KR20230032679A - Potato virus X-based recombinant plant expression vector containing heterologous viral suppressor of RNA silencing and uses thereof - Google Patents

Abstract

The present invention relates to a recombinant plant expression vector to increase the expression of a target protein in plants and use thereof. According to the present invention, the recombinant plant expression vector comprises: an RNA-dependent RNA polymerase (RdRp) coding sequence derived from potato virus; a triple gene blocks (TGBs) loss-of-function mutant coding sequences; an expression cassette 1 containing multiple cloning site (MCS) for insertion of a target protein coding sequence and a C-terminal sequence of a coat protein (CP) generate; and an expression cassette 2 containing a viral suppressor of RNA silencing coding sequence.

Description

Potato virus X-based recombinant plant expression vector containing heterologous viral suppressor of RNA silencing and uses thereof}

The present invention relates to a Potato virus X-based recombinant plant expression vector containing a heterologous viral RNA silencing protein and a use thereof.

Plants as bioreactors offer attractive alternatives to traditional expression systems because they are relatively simple, inexpensive and can rapidly mass-produce substances. In addition, since plants are essentially free of human pathogens, proteins produced by plant systems are considered safe.

The use of plant viruses as recombinant vectors is because viruses have been specifically evolved to utilize small, autonomously replicating genomes to induce rapid, high titer production of foreign proteins of interest in plants. Recently, as a second generation of plant viral vectors, deconstructed viral vectors have been developed in which nonessential viral sequences such as capsid coding genes have been removed, and the system is transiently introduced into plant hosts by Agrobacterium. These features have alleviated biosafety concerns related to the spread of recombinant viruses.

Potato virus X (PVX) is a plus-stranded RNA virus that allows easy and stable introduction of relatively large foreign genes (over 1 kb), so it is used as a recombinant protein expression vector in plants. It is rarely used as a vector. This is because a new deconstructed viral vector, which has a higher expression rate of foreign genes than PVX and does not have viral pathogenicity, was developed.

On the other hand, Korean Patent Publication No. 2003-0004305 discloses 'a method of increasing the expression of a transgene by expressing it together with a post-transcriptional gene silencing (PTGS) inhibitor', and Korean Patent Publication No. 2018-0084680 discloses ' A recombinant vector for expressing a target protein in plant cells is disclosed, but there is no disclosure of a 'potato virus X-based recombinant plant expression vector containing an RNA silencing protein and its use' of the present invention.

The present invention was derived from the above needs, and the inventors of the present invention, in order to develop a novel expression vector based on Potato virus X (PVX) capable of rapidly and in large quantities expressing foreign target proteins in plants, PVX RNA The polymerase function was maintained, genes not necessary for the expression of the target protein (related to the pathogenicity of the PVX virus) were removed, and a heterologous viral suppressor of RNA silencing was introduced to create a novel PVX-based Recombinant plant expression vectors were developed. Then, by inserting a fluorescent protein sequence into the expression vector, significantly higher fluorescence expression was achieved compared to the original expression of the fluorescent protein by the PVX vector while changing the type of RNA silencing suppressor protein derived from a heterologous virus and the expression orientation in the vector. A novel PVX-based expression vector shown was completed.

In order to solve the above problems, the present invention is Potato virus X derived RdRp (RNA-dependent RNA polymerase) coding sequence; triple gene blocks (TGBs) loss-of-function mutant coding sequences; Expression cassette 1 containing a multiple cloning site (MCS) for insertion of a coding sequence for a target protein and the C-terminal sequence of a coat protein ( CP ) gene; and a viral suppressor of RNA silencing coding sequence. A recombinant plant expression vector for increasing the expression of a protein of interest in a plant comprising expression cassette 2 is provided.

In addition, the present invention comprises the steps of inserting the target protein coding sequence into the MCS of the recombinant plant expression vector; and introducing a recombinant plant expression vector into plant cells into which the target protein coding sequence is inserted.

In addition, the present invention is an RdRp coding sequence derived from Potato virus X; TGBs loss-of-function mutant coding sequence; C-terminal sequence of CP gene; and a recombinant vector containing a viral suppressor of RNA silencing coding sequence as an active ingredient, for producing a target protein in a plant.

The novel recombinant vector of the present invention significantly improved the expression level of the target protein in plant cells compared to the vector without introducing the viral suppressor of RNA silencing derived from a heterologous virus, thereby inducing Agrobacterium It can be usefully used for the production of a target protein using a transient expression method.

1 is a schematic diagram of a novel PVX recombinant expression vector developed through modification of a PVX vector containing a heterologous viral RNA silencing protein (viral suppressor of RNA silencing, VSR).
Figure 2 compares the expression of P1 and P2 in Figure 1 with P0, and confirms that the GFP expression of these P1 and P2 is further increased by the VSR (P19, TCV CP, NS) of heterologous viruses cloned into other vectors. am. Through this, it was confirmed that co-expression of the heterologous virus-derived VSR in the P2 vector without TGBs and CP, which are PVX's own VSR, is the combination that can obtain the highest target protein expression.
FIG. 3 is a result of confirming the GFP expression level with fluorescence images and gel-stained photographs after heterologous virus-derived VSRs (P19, NSs, TCV CP) were introduced into P2 of FIG. 1 to express them in a single vector. From this, it can be seen from FIG. 2 that when P19, which induces high GFP expression, is introduced into the P2 vector and expressed, GFP expression is rather suppressed. P2T, in which TCV CP was introduced into P2, had higher expression than P2, but compared to the results of FIG. 2, TCV CP showed lower GFP expression than in the case of a separate independent vector. In the case of P2N into which NSs were introduced, higher GFP expression was induced than in the condition of FIG. 2 .
Figure 4 analyzes the expression change of GFP by reversing the expression direction of heterologous virus-derived VSR in the P2 vector to the PVX RdRp expression direction, and the expression of GFP in P3N and P3T vectors in opposite directions of NSs and TCV CP It can be seen that it is higher than the GFP expression in P2N and P2T vectors.
Figure 5 is a schematic diagram of the vector expressing the antigen in P3N constructed in the present invention and the transient expression test results, confirming the protein expression level by Western blot. From this, it can be confirmed that the antigen expressed by the P3N vector is expressed with high efficiency.

In order to achieve the object of the present invention, the present invention is Potato virus X derived RdRp (RNA-dependent RNA polymerase) coding sequence; triple gene blocks (TGBs) loss-of-function mutant coding sequences; Expression cassette 1 containing a multiple cloning site (MCS) for insertion of a coding sequence for a target protein and the C-terminal sequence of a coat protein ( CP ) gene; and a viral suppressor of RNA silencing coding sequence. A recombinant plant expression vector for increasing the expression of a protein of interest in a plant comprising expression cassette 2 is provided.

In the recombinant plant expression vector of the present invention, the expression cassette 1 includes a promoter; RdRp coding sequence from Potato virus X; TGBs loss-of-function mutant coding sequence; CP promoter; MCS for insertion of the target protein coding sequence; C-terminal sequence of CP gene; and a terminator may be operably linked.

In the recombinant plant expression vector of the present invention, the TGB loss-of-function mutant refers to a mutant in which normal TGB function is lost due to deletion of some bases in the TGB coding sequence.

In the present invention, "expression cassette" refers to a set of DNA to enable the expression of a target protein to be temporarily expressed using Agrobacterium in a plant, and includes the following three main elements. : i) a promoter; ii) "coding polynucleotide", or "coding sequence (coding sequence), which is operably linked to the promoter and whose transcription is directed by the promoter when the expression cassette is introduced into a plant; a polynucleotide that may be referred to as "a gene"; and iii) a terminator polynucleotide (also referred to as a transcription terminator) that directs the termination of transcription and is immediately downstream of the polynucleotide.

The term “promoter” refers to a region of DNA upstream from a structural gene and to which RNA polymerase binds to initiate transcription. The promoters of the expression cassette 1 according to the present invention are promoters suitable for transformation, preferably CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter, and more preferably CaMV 35S promoter. It may be, but is not limited thereto. A "constitutive promoter" is a promoter that is active under most environmental conditions and states of development or cell differentiation. A constitutive promoter may be preferred in the present invention to enable constitutive expression in plants through transient expression.

In addition, the TGBs loss-of-function mutant coding sequence is a sequence in which the 252nd to 1,084th nucleotide sequence of the TGBs coding sequence consisting of the nucleotide sequence of SEQ ID NO: 1 is deleted, and the C-terminal sequence of the CP gene is the nucleotide sequence of SEQ ID NO: 2 It may be the 654th to 714th nucleotide sequence of the CP coding sequence consisting of, but is not limited thereto.

In addition, a conventional terminator may be used as the terminator, and examples thereof include a Nopaline synthase (NOS) terminator, a rice α-amylase RAmy1 A terminator, a phaseolin terminator, and Agrobacterium tumefaciens ( Agrobacterium tumefaciens ), but octopine synthase (Octopine synthase) terminator and the like, but is not limited thereto. Regarding the need for terminators, it is generally understood that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly preferred in the context of the present invention.

In the present invention, "operably linked" means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. That is, the gene encoding the target protein can be ligated so that its expression can be controlled by a promoter in the vector.

In the recombinant plant expression vector of the present invention, the MCS means a DNA fragment having a restriction site, which is a site that is recognized and cut by various restriction enzymes, and the MCS is recognized and cut by specific restriction enzymes. It enables the insertion of the target protein coding sequence at the site of the cleaved MCS. As the MCS, any MCS known to those skilled in the art may be used without limitation. The MCS can be added to a restriction enzyme site generally available in the art. When inserting a target protein coding sequence into the MCS of the expression cassette 1 of the present invention, a restriction enzyme site that does not exist in the plant virus gene can be used. Plant virus genes have different sequences from each other, so the site of the restriction enzyme in the MCS used may vary depending on the plant virus.

In addition, in the recombinant plant expression vector of the present invention, the expression cassette 2 is a promoter; RNA silencing protein coding sequence; and a terminator may be operably linked, and the promoter and terminator of expression cassette 2 are as described above.

In the recombinant plant expression vector according to an embodiment of the present invention, the RNA silencing protein may be TSWV (Tomato spotted wilt virus)-derived non-structural proteins (NSs) or TCV (Turnip crinkle virus)-derived CP, and TSWV The derived NSs may be encoded by the nucleotide sequence of SEQ ID NO: 3, and the CP derived from TCV may be encoded by the nucleotide sequence of SEQ ID NO: 4, but is not limited thereto.

In addition, in the recombinant plant expression vector of the present invention, the expression cassette 2 may be linked to the expression cassette 1 in a sense or antisense orientation.

The term "vector" is used to refer to DNA fragment(s), nucleic acid molecules, which are delivered into cells. Vectors replicate DNA and can reproduce independently in host cells. The term “delivery vehicle” is often used interchangeably with “vector”. The recombinant plant expression vector of the present invention is a 'recombinant viral vector', which refers to a process that is changed differently from conventional viral vectors through disassembly and reassembly of viral components. Recombinant viral vectors can express proteins or gene fragments that are not expressed through the components of conventional vectors, either in sense or antisense form.

A recombinant vector may preferably include one or more selectable markers, but is not limited thereto. The marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, bleomycin, hygromycin or chloramphenicol. There is a resistance gene, but is not limited thereto.

The present invention also includes the steps of inserting the target protein coding sequence into the MCS of the recombinant plant expression vector; and introducing a recombinant plant expression vector into plant cells into which the target protein coding sequence is inserted.

The method for producing a target protein in a plant according to the present invention may be to infect a plant using Agrobacterium containing a recombinant plant expression vector into which a coding sequence for the target protein is inserted, and produce the target protein through transient expression. , but not limited thereto.

In the method of the present invention, the recombinant plant expression vector is RdRp (RNA-dependent RNA polymerase) coding sequence derived from Potato virus X; triple gene blocks (TGBs) loss-of-function mutant coding sequences; Expression cassette 1 containing a multiple cloning site (MCS) for insertion of a coding sequence for a target protein and the C-terminal sequence of a coat protein ( CP ) gene; and a viral suppressor of RNA silencing coding sequence. As a recombinant vector containing expression cassette 2, details are as described above.

In the present invention, the target protein that can be inserted into the MCS has a biological activity selected from the group consisting of medical, research and industrial proteins, for example, enzymes, antigens, antibodies, cell receptors, structural proteins, serum and cell proteins. It may be various types of proteins having, but is not limited thereto.

Methods for introducing the recombinant vector of the present invention into plant host cells include microinjection (Capecchi, M.R., Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, F.L. et al., Virology, 52:456 (1973)). )), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, T.K. et al., Gene, 10:87 (1980)) , DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 ( 1990)) and Agrobacterium-mediated transient expression (Bevan M., Nucleic Acids Res. 12:8711-8721 (1984); Sainsbuty F., Lomonossoff G.P., Plant Physiolgy, 148:1212-1218 (2008)) et al. It may, but is not limited thereto.

A "plant cell" used for plant transformation can be any plant cell. A plant cell may be any type of cultured cell, cultured tissue, cultured organ or whole plant, preferably a cultured cell, cultured tissue or cultured organ and more preferably a cultured cell. "Plant tissue" is a differentiated or undifferentiated plant tissue, such as but not limited to fruit, stem, leaf, pollen, microspores, seeds, cancer tissue, and various types of cells used in culture, i.e. single cells. , including protoplasts, shoots and callus tissue. Plant tissue may be in planta or may be in organ culture, tissue culture or cell culture.

In the present invention, the plant is tobacco, Arabidopsis, potato, eggplant, pepper, tomato, burdock, crown daisy, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, water parsley, parsley, radish, Chinese cabbage, cabbage, mustard It may be a dicotyledonous plant such as watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, or pea, or a monocotyledonous plant such as rice, barley, wheat, rye, corn, sugar cane, oats, and onion, preferably It may be a cigarette, but is not limited thereto.

The present invention also, RdRp (RNA-dependent RNA polymerase) coding sequence derived from Potato virus X; triple gene blocks (TGBs) loss-of-function mutant coding sequences; C-terminal sequence of coat protein ( CP ) gene; and a recombinant vector containing a viral suppressor of RNA silencing coding sequence as an active ingredient, for producing a target protein in a plant. The composition according to the present invention contains as an active ingredient a recombinant vector comprising a viral suppressor of RNA silencing coding sequence capable of increasing the expression level of a target protein as an active ingredient. When a protein is cloned and transformed into a plant cell, the target protein can be produced in large quantities in the plant.

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

Materials and Methods

1. Transient expression protocol

Tobacco ( Nicotiana benthamiana ) plants aged 4 weeks (± 3 days) after sowing were used, and were grown under a photoperiod of 16 hours light / 8 hours dark and a temperature of 23 ± 2 ° C.

The Agrobacterium GV3101 strain to be used for transient expression was cultured in LB broth for one day at 28°C with a stirring speed of 180-220 rpm. The culture medium was then centrifuged at 5,000 rpm for 8 minutes to recover cell pellets. Agrobacterium cells were suspended in an infiltration buffer (10 mM MES (pH 5.6), 10 mM MgCl ₂ , 200 μM acetosyringone) and incubated at room temperature for 3 hours to activate the cells. Then, the concentration of the cell suspension was adjusted to 0.4 at OD ₆₀₀ . Tobacco leaves were inoculated with Agrobacterium using a disposable syringe without a needle. After inoculating the bacteria, the plants were not immediately transferred to the culture room, but maintained for half a day at a temperature of ≤20° C. and then transferred to the culture room. After 3, 5, and 7 days of Agrobacterium inoculation, GFP fluorescence was confirmed using a UV lamp in UVP MultiDoc-It, and then sampling was performed. Leaf discs (1 cm in diameter) from different tobacco plants were sampled in triplicate tubes. The sampled tube was immediately put into liquid nitrogen to freeze the leaves, and the leaf samples were stored in a cryogenic freezer.

2. Western blot

Samples stored in a cryogenic freezer were transferred to liquid nitrogen, and three tobacco leaf disks were pulverized in a tube using a pestle to prevent the samples from melting. After adding 100 to 150 μl of NP-40 lysis buffer, the leaf samples were completely ground using a pestle motor (Kontes™).

Composition of NP-40 lysis buffer ingredient density Tris-HCl (pH 8.0) 50 mM NaCl 150 mM EDTA 0.5 mM NP-40 0.5% Protease inhibitor cocktail (11836153001 Roche) 1 tablet per 10ml

Thereafter, the reaction was performed on ice for 30 minutes, and only the supernatant was separated by centrifugation at 4° C. and 13,000 rpm for 20 minutes. The separated supernatant was mixed with 5X Sample buffer, denatured by boiling at 100 ° C for 5 to 10 minutes, and then cooled on ice.

Composition of 5X Sample buffer ingredient density Tris-HCl (pH 6.8) 60 mM Glycerol 25% SDS 2 % 2-mercaptoethanol 14.4 mM Bromophenol blue 0.1%

After loading 20-30 μg of protein on a 12% SDS PAGE gel, electrophoresis was performed at 65-120V for 2-3 hours. For protein transfer, the PVDF membrane was immersed in methanol for 5 minutes to activate it, and then protein transfer was performed at 200 mA for 2 hours by a wet transfer method. The membrane was stained with Ponceau S reagent to determine whether the protein was translocated. Then, staining of the membrane was completely removed with PBS-T buffer. After putting the membrane in blocking buffer (7% skim milk in PBS-T) and reacting at room temperature for 1 hour, washing the membrane with PBS-T, GFP antibody (sc-9996, Santa Cruz) was applied at 1:1,000 or 1: After treatment at a concentration of 2,000, it was reacted at 4°C for one day. After the reaction, the process of washing the membrane with PBS-T for 10 minutes was repeated three times, and the goat anti-mouse HRP (ab6789, Abcam) antibody was reacted at a ratio of 1:10,000 at room temperature for 1 hour. The process of washing the membrane with PBS-T for 10 minutes was repeated three times, and protein expression was confirmed using ECL (P65406271, Amersham ECL Prime) reagent.

Example 1. Construction of PVX-based plant expression vector for high expression of target protein

1-1. Removal of internal elements of PVX

The original PVX vector used in the present invention is the same as NovoPro's V005901 product. In order to develop a new viral vector with a combination of PVX and RNA silencing suppressor protein (VSR) of heterologous viruses, first, among the PVX viral element proteins, the envelope protein (CP) coding gene, which is known not to be involved in the expression of external proteins, and compared to other viruses A vector was prepared in which the TGBs (triple gene blocks) protein coding gene of PVX, which is known to have a relatively weak VSR function, was deleted. The CP gene deletion refers to a deletion of the 1st to 653rd base sequence of the CP coding sequence (SEQ ID NO: 2), and the TGBs deficiency is a form in which the 252nd to 1,084th base sequence of the TGBs coding sequence (SEQ ID NO: 1) is deleted. means After preparing each vector, a gene encoding the marker protein GFP was inserted, and changes in GFP expression were confirmed by fluorescence and Western blotting.

1-1-1) P1: PVX CP defect

SalI (G ^▼ TCGA _▲ C, cat.# R3138, NEB) in the MCS (multiple cloning site) of the original PVX vector and XhoI (C ^▼ TCGA _▲ G, cat.# R0146, NEB) restriction enzyme sites in the CP After cleaving the 1st to 653rd nucleotide sequences of the CP using T4 DNA Ligase (M0202, NEB), the P1 vector was constructed by attaching the cut surface. By disassembling and recombination, 653 bases of the CP gene were deleted, and the remaining 61 base sequences exist as non-coding sequences that cannot be translated into proteins.

1-1-2) P2: PVX CP, TGB defect

CP promoter and MCS synthesized through PCR after cleaving the 253-833 nucleotide sequence of TGB using XhoI in CP of the original PVX vector and BstZ17I (GTAːTAC, cat.# R3594, NEB) restriction enzyme site in TGB. was combined with the vector cut by In-Fusion ^® HD Cloning Kit (TaKaRa) to construct a P2 vector. The remaining 267nt sequence from the TGB coding sequence through in-fusion cloning does not function as a TGB.

As a result, it was observed that GFP expression was increased in P1 (CP deficient PVX vector) compared to P0 (original PVX vector) (FIG. 2A). In the case of the PVX vector (P2) deficient in both CP and TGBs, it was confirmed that the GFP expression level was significantly reduced compared to P0.

1-2. Introduction of heterologous virus VSR genes

The present inventors mixed the P0, P1 or P2 vectors and Agrobacterium strains each containing vectors containing heterologous virus-derived VSRs regulated by the 35S promoter at a 1:1 ratio, inoculated tobacco leaves, and then inoculated the GFP Expression levels were observed. The heterologous virus-derived VSRs are as follows: TBSV (Tomato bushy stunt virus)-derived P19, TSWV (Tomato spotted wilt virus)-derived NSs, or TCV (Turnip crinkle virus)-derived CP.

As a result, as shown in FIG. 2B, it was confirmed that the expression of GFP was increased in the experimental group inoculated with the vector containing each VSR compared to the experimental group inoculated with the PVX vector alone (P0, P1, and P2) (FIG. 2B). .

Based on the above results, the present inventors constructed new constructs by introducing heterologous virus-derived VSRs into the P2 vector (P2N and P2T in FIG. 1) and analyzed the GFP expression levels. As a result, as shown in FIG. 3, it was confirmed that GFP fluorescence was excellent in the vector (P2N and P2T, respectively) into which TSWV NSs or TCV CP was introduced into P2, respectively. In particular, the P2N vector introduced with TSWV NSs showed high GFP expression, similar to the mixed treatment condition of the individual vector containing VSR and the P2 vector, and the P2T vector introduced with TCV CP was confirmed under the mixed treatment condition of the VSR vector and P2 vector. showed lower fluorescence. However, in the case of P2P19, a construct in which TBSV-derived P19 was introduced into P2, the opposite result was obtained in which GFP expression was greatly reduced (FIG. 3). This shows that PVX-based protein expression varies greatly depending on which heterologous virus VSR is introduced.

1-3. Changes in the intra-vector transcriptional direction of TSWV NSs and TCV CP

The inventors cloned the introduced VSRs in the opposite direction to that of the new PVX vector (P2) and examined the effect of increasing GFP expression. As a result, it was observed that when TSWV NSs or TCV CP were cloned in the reverse direction of transcription of the PVX vector (P3N, P3T, respectively), GFP expression was significantly enhanced than when cloned in the same direction (P2N, P2T) ( Fig. 4).

Example 2. Verification of protein expression in plants of the vector developed through the expression of the target protein

Clonorchiasis is a common endemic disease in Asian countries such as China, Korea, and Vietnam, and about 35 million people in the region are known to be infected with Clonorchis sinensis. The development of a rapid diagnostic kit is required to easily check the infection of liver flukes in the field. For this purpose, the liver fluke fusion antigen gene (hereinafter Ag) was introduced into the P3N vector and expression was confirmed by western blotting in plants (FIG. 5).

<110> Korea Research Institute of Bioscience and Biotechnology BioApplications Inc. <120> Potato virus X-based recombinant plant expression vector containing heterologous viral suppressor of RNA silencing and uses it <130> PN21184 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 1100 <212> DNA <213> Potato virus x <400> 1 atggatattc tcatcagtag tttgaaaagt ttaggttat ctaggacttc caaatcttta 60 gattcaggac ctttggtagt acatgcagta gccggagccg gtaagtccac agccctaagg 120 aagttgatcc tcagacaccc aacattcacc gtgcatacac tcggtgtccc tgacaaggtg 180 agtatcagaa ctagaggcat acagaagcca ggacctattc ctgagggcaa cttcgcaatc 240 ctcgatgagt atactttgga caacaccaca aggaactcta accaggcact ttttgctgac 300 ccttatcagg caccggagtt tagcctagag ccccacttct acttggaaac atcatttcga 360 gttccgagga aagtggcaga tttgatagct ggctgtggct tcgatttcga gaccaactca 420 ccggaagaag ggcacttaga gatcactggc atattcaaag ggcccctact cggaaaggtg 480 atagccattg atgaggagtc tgagacaaca ctgtccaggc atggtgttga gtttgttaag 540 ccctgccaag tgacgggact tgagttcaaa gtagtcacta ttgtgtctgc cgcaccaata 600 gaggaaattg gccagtccac agctttctac aacgctatca ccaggtcaaa gggattgaca 660 tatgtccgcg cagggccata ggctgaccgc tccggtcaat tctgaaaaag tgtacatagt 720 attaggtcta tcatttgctt tagttcaat tacctttctg ctttctagaa atagcttacc 780 ccacgtcggt gacaacattc acagcttgcc acacggagga gcttacagag acggcaccaa 840 agcaatcttg tacaactccc caaatctagg gtcacgagtg agtctacaca acggaaagaa 900 cgcagcattt gctgccgttt tgctactgac tttgctgatc tatggaagta aatacatatc 960 tcaacgcaat catacttgtg cttgtggtaa caatcatagc agtcattagc acttccttag 1020 tgaggactga accttgtgtc atcaagatta ctggggaatc aatcacagtg ttggcttgca 1080 aactagatgc agaaaccata 1100 <210> 2 <211> 714 <212> DNA <213> Potato virus x <400> 2 atgtcagcac cagctagcac aacacagccc atagggtcaa ctacctcaac taccacaaaa 60 actgcaggcg caactcctgc cacagcttca ggcctgttca ccatcccgga tggggatttc 120 tttagtacag cccgtgccat agtagccagc aatgctgtcg caacaaatga ggacctcagc 180 aagattgagg ctatttggaa ggacatgaag gtgcccacag acactatggc acaggctgct 240 tgggacttag tcagacactg tgctgatgta ggatcatccg ctcaaacaga aatgatagat 300 acaggtccct attccaacgg catcagcaga gctagactgg cagcagcaat taaagaggtg 360 tgcacactta ggcaattttg catgaagtat gctccagtgg tatggaactg gatgttaact 420 aacaacagtc cacctgctaa ctggcaagca caaggtttca agcctgagca caaattcgct 480 gcattcgact tcttcaatgg agtcaccaac ccagctgcca tcatgcccaa agaggggctc 540 atccggccac cgtctgaagc tgaaatgaat gctgcccaaa ctgctgcctt tgtgaagatt 600 acaaaggcca gggcacaatc caacgacttt gccagcctag atgcagctgt cactcgaggt 660 cgtatcactg gaacaacaac cgctgaggct gttgtcactc taccaccacc ataa 714 <210> 3 <211> 1380 <212> DNA <213> Tomato spotted wilt virus <400> 3 atgagcaccg ccaagatgag cgctggtgag ttctctaaga cctacggcac caaggatagc 60 cggtctgtga atgattgcta cgccatctac agcggcaacg gcatcaactt catcaacctg 120 ttcatgcaca ccaacaccgg catcaagagc gccttctcta ttaacgacct tggccggaac 180 gaggacatta agcttcatga ggccgagatc atcaacagct tgcacccgta ctcattcttc 240 gacaagttcg gcctggatat catcctgtgc aaccacgtga tggacatcat cgtgactaag 300 cctggcctta agaacaccgg atgcaagttc cagatgcaca accagatctt caacccgaat 360 gaggatattc tggctaagac ccctggcatc atcagcgaag agaacttcta cgacctgagc 420 aagatcaagc ctaagggcat gacacctttc gactggtaca tcaacgagtg caagaagtac 480 gacttctaca tcagcgacaa cggcgacatc tctctggatt acggtttccc tgtgatgggc 540 aagaccacct cttattggag agagaacatc ccgcgagaga agatcatctc cgttaagcag 600 aagtgcctgc cgaacacctc tgctcttacc aacaggattc tgagcctttc taccgtgagg 660 gctatccaga ttgcttctga gctggcttct gaaaagaccg tggttcttgc ttctaggcag 720 aggctggatc tggacatcaa gtctcagtac cggatctcat tccctggtgt tcaagatgag 780 ggcgctttca ctaggacttt ctgcatccct atggacaaga ccgctaggat cgtttgcttc 840 tacgctaaga cctccgtgga cgtgtcaaat gagaggacca ccttgaccat caagatcgtc 900 aacaagaccg tcgagagcaa ctacatgggt cctgtgccta aggatcacat gtactgcgat 960 aagagcatcg gcgctaggat tggtcttgtg gatattgtga ggggcgaccc gaactacaat 1020 cagatgattg ctcgggaaat gatcagcgtc cacaccaact ttgctctgag gctttctgag 1080 gctctgaaga agccggtcat cgttttcaag atgtacgaga aagagctgaa cttcgagact 1140 tacgacctgt ccggtcggtc tttgtcttac cagaaggaca gcagcggcaa tatctacttc 1200 ctgtctagga cccttgagat cctgccgaag tctctttcta ccctgaccta cctgaagaac 1260 atctctcctg cctgctggaa agaatccatc agcatgcagc atttctacat cggggagcaa 1320 gaagagggac cttctggttc taatctggcc gagtctgaag agaaggtgca gactgcttag 1380 1380 <210> 4 <211> 1056 <212> DNA <213> Turnip crinkle virus <400> 4 atggaaaatg atcctagagt ccggaagttc gcatctgatg gcgcccaatg ggcgataaag 60 tggcagaaga agggctggtc aaccctaacc agcagacaga aacagaccgc ccgcgcagcg 120 atggggatca agctctctcc tgtggcgcaa cctgtgcaga aagtgactcg actgagtgct 180 ccggtggccc ttgcctaccg cgaggttacc acccagcctc gggtctctac tgccagggac 240 ggcataacca gaagcggttc tgaactgatc acaaccttga agaagaacac tgacactgaa 300 cctaagtaca ccacagctgt gcttaaccca agcgaacccg gaacattcaa ccagctcatt 360 aaggaggcgg cccagtatga aaaataccga ttcacgtcac tcagatttag gtactccccc 420 atgagccctt caaccaccgg aggcaaggtg gctctggcat tcgatcgaga tgccgccaaa 480 cctccgccca acgacctcgc ttccctctac aacatagagg gttgtgtatc tagcgtgccc 540 tggacagggt ttattttgac cgtcccaaca gattctactg accgctttgt ggcggatggt 600 atcagcgatc caaagcttgt cgatttcggc aagctcatca tggccaccta cggccaagga 660 gccaatgatg ccgcccaact cggtgaagtg cgagtcgagt acaccgtgca gctcaagaac 720 agaactggct caaccagcga cgcccagatt ggggacttcg caggtgttaa ggacggaccc 780 aggctggttt catggtccaa gaccaagggg acagctgggt gggagcacga ttgtcatttt 840 ctcggaaccg gaaacttctc gttgacattg ttctacgaga aggcgccggt ctcggggcta 900 gaaaacgcag acgcctctga cttctcggtc ctgggagaag ccgcagcagg tagtgtccaa 960 tgggcaggag tgaaggtagc agaaagggga caaggcgtga aaatggtcac aactgaggag 1020 cagccaaagg gtaaatggca agcactcaga atttag 1056

Claims (9)

Potato virus X-derived RdRp (RNA-dependent RNA polymerase) coding sequence; triple gene blocks (TGBs) loss-of-function mutant coding sequences; Expression cassette 1 containing a multiple cloning site (MCS) for insertion of a coding sequence for a target protein and the C-terminal sequence of a coat protein ( CP ) gene; and a viral suppressor of RNA silencing coding sequence. A recombinant plant expression vector for increasing the expression of a protein of interest in a plant, comprising expression cassette 2. According to claim 1, wherein the expression cassette 1, a promoter; RdRp coding sequence from Potato virus X; TGBs loss-of-function mutant coding sequence; CP promoter; MCS for insertion of the target protein coding sequence; C-terminal sequence of CP gene; and a terminator operably linked. The method of claim 2, wherein the TGBs loss-of-function mutant coding sequence is a sequence in which the 252nd to 1,084th nucleotide sequence of the TGBs coding sequence consisting of the nucleotide sequence of SEQ ID NO: 1 is deleted, and the C-terminal sequence of the CP gene is SEQ ID NO: A recombinant plant expression vector, characterized in that it is the 654th to 714th nucleotide sequence of the CP coding sequence consisting of the nucleotide sequence of 2. According to claim 1, wherein the expression cassette 2, a promoter; RNA silencing protein coding sequence; and a terminator operably linked. The recombinant plant expression vector according to claim 4, wherein the RNA silencing suppressor protein is a non-structural protein (NSs) derived from tomato spotted wilt virus (TSWV) or a coat protein (CP) derived from turnip crinkle virus (TCV). The recombinant plant expression vector according to claim 5, wherein the TSWV-derived NSs is encoded by the nucleotide sequence of SEQ ID NO: 3, and the TCV-derived CP is encoded by the nucleotide sequence of SEQ ID NO: 4. The recombinant plant expression vector according to claim 1, wherein expression cassette 2 is linked to expression cassette 1 in a sense or antisense direction. Inserting a target protein coding sequence into the MCS of the recombinant plant expression vector according to any one of claims 1 to 7; and
A method for producing a target protein in a plant comprising introducing a recombinant plant expression vector into a plant cell into which the target protein coding sequence is inserted. Potato virus X-derived RdRp (RNA-dependent RNA polymerase) coding sequence; triple gene blocks (TGBs) loss-of-function mutant coding sequences; C-terminal sequence of coat protein ( CP ) gene; and a recombinant vector containing a viral suppressor of RNA silencing coding sequence as an active ingredient, for production of a target protein in a plant.

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