vector containing the corresponding gene. It is one type of post-transcriptional gene silencing (PTGS) and has characteristics of being post transcriptional and RNA turnover- and nucleotide sequence-specific. Therefore, by using VIGS, large scale analysis of an introduced gene can be achieved both quickly and conveniently.

According to the recombinant VIGS vector of the present invention, the promoter is a promoter which is suitable for in vitro transcription. Preferred examples of the promoter include T7 promoter, SP6 promoter, and 35S promoter, but not limited thereto. The term “promoter”indicates a region of DNA located upstream of a structure gene, and it corresponds to a DNA molecule to which an RNA polymerase binds to initiate transcription.

In the recombinant VIGS vector of the present invention, the restriction enzyme site is a place at which a foreign gene can be introduced. It is located within the gene encoding the coat protein, and preferably at the 3’-end of the gene encoding the coat protein. As a potent RNA silencing suppressor, a coat protein, i.e., P38 protein, is known from turnip crinkle virus (TCV) belonging to carmovirus like SYMMV. Thus, to inhibit the function of a RNA silencing suppressor, a foreign gene is inserted within a gene encoding the coat protein.

The term “recombinant” indicates a cell which replicates a heterogeneous nucleotide or expresses said nucleotide, a peptide, a heterogeneous peptide, or a protein encoded by a heterogeneous nucleotide. Recombinant cell can express a gene or a gene fragment in a form of a sense or anti-sense, that are not found in natural state of cell. In addition, a recombinant cell can express a gene that is found in natural state, provided that said gene is modified and re-introduced into the cell by an artificial means.

The term “vector” is used herein to refer DNA fragment (s) and nucleotide molecules that are delivered to a cell. Vector can replicate DNA and be independently reproduced in a host cell. The terms “vehicle”and “vector” are often interchangeably used.

It is also provided by the present invention a recombinant VIGS vector with inserted PDS fragment which is characterized in that PDS gene is inserted in an anti-sense direction to the 3’-end of a gene encoding the coat protein in the recombinant VIGS vector. The recombinant VIGS vector inserted with PDS fragment may be the vector illustrated in FIG. 5 and FIG. 10., i.e., pSYMMT7-fullIC-PDS3 vector or pSYMM35S-fullIC-PDS3 vector, but not limited thereto.

According to one embodiment of the present invention, PDS gene, which is a gene related to photosynthesis, was used as a marker to determine successful VIGS. As PDS gene encodes an enzyme which is involved with carotenoid biosynthesis, photo-bleaching of plant leaves will be observed when this gene is inhibited. According to the present invention, the PDS gene fragment was inserted to the restriction enzyme site of the recombinant VIGS vector, and after transfecting a plant with the vector, any change in phenotype of the plant (i.e., photo-bleaching) was examined.

The present invention also relates to a method for functional analysis of a foreign gene comprising:

- inserting a foreign gene whose function is to be analyzed to the recombinant VIGS vector of the present invention;

- digesting the recombinant VIGS vector having the inserted foreign gene and performing in vitro transcription to produce an RNA transcript;

- inoculating the RNA transcript into a plant for silencing of the foreign gene; and

- analyzing the phenotype of the plant having the silenced foreign gene.

The method for functional analysis of a foreign gene comprises inserting a foreign gene whose function is to be analyzed to the recombinant VIGS vector of the present invention. In this case, the foreign gene is preferably inserted within the gene encoding the coat protein. More preferably, it is inserted to 3’-end of the gene encoding the coat protein. With regard to the direction of the insertion, it may be either a sense direction or an anti-sense direction. Preferably, it is inserted in an anti-sense direction.

According to the method for functional analysis of the foreign gene of the present invention, T7 RNA polymerase, etc. may be used for the in vitro transcription. However, the present invention is not limited thereto.

The method for functional analysis of a foreign gene of the invention comprises inoculating the RNA transcript into a plant for silencing of the foreign gene.

The term “silence(silencing)”generally means that gene expression is down-regulated or completely suppressed. The plant to be transfected with SYMMV is not specifically limited, however, it is preferably soybean (Glycine max).

Examples of a useful gene which can be analyzed by the recombinant VIGS vector of the present invention include a gene related to plant growth, a gene related to disease resistance, and a gene related to environmental stress, etc., but not limited thereto.

The present invention also provides a method for production of a plant having a silenced target gene comprising:

- inserting a foreign gene to be silenced to the recombinant VIGS vector of the present invention;

- digesting the recombinant VIGS vector having the inserted foreign gene and performing in vitro transcription to produce an RNA transcript; and

- inoculating the RNA transcript into a plant for silencing of the foreign gene.

Regarding the method for production of a plant having a silenced target gene of the present invention, each step is the same as the steps above described in relation to the analysis of a foreign gene function.

The present invention also provides a plant having a silenced target gene which is obtained by the method above, and seeds of the plant. The plant to be transfected with SYMMV is not specifically limited, however, it is preferably soybean (Glycine max).

The present invention also provides a combination of primers having SEQ ID NO: 1 to SEQ ID NO: 4 for amplifying a SYMMV infectious clone comprising T7 promoter and the full length SYMMV nucleotide sequence. The combination of primers consists of a primer set having SEQ ID Nos. 1 and 2 and a primer set having SEQ ID Nos. 3 and 4. The primer set having SEQ ID Nos. 1 and 2 is responsible for amplification of T7 promoter and a region including 5’-end of SYMMV and the primer set having SEQ ID Nos. 3 and 4 is responsible for amplification of a region including 3’-end of SYMMV.

In the present specification, the term “primer” means a single-stranded oligonucleotide sequence which is complementary to a nucleotide sequence to be copied, and it may serve as an initiation point for synthesis of a primer-extended product. The length and sequence of the primer should be selected so as to allow the initiation of the synthesis of a primer-extended product. Specific length and sequence of the primer depends not only on the complexity of a desired target DNA or RNA but also on the condition for use of primers like temperature and ionic strength, etc.

In the present specification, it is understood that the oligonucleotide used as a primer may further comprise a nucleotide analogue, for example, phosphorothioate, alkyl phosphorothioate, or peptide nucleic acid, and also an intercalating agent.

Herein below, the present invention is explained in greater detail in view of the examples. However, it is only to exemplify the present invention and in no case the scope of the present invention is construed to be limited by these examples.

Example 1: Determination of entire nucleotide sequence of soybean yellow mottle mosaic virus as a new virus isolated from soybean

SYMMV was isolated from soybean for the first time in Korea and the entire nucleotide sequence of the virus was analyzed. SYMMV was found to have a single-stranded RNA genome, consisting of 4009 bp in total (GenBank FJ457015.1). The entire ORF (open reading frame) consists of six parts. Specifically, ORF1 (nt 40-723) and ORF2 (nt 724-2280) encode 28 kDa protein and 56 kDa protein, respectively, and are related with viral replication, ORF3 (nt 2280-2486) and ORF4 (nt 238302667) encode 7 kDa protein and 10 kDa protein, respectively, and are related with the motility of the virus, ORF5 (nt 2664-3728) encodes 38 kDa protein and is responsible for production of viral coat protein, and ORF6 (nt 2668-3339) encodes 25 kDa protein and its function remains unknown (FIG. 1).

Example 2: Production of infectious clone using SYMMV

To produce an infectious clone by using SYMMV, two overlapping fragments were produced around SalI site by polymerase chain reaction (PCR). Each of the two fragments consists of 5'-fragment and 3'-fragment, having a size of 2.4 kb and 1.6 kb, respectively. 5'-Fragment was amplified by using primers of SYMMV-T7-EcoRI-U (5'-AAAGAATTCTAATACGACTCACTATACGGTAACCCAGCCAGTTATC-3'; SEQ ID NO: 1) and SYMM-R (5'-GATCACCGCCAGGTGGTTTA-3'; SEQ ID NO: 2), and it contains EcoRI site and T7 promoter. 3'-Fragment was amplified by using primers of SYMM-F (5'-GCCAGAGAACAAGCAGATTCA-3'; SEQ ID NO: 3) and SYMM-End2-PstI-L (5'-AAACTGCAGGGGGGGGGCCCCGCATGCCC-3'; SEQ ID NO: 4), and it contains PstI site. Each of the two fragments was cloned in pUC19 vector, and with SalI site, it was established as a single full-length SYMMV infectious clone (FIG. 2).

The SYMMV infectious clone was prepared as a single-stranded plasmid by using PstI, and RNA was synthesized by using T7-RNA polymerase. The synthesized RNA was used as an inoculant. The RNA was inoculated into two main leaves of soybean plant ten days after sowing. As shown in FIG. 3, a buffer solution was inoculated for (a) and (b), SYMMV standard type strain was inoculated for (c) and (d), and SYMMV infections clone (pSYMMT7-fullIC) was inoculated for (e) and (f). One week after the inoculation, it started to show a symptom of disease. Fifteen days after the inoculation, the symptom of disease was very clear. Meanwhile, the soybean inoculated with the buffer solution did not show any symptom of disease. Although there is a slight difference in the pattern of disease symptom, a clear symptom of disease was identified from the plant which had been inoculated with SYMMV standard type strain or SYMMV infections clone (FIG. 3). In addition, considering that the viruses were detected by PCR from the upper leaves to which no inoculation has been made, it was recognized that the infectious clone operates normally.

Example 3: Construction of VIGS vector using SYMMV infectious clone

To determine the usefulness of SYMMV infectious clone as VIGS vector, a PDS gene fragment was inserted to the SYMMV infectious clone. As a potent RNA silencing suppressor, a coat protein, i.e., P38 protein, is known from TCV belonging to carmovirus like SYMMV. Thus, to inhibit the function of a RNA silencing suppressor, one PDS fragment was inserted in the middle of SYMMV coat protein and the other fragment was inserted to the end of the coat protein. Both gmPDS fragments were amplified from soybean by RT-PCR. gmPDS1 fragment was amplified by using gmPDS1-MluI-U (5'-AAAACGCGTAACGGTGAAGAGCTTCTTACTA-3'; SEQ ID NO: 5) and gmPDS1-MluI-L (5'-AAAACGCGTACCAACTCTAACATTGACTGGT-3'; SEQ ID NO: 6), while gmPDS3 was amplified by using gmPDS3-XbaI-U (5'-AAATCTAGAACGGTGAAGAGCTTCTTACTAA-3'; SEQ ID NO: 7) and gmPDS3-XbaI-L (5'-AAATCTAGAAACCAACTCTAACATTGACTGGTT-3'; SEQ ID NO: 8). For inserting the gmPDS fragments to the SYMMV clone, site-directed mutagenesis was introduced at the sites of C3575A and C3725A of SYMMV 3'-clone, thus yielding restriction enzyme sites to which the insertion can be made. Then, gmPDS1 and gmPDS3 were inserted to the restriction enzyme site therefor, as shown in the drawings. gmPDS1 and gmPDS3 were inserted in a forward direction and a reverse direction, respectively (FIG. 4).

SYMMV 3' clone having inserted gmPDS was again replaced with the same region of SYMMV infectious clone. SYMMV 3' clone having inserted gmPDS was replaced by using SalI and PstI, and finally, a clone comprising gmPDS fragment which is inserted to the coat protein region of SYMMV infectious clone was produced (FIG. 5).

For the synthesis of RNA of SYMMV infectious clone having inserted PDS, in vitro transcription was carried out. Specifically, with about 10㎍ of the SYMMV infectious clone having inserted PDS, the plasmid was prepared to have a single-strand by using PstI. With the plasmid prepared to be in single-stranded state, RNA was synthesized for 2 hours at 37℃ by using T7 RNA polymerase. After that, residual DNA was removed by DNAseI. Then, the RNA was purified by phenol/chloroform extraction and the resulting RNA was used as an inoculant (FIG. 6).

Example 4: Determination of usefulness of pSYMMT7-PDS3 as VIGS vector

Buffer solution, pSYMMT7-fullIC, or pSYMMT7-PDS3 was inoculated to the main leaves of soybean plant ten days after sowing. The soybean inoculated with the buffer solution did not show any symptom of disease. One week after the inoculation, the soybean inoculated with pSYMMT7-fullIC showed a weak symptom of disease. About fifteen days later, a severe symptom of disease was shown from the entire part of the plant. The soybean inoculated with pSYMMT7-PDS3 showed a little slow expression of symptom of disease, i.e., about eighteen days later, a symptom of viral disease was identified, and the photo bleaching, which is a typical sign of PDS, was observed about 23 days later (FIG. 7).

Further, to verify the normal silencing by PDS gene, RT-PCR was performed. According to RT-PCR together with gmActin gene, it was able to confirm that the same amount of RNA was used. For the amplification of gmPDS, primers of gmPDS probe-U (5'-CACCAGCCGAAGAATGGATTT-3'; SEQ ID NO: 9) and gmPDS probe-L (5'-CTGTACAATAGCCTGTGCACA-3'; SEQ ID NO: 10) were used. As illustrated in FIG. 8, there is a significant difference in the amount of amplified PDS gene compared to the inoculation of buffer solution or SYMMV. Based on this result, it was confirmed that the PDS fragment inserted in a vector based on VIGS using SYMMV causes the silencing of gmPDS gene that is usually expressed in a plant cell. Thus, it was recognized that, as the VIGS vector using SYMMV induces the silencing of a specific gene of soybean, it can be used for large scale analysis of gene function.

Example 5: Construction of VIGS vector using SYMMV infectious clone

To establish a VIGS system using SYMMV, 35S promoter was attached to the 5’-end site of SYMMV by single-joint PCR. 35S promoter was amplified using pSNU1 vector (kindly provided by Prof. Kookhyung Kim at Seoul National University), and the 5’fragment of SYMMV including part of 35S promoter was amplified by PCR. Each of the above two fragments was linked by the second PCR, and as a result, it was possible to obtain a fragment in which 35 promoter is linked to the 5’-end of SYMMV via PSNU35S-SJ2-EcoR-1U primer (5'-AAAGAATTCAAGCTTGCATGCCTGCTGGT-3'; SEQ ID NO: 11) and SYMMV-R primer (5'-GATCACCGCCAGGTGGTTTA-3'; SEQ ID NO: 12). This fragment was again linked to the SYMMV 3’-end fragment to which Rz-NOS (cis-cleaving ribozyme sequence - NOS terminator) has been attached, and then cloned in binary vector pPZP211 (FIG. 9).

Example 6: Determination of gene silencing effect of pSYMMV as VIGS vector using SYMMV

To determine the gene silencing effect of VIGS vector by using pSYMMV, a restriction enzyme site (XbaI) was created behind CP of SYMMV by single point mutation, and gmPDS fragment amplified from soybean was inserted thereto in a reverse direction. Agrobacterium was transformed with each of pSYMMV-full as SYMMV VIGS vector and pSYMMV-PDS clone. By using acetocyringon, the pathogenic gene (vir gene) located within the binary vector was activated, and then inoculated into the backside of the soybean leaves using a syringe. Regarding the inoculation, one group was inoculated with a buffer as a control and the other group was inoculated with SYMMV virus vector without any inserted PDS gene. About two weeks after the inoculation, a slight symptom of viral disease was observed from the plant inoculated with pSYMMV-full. Three weeks later, the silencing effect of PDS started to appear in the plant inoculated with pSYMMV-PDS. About four weeks after the inoculation, the photo bleaching, i.e., a phenomenon of discoloration of soybean leaves to show white color, was detected (FIG. 10). These results indicate that, by using pSYMMV which is a VIGS vector constructed by using SYMMV, large scale identification of useful genes of soybean can be made.

Claims

A recombinant virus-induced gene silencing (VIGS) vector from soybean yellow mottle mosaic virus (SYMMV), characterized in that it comprises a promoter and the full-length nucleic acid sequence of SYMMV, wherein a restriction enzyme site for inserting a foreign gene is included within the gene encoding the coat protein in the full-length nucleic acid sequence of SYMMV.
The recombinant VIGS vector according to Claim 1, characterized in that the promoter is T7 promoter, SP6 promoter, or 35S promoter.
The recombinant VIGS vector according to Claim 1, characterized in that the restriction enzyme site is located at the 3’-end of a gene encoding the coat protein.
A recombinant VIGS vector having inserted phytoene desaturase (PDS) fragment, characterized in that PDS gene is inserted in an anti-sense direction at the 3’-end of a gene encoding the coat protein in the recombinant VIGS vector of Claim 1.
The recombinant VIGS vector having inserted PDS fragment according to Claim 4, characterized in that it is pSYMMT7-fullIC-PDS3 vector shown in FIG. 5 or pSYMM35S-fullIC-PDS3 vector shown in FIG. 10.
A method for functional analysis of a foreign gene comprising:

- inserting a foreign gene whose function is to be analyzed to the recombinant VIGS vector of Claim 1;

- digesting the recombinant VIGS vector having the inserted foreign gene and performing in vitro transcription to produce an RNA transcript;

- inoculating the RNA transcript into a plant for silencing of the foreign gene; and

- analyzing the phenotype of the plant having the silenced foreign gene.
The method according to Claim 6, characterized in that the foreign gene is inserted within the gene encoding the coat protein in an anti-sense direction.
The method according to Claim 6, characterized in that the plant is soybean.
A method for production of a plant having a silenced target gene comprising:

- inserting a foreign gene to be silenced to the recombinant VIGS vector of Claim 1;

- digesting the recombinant VIGS vector having the inserted foreign gene and performing in vitro transcription to produce an RNA transcript; and

- inoculating the RNA transcript into a plant for silencing of the foreign gene.
A plant having a silenced target gene produced by the method of Claim 9.
The plant according to Claim 10, characterized in that the plant is soybean.
Seeds of the plant according to Claim 10.
Combination of primers of SEQ ID NO: 1 to SEQ ID NO: 4 for amplifying SYMMV infectious clone comprising T7 promoter and full length SYMMV nucleotide sequence.