JP5266489B2 - Gene silencing suppression technology - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for constructing an expression vector in a good efficiency by shortening a gene region encoding a gene-silencing inhibitory protein. <P>SOLUTION: (a) This nucleic acid sequence encoding a water melon green spot mosaic virus 129K protein or the fragment of a nucleic acid sequence encoding a corresponding protein to the above and containing at least a specific sequence, (b) the nucleic acid sequence containing one or several substitutions, additions or deletions to the sequence (a), or (c) a nucleic acid containing a nucleic acid sequence hybridizing with the nucleic acid having the sequence (a) under a stringent condition and encoding a product having a function of inhibiting the gene-silencing on its expression are provided. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to a gene silencing technique (particularly, plant gene silencing). Specifically, the present invention relates to a technique for suppressing gene silencing.

  RNA silencing has become an obstacle in the technology for obtaining large amounts of useful proteins and substances by introducing foreign genes into eukaryotes. For this reason, when silencing occurs in a gene-introduced organism, a silencing suppression protein may be used, and some of them are still used (Non-Patent Document 1 and Non-Patent Document 2).

  Conventionally, the silencing suppression technique uses a full-length protein and does not use only a short part. For example, tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) belonging to Tobamovirus have been used as full-length silencing suppression proteins (Non-Patent Document 3 and Non-Patent Document 4).

However, when the full-length protein is used for silencing suppression technology, the gene region to be encoded becomes long, so there is a problem that it takes time to construct an expression vector.
Atsushi Takeda, Kazuyuki Mise, Tetsuro Kanno “Plant Virus and RNAi”, Chemistry and Biology. 43 (7); 468-475, 2005 Vonet O, Rivas S, Mestre P, Baulcombe D. et al. “An enhanced transgenic expression system in plants based on suppression of the gene silencing by the p19 protein of tomato bush. 2003 Mar; 33 (5): 949-56. Kubota et al. “Tomato mosaic virus replication protein supplements virus-targeted posttranslational gene silencing”, Journal of Virology 2003 Oct; Ding et al. “The Tobacco mosaic virus 126-kDa Protein Associated with Virus Replication and Movement Suppresses RNA Silencing”, MPMI Vol. 17, no. 6, 2004, 583-592

As described above, conventionally, a full-length silencing suppression protein has been used to suppress silencing. This method is not an efficient method because the gene region encoding the silencing suppressor protein becomes long and it takes time to construct an expression vector.
In the technology for obtaining a large amount of useful proteins and substances by introducing foreign genes into eukaryotes, RNA silencing occurs and becomes an obstacle, and this suppressor protein is sometimes used. However, since the silencing suppressor protein is a full-length protein, the encoded gene region becomes long, and it takes time to construct an expression vector. The problem is to find a method for solving this problem.

  We are not the full-length 129K protein of watermelon green spot mosaic virus (CGMMV SH strain) (Ugaki et al. Journal of General Virology 1991, 72: 1487-1495), but only a short protein with a methyltransferase domain The above problem was solved by finding that silencing can be suppressed (high expression of a gene) simply by expressing. Thus, the above-described problems were solved by finding a method for efficiently constructing an expression vector by shortening the gene region encoded by the silencing suppressor protein.

  Therefore, the present invention also provides a technique for suppressing silencing of a gene that becomes an obstacle in producing a large amount of a useful protein or useful substance in an organism by gene transfer.

  Thus, since it was found that silencing is suppressed even when a part of silencing suppression protein is used, genes that interfere with mass production of useful proteins or useful substances in organisms by gene transfer Helps reduce silencing.

In order to achieve the above object, the present invention provides, for example, the following means.
(Item 1)
(A) a nucleic acid sequence shown in SEQ ID NO: 1 encoding a watermelon green spot mosaic virus 129K protein or a fragment of a nucleic acid sequence encoding a protein corresponding thereto, the sequence shown in at least positions 1 to 912 of SEQ ID NO: 1 or A nucleic acid sequence comprising the corresponding sequence;
(B) a nucleic acid sequence comprising one or several substitutions, additions or deletions to the sequence of (a); or (c) hybridizing under stringent conditions to a nucleic acid having the sequence of (a) And a nucleic acid encoding a product having a function of suppressing gene silencing when expressed.
(Item 2)
2. The nucleic acid according to item 1, wherein when the fragment is longer than 1440 nucleotides, the amino acid encoded by nucleotides 1438 to 1440 of SEQ ID NO: 1 or the corresponding amino acid is glutamine.
(Item 3)
24. The nucleic acid according to item 1, wherein the corresponding protein consists of the amino acid sequence shown in any one of SEQ ID NOs: 10 to 23.
(Item 4)
The nucleic acid according to item 1, comprising the nucleic acid sequence shown in SEQ ID NO: 3.
(Item 5)
The nucleic acid according to item 1, consisting of the nucleic acid sequence shown in SEQ ID NO: 3.
(Item 6)
The nucleic acid according to item 1, wherein the nucleic acid is shorter than the full length (SEQ ID NO: 1) of the 129K protein of watermelon green spot mosaic virus (CGMMV).
(Item 7)
An isolated nucleic acid construct comprising the nucleic acid of item 1.
(Item 8)
The nucleic acid construct according to item 7, further comprising a promoter sequence.
(Item 9)
Item 9. The nucleic acid construct according to Item 8, wherein the promoter sequence is derived from a promoter selected from the group consisting of a 35S promoter and a ubiquitin promoter.
(Item 10)
The nucleic acid construct according to item 7, further comprising a terminator sequence.
(Item 11)
Item 11. The nucleic acid construct according to Item 10, wherein the terminator sequence is derived from a terminator selected from the group consisting of a Nos terminator and a 35S terminator.
(Item 12)
8. The nucleic acid construct according to item 7, further comprising a 35S promoter sequence and a Nos terminator sequence.
(Item 13)
A method for producing a nucleic acid construct comprising:
A) providing a nucleic acid construct in which a promoter is operably linked to the nucleic acid according to item 1;
B) transforming an organism with the nucleic acid construct; and C) selecting a transformed gene silencing-suppressed organism for the transformed organism.
(Item 14)
A vector comprising the nucleic acid according to item 1.
(Item 15)
Item 15. The vector according to Item 14, further comprising a promoter sequence.
(Item 16)
Item 15. The vector according to Item 14, further comprising a terminator sequence.
(Item 17)
An organism comprising the nucleic acid according to item 1.
(Item 18)
The organism according to item 17, wherein the organism is a plant.
(Item 19)
A method for suppressing gene silencing caused by gene transfer, which comprises the following:
a) providing a nucleic acid construct comprising the nucleic acid of item 1; and b) transforming an organism in which the gene silencing has occurred with the nucleic acid construct.
(Item 20)
A kit for suppressing gene silencing caused by gene transfer, the kit comprising:
The nucleic acid according to item 1;
Means for introducing the nucleic acid into the organism in which the gene silencing has occurred;
A kit comprising:
(Item 21)
A method for producing a polypeptide of a foreign gene in an organism, wherein gene silencing occurs in the organism during the production,
a) introducing a nucleic acid molecule encoding the foreign gene into the organism;
b) providing a nucleic acid construct comprising the nucleic acid of item 1;
c) transforming the organism with the nucleic acid construct; and d) subjecting the organism to conditions under which the polypeptide is expressed,
Including the method.

  According to the present invention, silencing can be suppressed (that is, high gene expression) by expressing only a short protein having a methyltransferase domain, not a full-length 129K protein.

  The present invention has also achieved the suppression of gene silencing, which is an obstacle in producing large amounts of useful proteins or substances in living organisms by gene transfer. This provides a technique that leads to expression of a desired protein or substance in large quantities by an organism that constantly expresses a silenced gene.

  The present invention will be described below with reference to the best mode. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the above field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  Hereinafter, definitions of terms particularly used in the present specification will be described as appropriate.

  As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a nucleotide polymer of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. A “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2′-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphate in an oligonucleotide. Derivative oligonucleotide in which diester bond is converted to N3′-P5 ′ phosphoramidate bond, derivative oligonucleotide in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide Oligonucleotide substituted with C-5 propynyluracil, derivative oligonucleotide substituted with C-5 thiazo-ruuracil in uracil in oligonucleotide, cytosine in oligonucleotide is C-5 propynylcytosine Substituted derivative oligonucleotides, cytosines in oligonucleotides substituted with phenoxazine-modified cytosine, ribose in DNA replaced with 2'-O-propyl ribose Examples include derivative oligonucleotides and derivative oligonucleotides in which the ribose in the oligonucleotide is substituted with 2'-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-). 98 (1994)).

  As used herein, “nucleic acid molecule” is also used interchangeably with nucleic acid, oligonucleotide, and polynucleotide, and includes cDNA, mRNA, genomic DNA, and the like. As used herein, nucleic acids and nucleic acid molecules can be included within the concept of the term “gene”. Nucleic acid molecules encoding certain gene sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition.

  As used herein, an “isolated” biological factor (eg, a nucleic acid or protein, etc.) refers to other biological factors within the cells of the organism in which it is naturally occurring (eg, When it is a nucleic acid, it is substantially separated from a non-nucleic acid and a nucleic acid containing a nucleic acid sequence other than the target nucleic acid; Or it is purified. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

  As used herein, a “purified” biological agent (such as a nucleic acid or protein) refers to a product from which at least part of the factors naturally associated with the biological agent has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged on a chromosome in a certain order, but extrachromosomal ones fall within the category of genes as long as they define the inheritance. It is called a structural gene that defines the primary structure of a protein, and a regulatory gene that affects its expression.

  As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein” “polypeptide”, “oligopeptide” and “peptide”. As used herein, “gene product” also refers to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide” expressed by a gene. " A person skilled in the art can understand what a gene product is, depending on the situation.

  As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, if there is a conservative substitution, identity and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, identity and similarity indicate the same numerical value.

  As used herein, “stringent hybridization conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, polynucleotides that hybridize under stringent conditions are hybridized at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA is immobilized. Then, a 0.1- to 2-fold concentration of SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride and 15 mM sodium citrate) under the conditions of 65 ° C. It means a polynucleotide that can be identified by washing the filter. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. The “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, the hybridizable polynucleotide is a polynucleotide having at least 60% homology with the base sequence of DNA encoding a polypeptide having the amino acid sequence specifically shown in the present invention, preferably 80% The polynucleotide which has the above homology, More preferably, the polynucleotide which has 95% or more of homology can be mentioned.

  In this specification, comparison of base sequence similarity, identity and homology is performed using NCBI BLAST 2.2.9 (issued 2004.5.12), which is a tool for sequence analysis, as a default parameter. Is calculated using In the present specification, the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.

  As used herein, the term “foreign gene” refers to a gene that does not exist naturally in an organism. Such a foreign gene may be a modified gene that exists naturally in the organism, may be a gene that naturally exists in another plant, or may be a gene that is artificially synthesized. It may be a complex (for example, a fusion) thereof. Organisms containing such foreign genes can express gene products that are not naturally expressed.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. etal. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  In the present specification, the “foreign gene” may be any gene as long as it can be expressed in an organism. Accordingly, in one embodiment, the “foreign gene” may be anything that encodes a useful protein that is intended to be expressed in large quantities, and such is also the present invention. It is included in the range. Examples of such foreign genes include pharmaceutically active peptides (eg, cytokines (interleukins, chemokines, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF)). Hematopoietic factors such as granulocyte colony stimulating factor (G-CSF), multi-CSF (IL-3), erythropoietin (EPO), leukemia inhibitory factor (LIF), c-kit ligand (SCF), tumor necrosis factor, Inter-ferons, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), etc.), hormones (Insulin, growth hormone, thyroid hormone, etc.)), vaccine antigens, blood products, agriculture Peptides useful in industry, such as antibacterial proteins, various enzymes and hydrolases that synthesize secondary metabolites with physiological and pharmacological effects, inhibitors that regulate enzymatic reactions, soybean glycinin, which is said to have blood pressure effects, Another example is an artificial protein designed so that a bioactive peptide is cut out by enzymatic degradation in the digestive tract, but is not limited thereto. Examples of nutritionally significant substances include, but are not limited to, casein, leguminous albumins and globulins, and vitamins, sugars and lipid synthases. Furthermore, as a protein involved in processing characteristics as raw materials for various processed foods, for example, wheat glutenin (baking), soybean globulin group (tofu), milk casein group (cheese), etc., and to enhance the palatability and functionality of food Enzymatic digestion of proteins such as cyclodextrins, oligosaccharides, special sugar and amino acid synthases such as γ-aminoacetic acid, pigment synthases that improve the appearance and proteins involved in taste component synthesis, or digestive tracts An artificial protein or the like designed so that a peptide having a physiological action (for example, an angiotensin converting enzyme inhibitory peptide having a blood pressure effect action, etc.) can be cut out by being received is not limited thereto.

  In the present specification, “expression” of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form, but transcription to produce mRNA can also be a form of expression. More preferably, such polypeptide forms may be post-translationally processed.

  Therefore, in the present specification, “reduction” of “expression” of genes, polynucleotides, polypeptides, etc. means that the amount of expression is significantly reduced when the factor of the present invention is applied, rather than when it is not applied. That means. Preferably, the decrease in expression includes a decrease in the expression level of the polypeptide. More specifically, a decrease in the amount of expression refers to a decrease in expression after action of at least about 10% compared to before action of the factor, more preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 75%, more preferably at least about 90%. In this specification, “increase” in “expression” of a gene, polynucleotide, polypeptide, etc. means that the amount of expression increases significantly when the factor of the present invention is applied, rather than when it is not. Say. Preferably, the increase in expression includes an increase in the expression level of the polypeptide. More specifically, increasing the amount of expression refers to an increase in expression after action of at least about 10% compared to before action of the factor, more preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 75%, more preferably at least about 90%, increased or expressed prior to action It means that something that did not exist will be expressed.

  In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art. The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention. The term “unnatural amino acid” refers to an amino acid that is not normally found in proteins in nature. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine. “Amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code.

  As used herein, a “corresponding” amino acid or nucleic acid has or has the same action as a predetermined amino acid or nucleotide in a reference polypeptide or polynucleotide in a polypeptide molecule or polynucleotide molecule. In particular, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes similarly to the catalytic activity. For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain.

  As used herein, a “corresponding” gene (eg, a polypeptide molecule or a polynucleotide molecule) has or is expected to have the same effect as a given gene in a species that is the basis for comparison in a species. A gene in another species, often a region where a particular amino acid sequence is highly conserved. Such a feature is that they evolutionarily have the same ancestor, and the corresponding gene of a gene can be an ortholog of that gene. Corresponding genes include cognate genes. Corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in an organism can be found by searching the sequence database of that organism using the sequence of the gene serving as a reference for the corresponding gene as a query sequence.

  In the present invention, a gene corresponding to methyltransferase in the full length of the 129K protein of watermelon green spot mosaic virus (CGMMV) can be exemplified as shown in FIG. In FIG. 5, Tobacco mosaic virus Korean (TMV-KR), Tobacco mosaic virus Rakkyo strain (TMV-RAK), Tobacco mosaic virMuirVir (TMV), green mosaic virus (TMGMV), Tobacco mosaic virus Ob strain (TMV-OB), Odontoglossum ringspot virus (ORSV), Turn venin clearing virus (TVCbm, VCR) inese rape mosaic virus (CRMV), Tobacco mosaic virus cg Ltd. (TMV-CG), Cucumber green mottle mosaic virus (CGMMV), Sun-hemp mosaic virus (SHMV) is shown.

  In the present specification, the “nucleotide” may be natural or non-natural. A “derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include phosphorothioate, phosphoramidate, methyl phosphonate, chiral methyl phosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA). However, it is not limited to these.

  In the present specification, the “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above. Alternatively, the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.

  As used herein, “biological activity” refers to an activity that a certain factor (eg, polypeptide or protein) may have in vivo, and includes an activity that exhibits various functions. For example, when a certain factor is an antisense molecule, its biological activity includes binding to a nucleic acid molecule of interest, thereby suppressing expression. For example, when a factor is an enzyme, the biological activity includes the enzyme activity. In another example, when an agent is a ligand, the ligand includes binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art.

  As used herein, “antisense activity” refers to an activity that can specifically suppress or reduce the expression of a target gene. More specifically, depending on a certain nucleotide sequence introduced into the cell, an activity that can reduce the protein expression level by specifically reducing the mRNA level of a gene having a nucleotide sequence region complementary to that sequence. Say. As a technique, there are a method of directly introducing an RNA molecule complementary to mRNA produced from a target gene into a cell, and a method of introducing a construction vector capable of expressing RNA complementary to a target gene in the cell. Although broadly divided, the latter is more common in plants.

  Antisense activity is usually achieved by a nucleic acid sequence of at least 8 contiguous nucleotides that is complementary to the nucleic acid sequence of the gene of interest. Such a nucleic acid sequence is preferably at least 9 contiguous nucleotides long, more preferably 10 contiguous nucleotides long, even more preferably 11 contiguous nucleotides long, 12 contiguous nucleotides long, 13 14 contiguous nucleotide lengths, 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 20 contiguous nucleotide lengths, 25 contiguous nucleotide lengths, 30 contiguous nucleotide lengths, 40 contiguous nucleotide lengths It can be a nucleic acid sequence that is 50 contiguous nucleotides in length. Such nucleic acid sequences include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, 95% homologous to the sequences described above. Such antisense activity is preferably complementary to a sequence at the 5 'end of the nucleic acid sequence of the gene of interest. Such antisense nucleic acid sequences also include those having one, several or one or more nucleotide substitutions, additions and / or deletions relative to the sequences described above. Therefore, as used herein, “antisense activity” includes, but is not limited to, a decrease in gene expression.

  General antisense technology is described in textbooks (Murray, JAH eds., Antisense RNA and DNA, Wiley-Liss Inc, 1992). Furthermore, the latest research revealed a phenomenon called RNA interference (RNAi), which led to the development of antisense technology. When RNAi introduces a short double-stranded RNA (about 20 bases) having a sequence homologous to the target gene into the cell, the mRNA of the target gene homologous to the RNA sequence is specifically degraded. This is a phenomenon in which the expression level decreases. This phenomenon, which was first discovered in nematodes, has been found to be a universal phenomenon in organisms including plants, and the molecular mechanism by which the expression of target genes is suppressed by antisense technology is It has been elucidated that it goes through a similar process. Conventionally, one DNA sequence that is complementary to the nucleotide sequence of the target gene is linked to an appropriate promoter, and an expression vector that expresses artificial mRNA under the control of the DNA is constructed and introduced into the cell. Was done. In recent knowledge, an expression vector designed so that a double-stranded RNA can be constructed in a cell is used. The basic structure is created by linking one DNA sequence complementary to a target gene, one under the promoter, and the other in the opposite direction. In the single-stranded mRNA transcribed from this constructed gene, a pair of nucleotide sequences connected in the opposite direction are in a complementary relationship, and thus are paired to form a double-stranded RNA state having a hairpin-like secondary structure. This causes mRNA degradation of the target gene according to the RNAi mechanism. In plants, an example used in Arabidopsis thaliana has been reported (Smith, NA et al., Nature 407.319-320, 2000). Also, RNAi in general is summarized in a recent review (Morita and Yoshida, Protein / Nucleic Acid / Enzyme 47, 1939-1945, 2002). The contents described in these documents are incorporated herein by reference in their entirety.

  In the present specification, “RNAi” is an abbreviation for RNA interference, and by introducing a factor that causes RNAi such as double-stranded RNA (also referred to as dsRNA) into cells, homologous mRNA is specifically degraded, It refers to a phenomenon in which the synthesis of a gene product is suppressed and the technology used for it. As used herein, RNAi can also be used interchangeably with an agent that causes RNAi in some cases. In the present specification, “post-transcriptional gene silencing (PTGS)” can also be used interchangeably with “RNAi”. As used herein, “gene silencing” and “RNA silencing” can also be used interchangeably for “RNAi”.

  As used herein, “factor causing RNAi” refers to any factor capable of causing RNAi. In the present specification, the “factor causing RNAi” with respect to “gene” means that RNAi related to the gene is caused and an effect brought about by RNAi (for example, suppression of expression of the gene) is achieved. Factors that cause such RNAi include, for example, at least 10 nucleotides, including sequences having at least about 70% homology to a portion of the nucleic acid sequence of the target gene or sequences that hybridize under stringent conditions Examples include, but are not limited to, RNAs containing long double-stranded portions or variants thereof. Here, the factor preferably comprises a 3 'overhang, more preferably the 3' overhang can be 2 or more nucleotides long DNA (eg 2 to 4 nucleotides long DNA).

  Alternatively, examples of RNAi used in the present invention include, but are not limited to, a pair of short complementary sequences (for example, 15 bp or more, for example, 24 bp).

  Without being bound by theory, one of the possible mechanisms for RNAi to work is that when a molecule that causes RNAi, such as dsRNA, is introduced into a cell, a relatively long (eg, 40 base pairs or more) RNA, helicase In the presence of ATP, an RNaseIII-like nuclease called Dicer having a domain excises the molecule from the 3 ′ end by about 20 base pairs to produce a short dsRNA (also called siRNA). As used herein, “siRNA” is an abbreviation for short interfering RNA, which is artificially chemically synthesized, biochemically synthesized, synthesized in an organism, or about 40 This refers to short double-stranded RNA of 10 base pairs or more formed by decomposing double-stranded RNA of bases or more in the body, and usually has a 5′-phosphate, 3′-OH structure. 'The end protrudes about 2 bases. A protein specific to the siRNA binds to form RISC (RNA-induced-silencing-complex). This complex recognizes and binds to mRNA having the same sequence as siRNA, and cleaves mRNA at the center of siRNA by RNaseIII-like enzyme activity. The relationship between the siRNA sequence and the mRNA sequence cleaved as a target is preferably 100% identical. However, with respect to the base mutation at a position off the center of siRNA, the cleavage activity by RNAi is not completely lost, but a partial activity remains. On the other hand, the mutation of the base at the center of siRNA has a large effect, and the cleavage activity of mRNA by RNAi is extremely reduced. Utilizing such properties, for mRNA having a mutation, only siRNA having the mutation can be specifically decomposed by synthesizing siRNA having the mutation in the center and introducing it into the cell. Therefore, in the present invention, siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, a dsRNA typically having about 40 bases or more) can be used as such a factor. it can.

  Moreover, although not wishing to be bound by theory, siRNA is synthesized separately from the above pathway, and the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP). It is also contemplated that this dsRNA becomes Dicer's substrate again, generating new siRNA and amplifying the action. Thus, in the present invention, siRNA itself and factors that produce siRNA are also useful. In fact, in insects and the like, for example, 35 dsRNA molecules almost completely degrade the mRNA in a cell having 1,000 copies or more, so it is understood that siRNA itself and factors that generate siRNA are useful. Is done.

  In the present invention, double-stranded RNA called siRNA having a length of about 20 bases (for example, typically about 21 to 23 bases in length) or less can be used. Such siRNA can be used for treatment, prevention, prognosis, etc. of a disease because it suppresses gene expression by expressing in a cell and suppresses expression of a pathogenic gene targeted by the siRNA.

  The siRNA used in the present invention may take any form as long as it can cause RNAi.

  In another embodiment, the factor causing RNAi of the present invention may be a short hairpin structure (shRNA; short hairpin RNA) having an overhang at the 3 'end. As used herein, “shRNA” is a single-stranded RNA that includes a partially palindromic base sequence, and thus has a double-stranded structure within the molecule, resulting in a hairpin-like structure. The above molecules. Such shRNA is artificially chemically synthesized. Alternatively, such shRNA can be generated by synthesizing RNA in vitro using T7 RNA polymerase with hairpin structure DNA in which the DNA sequences of the sense strand and antisense strand are ligated in the opposite direction. Although not wishing to be bound by theory, such shRNAs are approximately 20 bases in length (typically 21 bases, 22 bases, 23 bases, etc.) in length after being introduced into the cell. It should be understood that it is degraded to cause RNAi as well as siRNA and has the therapeutic effect of the present invention. It should be understood that such effects are exerted in a wide range of organisms such as insects, plants, animals (including mammals). Thus, since shRNA causes RNAi similarly to siRNA, it can be used as an active ingredient of the present invention. The shRNA can also preferably have a 3 'overhang. The length of the double-stranded part is not particularly limited, but may preferably be about 10 nucleotides or more, more preferably about 20 nucleotides or more. Here, the 3 'protruding end may be preferably DNA, more preferably DNA having a length of at least 2 nucleotides, and further preferably DNA having a length of 2 to 4 nucleotides. The factor causing RNAi used in the present invention can be either artificially synthesized (for example, chemical or biochemical) or naturally occurring, and the effect of the present invention can be achieved between the two. There is no essential difference. Those chemically synthesized are preferably purified by liquid chromatography or the like.

  The factor that causes RNAi used in the present invention can also be synthesized in vitro. In this synthesis system, antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is caused through the mechanism described above, and the effects of the present invention are achieved. Here, for example, such RNA can be introduced into cells by the calcium phosphate method.

  Factors causing RNAi of the present invention also include factors such as single strands that can hybridize to mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the treatment methods and compositions of the invention.

  As used herein, “suppression of gene silencing” refers to inhibition of gene silencing or RNAi. As a result, the expression of the protein (or other gene expression product) whose expression was suppressed by gene silencing or RNAi is enhanced, and preferably returns to the original (normal) expression level.

  As used herein, “silencing suppression protein” refers to a protein that suppresses RNAi.

  In this specification, the “129K protein” is a replication enzyme of watermelon green spot mosaic virus (CGMMV), and is a 3435 bp domain composed of a methyltransferase region (MT) and a helicase region. The methyltransferase (MT) region of watermelon green spot mosaic virus (CGMMV) is 1899 bp.

  In the present invention, among them, (a) a nucleic acid sequence shown in SEQ ID NO: 1 encoding a watermelon green spot mosaic virus 129K protein or a fragment of a nucleic acid sequence encoding a corresponding protein, at least one of SEQ ID NO: 1 It is understood that the region comprising the sequence shown at positions 912 or the corresponding sequence is very important. Accordingly, a nucleic acid sequence shown in SEQ ID NO: 1 encoding a watermelon green spot mosaic virus 129K protein or a fragment of a nucleic acid sequence encoding a protein corresponding thereto, at least a sequence shown in positions 1 to 912 of SEQ ID NO: 1 or a sequence thereof It has been found useful to include a region containing the corresponding sequence and have a function of suppressing gene silencing when expressed.

  The nucleic acid of the present invention includes (b) a nucleic acid sequence containing one or several substitutions, additions or deletions to the sequence (a); or (c) a nucleic acid having the sequence (a) A nucleic acid that contains a nucleic acid sequence that hybridizes under stringent conditions and that encodes a product that, when expressed, has the function of inhibiting gene silencing.

  In the present invention, when the fragment is longer than 1440 nucleotides, the methyltransferase is an amino acid encoded by nucleotides 1438 to 1440 of SEQ ID NO: 1 or correspondingly glutamine. A methyltransferase in which the corresponding protein has the amino acid sequence shown in any one of SEQ ID NOs: 10 to 23 can also be used in the present invention. In the present invention, the methyltransferase may include, for example, the nucleic acid sequence shown in SEQ ID NO: 3 or may consist of the nucleic acid sequence shown in SEQ ID NO: 3.

In the present invention, as methyltransferase, for example, Shintaku, M .; H. et al. (Virogy 221: 218-225,1996) variants may be used which is prepared by introducing the cDNA into site-specific mutations in M strains have been reported ^{(M IC)} by (MPMI Vol. 17, No. 6, 583-592, 2004). These can be made with reference to the table below.

  In this specification, as a method for producing a polypeptide of a foreign gene, for example, a gene-encoding technique is used to incorporate a gene encoding the polypeptide into an appropriate expression vector, and this is used to transform an expression host. The recombinant polypeptide can be obtained from the culture supernatant of the transformed cells. The host cell is not particularly limited as long as it expresses a polypeptide that retains at least one physiological activity of a foreign gene. Various host cells conventionally used in gene manipulation (for example, plant cells and the like) Escherichia coli, yeast, animal cells, etc.) can be used. As long as the polypeptide derived from the cells thus obtained has substantially the same action as the native polypeptide, one or more amino acids in the amino acid sequence are substituted, added and / or deleted. The sugar chain may be substituted, added and / or deleted.

  Certain amino acids can be substituted for other amino acids in a protein structure, such as, for example, a cationic region or a binding site of a substrate molecule, without an apparent reduction or loss of interaction binding capacity. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is well known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, an equivalent protein in enzymatic activity) It is. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.

  In protein modification, the hydrophilicity index can also be considered. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present invention, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above. Examples of conservative substitution include those having a hydrophilicity index or a hydrophobicity index within ± 2, preferably within ± 1, and more preferably within ± 0.5. But not limited to them. Thus, examples of conservative substitutions are well known to those skilled in the art, for example, substitutions within each of the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine , And isoleucine, but are not limited to these.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. Such allelic variants usually have the same or very similar sequence as their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, in the case of the hemoglobin gene family with multigene structure, the human and mouse α hemoglobin genes are orthologs, but the human α and β hemoglobin genes are paralogs (genes generated by gene duplication). is there. In addition, when comparing human cystatin A, which is a cysteine protease inhibitor, with rice oryzacystatin, only three short amino acid motifs that are considered important for the interaction with the target protease are conserved. Therefore, the commonality of amino acids in other parts is very low. However, since both belong to the cystatin gene superfamily and are said to have a common ancestor gene, not only the overall amino acid homology, but also the amino acid sequence having high local homology in common. If it exists, it can be an ortholog. Thus, orthologs of the present invention can also be useful in the present invention, since orthologs can usually perform the same function as the original species in another species. The prolamin targeted in the present invention forms a family having a large number of members, and such can also be referred to as a conservatively modified variant.

  As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide. Such base sequence modification methods include restriction enzyme digestion, DNA polymerase, Klenow fragment, DNA ligase treatment and other ligation treatments, site-specific base substitution methods using synthetic oligonucleotides (specification) Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)). it can.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. The addition of amino acids means that one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids are added to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, the term “peptide analog” or “peptide derivative” refers to a compound that is different from a peptide but is equivalent to at least one chemical or biological function of the peptide. Thus, peptide analogs include those in which one or more amino acid analogs or amino acid derivatives are added or substituted to the original peptide. Peptide analogs have the same function as the original peptide (for example, similar pKa values, similar functional groups, similar binding modes with other molecules, Such additions or substitutions are made so as to be substantially similar to (eg, similarity in sex). Such peptide analogs can be made using techniques well known in the art. Thus, a peptide analog can be a polymer containing an amino acid analog.

  In the present specification, “polynucleotide analog” and “nucleic acid analog” are used interchangeably and are different compounds from a polynucleotide or nucleic acid, but with a polynucleotide or nucleic acid and at least one chemical function or biological. A function is equivalent. Thus, polynucleotide analogs or nucleic acid analogs include those in which one or more nucleotide analogs or nucleotide derivatives are added or substituted to the original peptide.

  As used herein, a nucleic acid molecule may have a portion of its nucleic acid sequence deleted or otherwise as long as the expressed polypeptide has substantially the same activity as the native polypeptide. It may be substituted with a base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Alternatively, it may be a nucleic acid molecule that hybridizes under stringent conditions with a gene encoding a polypeptide and encodes a polypeptide having substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. For example, a site-specific displacement induction method, a hybridization method, or the like may be combined with these methods.

  As used herein, “substitution, addition and / or deletion” of a polypeptide or polynucleotide refers to an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, relative to the original polypeptide or polynucleotide. , Replacing, adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such number is a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, it can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  In the present specification, the expression "specifically expresses" a gene means that the gene is expressed at a different (preferably higher) level in a specific part or time of the plant than other parts or time. . With specific expression, it may be expressed only at a certain site (specific site) or may be expressed at other sites. The expression specifically preferably means that it is expressed only at a certain site.

  In this specification, a gene is “specifically repressed” means that the gene is repressed at a level (preferably higher) at a specific site or time of the plant that is different (preferably higher) from the other sites or time. Say. “Specific expression” may be suppressed only at a certain site (specific site) or may be suppressed at other sites. Preferably being specifically suppressed means that it is suppressed only at a certain site.

  In the present specification, “gene construct”, “nucleic acid construct” or “gene cassette” are used interchangeably, and operate according to a nucleic acid molecule (eg, DNA, RNA) encoding a gene. A nucleic acid sequence comprising a control sequence (eg, a promoter) linked to (possibly so as to control the expression of the nucleic acid), and optionally a control sequence (eg, a promoter) and act on it. A nucleic acid molecule comprising a heterologous gene operably linked (ie, in frame). It is also within the scope of the present invention to use this cassette or construct in combination with other regulatory elements as required. Preferred expression cassettes are gene cassettes or nucleic acid constructs that can be cleaved with specific restriction enzymes and easily recovered.

  As a method for introducing such a gene construct or vector, any method for introducing a nucleic acid molecule such as DNA into a cell can be used, and examples thereof include transfection, transduction, and transformation (for example, Electroporation method, particle gun (gene gun) method, etc.). Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  In the present specification, when referring to a gene, “vector” or “recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence to a target cell. Such vectors can be autonomously replicated in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals and individual plants, or can be integrated into chromosomes. Examples include those containing a promoter at a position suitable for nucleotide transcription. Examples of the vector that can be used in the present invention include, but are not limited to, pBI221 and pBI121. In the present invention, any vector can be used as long as the target polynucleotide sequence can be transferred to the target cell.

  As a method for introducing a vector, any of the above-described methods for introducing a nucleic acid molecule such as DNA into a cell can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method , Liposome method, DEAE dextran method, electroporation method [Methods. Enzymol., 194, 182 (1990)], method using particle gun (gene gun), etc.), lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol. , 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

  As used herein, “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. Examples of the terminator include NOS terminator, 35S terminator and the like, but are not limited thereto. In the present invention, any sequence can be used as long as it exhibits terminator activity in an organism.

  As used herein, the term “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is a base sequence that initiates transcription upon binding of RNA polymerase. Since the promoter region is usually found within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic base sequence is predicted using DNA analysis software, The promoter region can be estimated. The putative promoter region varies from structural gene to structural gene, but is usually upstream of the structural gene, but is not limited thereto and may be present in or downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point, but is not limited thereto, and other promoters are present. Preferably, in the present invention, a promoter that is specifically expressed can be used. Such a specific promoter is preferably a promoter that drives specific expression in a storage protein, but is not limited thereto. More preferably, in the present invention, the promoter may be a 35S promoter, a promoter of ubiquitin such as corn, and the like, but is not limited thereto.

  In the present specification, the expression of the promoter of the present invention is “constitutive” in an almost constant amount in all tissues of an organism, whether in the juvenile or mature phase of the organism. It refers to the property that is expressed.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique may be used for introducing a nucleic acid into a cell, and examples thereof include transformation, transduction, transfection, and the like. Such a nucleic acid introduction technique is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its 3rd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  As a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, particle gun (gene gun) ), Calcium phosphate method, liposome method, DEAE dextran method, electroporation method, etc.), lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol. , 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

  In the present specification, the “gene introduction reagent” refers to a reagent used for promoting the introduction efficiency in the gene introduction method. Examples of such gene transfer reagents include, but are not limited to, cationic polymers, cationic lipids, polyamine reagents, polyimine reagents, calcium phosphate, and the like. Specific examples of reagents used in the transfection include reagents commercially available from various sources, such as Effectene Transfection Reagent (cat. No. 301415, Qiagen, CA), TransFast ™ Transfection Reagent. (E2431, Promega, WI), TfxTM-20 Reagent (E2391, Promega, WI), SuperFect Transfection Reagent (301305, Qiagen, CA), PolyFect Transfection Reagent (301105, Eagen, CA9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E9, Q9, E1 Invit rogen corporation, CA), JetPEI (× 4) conc. (101-30, Polyplus-translation, France) and ExGen 500 (R0511, Fermentas Inc., MD) and the like.

  When the present invention is used in plants, plant expression vectors can be introduced into plant cells by methods well known to those skilled in the art, for example, methods involving Agrobacterium and methods for direct introduction into cells. As a method using Agrobacterium, for example, the method of Nagel et al. (Nagel et al. (1990), Microbiol. Lett., 67, 325) can be used. This method involves first transforming Agrobacterium by electroporation with, for example, an expression vector appropriate for a plant, and then transforming the transformed Agrobacterium into Gelvin et al. (Edited by Gelvin et al. (1994), Plant Molecular Biology). It is a method of introducing into plant cells by the method described in Manual (Kluwer Academic Press Publishers). Methods for introducing plant expression vectors directly into cells include the particle gun method (see Christou et al. (1991), Bio / Technology 9: 957-962) and the polyethylene glycol (PEG) method (Datta et al. ( 1990), Bio / Technology 8: 736-740), electroporation methods (Shimamoto et al. (1989), Nature, 338: 274-276; and Rhodes et al. (1989), Science, 240: 204-. 207). These methods are well known in the art, and a method suitable for the plant to be transformed can be appropriately selected by those skilled in the art.

  Although the present invention has been shown to be particularly useful in plants, it can also be used in other organisms. The molecular biology techniques used in the present invention are well known and commonly used in the art, see, for example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J, et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like.

  As used herein, “transformant” refers to all or part of a living organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject, and encompasses all of these forms herein, but may refer to a particular form in a particular context. .

  In the method of performing transformation, physical methods include a particle gun method, a polyethylene glycol method (PEG method), an electroporation method, and a microinjection method.

  In the present invention, in the transformant, the target nucleic acid molecule (transgene) may or may not be introduced into the chromosome. Preferably, the nucleic acid molecule of interest (transgene) has been introduced into a chromosome, and more preferably has been introduced into both two chromosomes.

  As used herein, “plant” is a general term for organisms belonging to the plant kingdom, and is characterized by chlorophyll, hard cell walls, the presence of abundant permanent embryonic tissues, and organisms that are not capable of motility. Typically, a plant refers to a flowering plant having cell wall formation and anabolism by chlorophyll. “Plant” includes both monocotyledonous and dicotyledonous plants. Examples of preferable plants include rice, onion, tobacco, tomato, melon, and watermelon. Preferred plants are not limited to crops, but also include flowers, trees, lawns, weeds and the like. Unless otherwise indicated, a plant means any plant, plant organ, plant tissue, plant cell, and seed. Examples of plant organs include roots, leaves, stems and flowers. Examples of plant cells include callus and suspension culture cells.

  In the present specification, the “organism” (or “plant” in the case of plants) is used in the broadest sense in the field and refers to an organism (or plant) that operates a biological phenomenon, and typically has a cell structure. It has various characteristics such as proliferation (self-reproduction), growth, regulation, substance metabolism, repair ability, etc., and usually has inheritance governed by nucleic acid and proliferation involved in metabolism governed by protein as basic attributes. Living organisms include prokaryotes, eukaryotes (plants, animals, etc.) and the like. Preferably, in the present invention, the organism may be a plant. As used herein, preferably such a plant may be fertile. More preferably, such a plant can produce seeds.

  As used herein, “transgenic” refers to an organism (for example, a plant (including rice, onion, etc.)) that is incorporated into an organism in which a specific gene is present.

(Description of Preferred Embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(Nucleic acid)
In one aspect, the present invention provides (a) a nucleic acid sequence shown in SEQ ID NO: 1 encoding a watermelon green spot mosaic virus 129K protein or a fragment of a nucleic acid sequence encoding a corresponding protein, wherein at least one of SEQ ID NO: 1 A nucleic acid sequence comprising the sequence shown at positions 1 to 912 or a sequence corresponding thereto; (b) a nucleic acid sequence comprising one or several substitutions, additions or deletions to the sequence of (a); or (c) A nucleic acid (also referred to as a gene silencing-suppressing nucleic acid) comprising a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid having the sequence (a) and having a function of suppressing gene silencing when expressed. ) Gene silencing here can target any gene (RNA) (whether it encodes a protein or it encodes like microRNA or tRNA, as long as it is transcribed). The reason why such a suppressor protein suppresses silencing is to act on (combine with) RNA alone or a complex (riboprotein) of RNA and the protein bound to it, thereby inhibiting the RNA degradation process. It is thought that it is done.

  In one embodiment, when the fragment is longer than 1440 nucleotides, the nucleic acid of the present invention is an amino acid encoded by nucleotides 1438 to 1440 of SEQ ID NO: 1 or correspondingly glutamine.

  In another embodiment, in the nucleic acid of the present invention, the corresponding protein consists of the amino acid sequence shown in any one of SEQ ID NOs: 10-23.

  Preferably, the present invention provides a nucleic acid comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO: 3. This nucleic acid is characterized by being shorter than the full length of the 129K protein of watermelon green spot mosaic virus (CGMMV).

(Nucleic acid construct)
In another aspect, the present invention provides an isolated nucleic acid construct comprising the gene silencing-suppressing nucleic acid of the present invention (preferably the nucleic acid sequence shown in at least SEQ ID NO: 3).

  In one embodiment, the nucleic acid construct of the present invention may further comprise a promoter sequence. Examples of the promoter sequence include, but are not limited to, a 35S promoter and a promoter of ubiquitin such as corn. The promoter sequence that can be used in the present invention is not particularly limited as long as it determines the initiation site of transcription of the nucleic acid of the present invention and can directly control the frequency thereof. The nucleic acid construct of the present invention may or may not contain a spacer sequence between the nucleic acid of the present invention and the promoter sequence.

  In other embodiments, the nucleic acid construct of the present invention may further comprise a terminator sequence. Examples of the terminator sequence include, but are not limited to, a Nos terminator and a 35S terminator. Any terminator sequence may be used in the present invention as long as it can terminate the transcription of the nucleic acid of the present invention. The nucleic acid construct of the present invention may or may not contain a spacer sequence between the nucleic acid of the present invention and the promoter sequence.

  In a preferred embodiment, the nucleic acid construct of the present invention may further comprise a 35S promoter sequence and a Nos terminator sequence.

(Method for producing nucleic acid construct)
In another aspect, the present invention provides a method for producing a nucleic acid construct. This method comprises the following steps: A) providing a nucleic acid construct in which a promoter is operably linked to at least the gene silencing-suppressing nucleic acid of the present invention (preferably at least the nucleic acid sequence shown in SEQ ID NO: 3); B) the nucleic acid Transforming the organism with the construct; and C) selecting, for the transformed organism, a gene with suppressed gene silencing. Here, as the nucleic acid and the nucleic acid construct, any form described in the above (nucleic acid) and (nucleic acid construct) can be used.

(vector)
In another aspect, the present invention provides a vector comprising at least the gene silencing-suppressing nucleic acid of the present invention (preferably at least the nucleic acid sequence shown in SEQ ID NO: 3).

  In one embodiment, the vector of the present invention may further comprise a promoter sequence. Preferably, examples of the promoter sequence include, but are not limited to, a 35S promoter and a sequence derived from a ubiquitin promoter such as corn. The promoter sequence that can be used in the vector of the present invention is not particularly limited as long as it determines the transcription initiation site of the nucleic acid of the present invention and can directly control the frequency thereof. The vector of the present invention may or may not contain a spacer sequence between the nucleic acid of the present invention and the promoter sequence.

  In another embodiment, the vector of the present invention may further comprise a terminator. Examples of the terminator sequence include, but are not limited to, a Nos terminator and a 35S terminator. The terminator sequence that can be used in the vector of the present invention may be any as long as it can terminate the transcription of the nucleic acid of the present invention. The vector of the present invention may or may not contain a spacer sequence between the nucleic acid of the present invention and the promoter sequence.

  In a preferred embodiment, the vector sequence of the present invention may include both a 35S promoter sequence and a Nos terminator sequence. Here, as the nucleic acid and the nucleic acid construct, any form described in the above (nucleic acid) and (nucleic acid construct) can be used.

(Transformant)
In another aspect, the present invention provides an organism comprising at least the gene silencing-suppressing nucleic acid of the present invention (preferably at least the nucleic acid sequence shown in SEQ ID NO: 3). The organisms provided in the present invention include plants (eg, rice, onions, tobacco, tomatoes, melons, watermelons, etc.), fungi (eg, red mold, yeasts, etc.), animals (eg, mites, nematodes, Drosophila, mice, etc.) ).

  Currently, as an RNAi suppressor, a gene product called HC-Pro (helper component-protease) in the genome of Potyvirus (a genus name of a group represented by Potato virus Y (PVY) or Tobacco etch virus (TEV)) Tombusvirus (Pat, which has been identified in the genome of Tokubashi stunt virus (TBSV), Cymbidium ringspot virus (CymRSV) or Carnation Italian ringspot virus (CIRV)) Tetsuro “Plant Virus and RNAi”, Chemistry and Biology.43 (7); 468 475,2005 see.). Potyvirus HC-Pco may inhibit double-stranded RNA cleavage, siRNA and miRNA incorporation into RISC. This HC-Pco suppresses RNAi not only in plants but also in Drosophila (B. Reavy et al., BMC Biotechnol, 4, 18, 2004). Tombusvirus P19 forms a dimer between molecules and specifically binds to 21 nt double-stranded siRNA to inhibit siRNA incorporation into RISC and suppress RNAi. P19 also suppresses RNAi in Drosophila and human cells (L. Lakatos et al., EMBO J., 23, 876, 2004 and P. Dunoyer et al., Plant Cell, 16, 1235, 2004).

  Thus, it is understood that the mechanism that suppresses RNAi exists as a common mechanism not only in plants such as fungi, rice, and onions, but also in animals such as Drosophila and humans. Therefore, when a fungus or animal is transformed with the gene silencing-suppressing nucleic acid of the present invention (preferably, the nucleic acid sequence shown in at least SEQ ID NO: 3), an organism containing the gene silencing-suppressing nucleic acid can be obtained as in the case of plants. It is possible to produce.

  In a preferred embodiment, the organism provided in the present invention is a plant. Examples of this plant include, but are not limited to, rice, onion, tobacco, tomato, melon, and watermelon. Rice and onion are preferable. Here, as the nucleic acid and the nucleic acid construct, any form described in the above (nucleic acid) and (nucleic acid construct) can be used.

(Method to suppress gene silencing caused by gene transfer)
In another aspect, the present invention provides a method for expressing a protein whose expression is suppressed by gene silencing. The method comprises the following steps: a) providing a nucleic acid construct comprising a gene silencing-inhibiting nucleic acid of the invention (preferably at least the nucleic acid sequence shown in SEQ ID NO: 3); and b) using the nucleic acid construct to A step of transforming an organism in which singing has occurred. Here, as the nucleic acid and the nucleic acid construct, any form described in the above (nucleic acid) and (nucleic acid construct) can be used.

(kit)
In another aspect, the present invention provides a kit for suppressing gene silencing caused by gene transfer. This kit comprises at least the gene silencing-suppressing nucleic acid of the present invention (preferably at least the nucleic acid sequence shown in SEQ ID NO: 3); means for introducing the nucleic acid into the organism in which the gene silencing has occurred. Here, as the nucleic acid and the nucleic acid construct, any form described in the above (nucleic acid) and (nucleic acid construct) can be used. Examples of proteins whose expression is suppressed by gene silencing include, but are not limited to, various proteins that are suppressed by the expression of GFP protein, β-glucuronidase (GUS) gene, and the like.

  As a means for introducing a nucleic acid into an organism in which gene silencing has occurred, any gene introduction means can be considered, and for example, a vector, a plasmid, and a gene gun can be considered.

(Method for producing a polypeptide of a foreign gene in an organism)
In another aspect, the present invention provides a method for producing a polypeptide of a foreign gene in an organism. In this method, gene silencing occurs in the organism during the production, the method comprising: a) introducing a nucleic acid molecule encoding the foreign gene into the organism; b) the gene silencing-suppressing nucleic acid of the present invention (preferably Providing a nucleic acid construct comprising at least the nucleic acid sequence shown in SEQ ID NO: 3; c) transforming the organism with the nucleic acid construct; and d) conditions under which the polypeptide expresses the organism Including a step. Here, as the nucleic acid and the nucleic acid construct, any form described in the above (nucleic acid) and (nucleic acid construct) can be used. When a polypeptide is expressed using a foreign gene and produced in large quantities, gene silencing frequently occurs and normal growth may not occur. The present invention can be used in such a case.

  For example, this method can eliminate the gene silencing that occurs when a polypeptide of a foreign gene is produced in an organism, thereby eliminating the disadvantage that a necessary gene is silenced, or a desired foreign gene. Can be used for such purposes as more efficient production. For example, this method can be used to produce a plant vaccine that does not require injection (for example, to produce a vaccine by introducing a gene encoding an antigen such as cholera or hepatitis virus into a banana), and to produce a plant that is rich in the desired substance. (For example, to produce high lysine, high β-carotene rice and high iron rice for the purpose of improving nutritional status) (Edible Vaccines; September 2000; Scientific American Magazine; by William HR Langridge, by Ricki Rusting; 6 Page (s); Ye et al. 2000. Engineering the provitamin A (beta-carotenene) biosynthetic pathway into (carotenoid-free) rice endosperm.Science 287 (5451): 303-305 PMID 10634784; Journal of the American College of Nutrition, Vol. Iron-Rich Rice Paula Lucca, see Richard Hurrell and Ingo Potrykus) and the like.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

  Hereinafter, although an example explains the composition of the present invention in detail, the present invention is not limited to this. With the exception of exceptions, reagents and supports used in the following examples were those commercially available from Sigma (St. Louis, USA), Wako Pure Chemicals, BIO-RAD and the like.

Example 1
(1. Cloning)
(1.1 PCR amplification)
Different fragments of CGMMV were amplified with the respective primer pairs shown in Table 1. PCR reaction mixtures were prepared as shown in Table 2. The PCR cycle procedure was performed on an iCycler Thermal Cycler (BIO-RAD Co., LTD, # 170-8720JA) as shown in Table 3. The PCR amplified product was digested with BamHI and SacI, extracted with a QIAEX II kit (Qiagen (Qiagen) # 20021) and purified with a StrataPrep® PCR purification kit (Stratagene (Stratagene) # 400771).

(1.2 Agarose gel extraction for PCR fragments)
After amplification of the target PCR fragment, it was extracted with a QIAEX II agarose gel extraction kit (Qiagen # 20021) according to the following procedure.

  (I) The digested product (50 μl) was run on a 1% Seplaque agarose gel (FIG. 1 top). Thereafter, a DNA band was cut out from the agarose gel using a clean sharp scalpel. The size of the gel slice was minimized by removing excess agarose. A 1.5 ml centrifuge tube was used to process the agarose to 250 mg.

  (Ii) A gel slice was placed in a colorless tube and weighed. For 1 volume of gel per 100 bp-4 kb DNA fragment, 3 volumes of buffer QX1 were added; otherwise, Table 4 below was followed. For example, 300 μl of buffer QX1 was added to 100 mg of each gel.

  (Iii) QIAEX II was resuspended by vortexing for 30 seconds. According to Table 5 below, QIAEX II was added to the sample and mixed.

  (Iv) To dissolve the agarose, it was incubated at 50 ° C. for 10 minutes and the DNA was allowed to bind. The tube was inverted and mixed by gently flicking. QIAEX II in suspension was maintained for 2 minutes each. It was confirmed that the color of this mixture was yellow. If the color of the mixture was orange or purple, 3M sodium acetate (pH 5.0) (10 μl) was added and mixed. The color of the mixture turned yellow. Incubation should then continue for at least another 5 minutes.

  (V) The sample was centrifuged for 30 seconds and the supernatant was carefully removed with a pipette.

  (Vi) The pellet was washed with buffer QX1 (500 μl). The pellet was resuspended by inverting the tube and flicking gently. Samples were centrifuged for 30 seconds and all traces of supernatant were removed. This washing step removed the remaining agarose contamination.

  (Vii) The pellet was washed twice with buffer PE (500 μl). The pellet was resuspended by inverting the tube and flicking gently. Samples were centrifuged for 30 seconds and all traces of supernatant were removed. The remaining salt contamination was removed by this washing step.

  (Viii) The pellets were air dried for 10-15 minutes or until the pellets were white. When 30 μl of QIAEX II suspension was used, the pellets were air dried for about 30 minutes. Since there was a possibility of overdrying, vacuum drying was not performed. When the QIAEX II pellet is overdried, the elution efficiency may be reduced.

(Ix) To elute the DNA, 20 μl of 10 mM Tris · Cl (pH 8.5) or H ₂ O was added, the tube was inverted and the pellet was resuspended by flicking. Incubated according to Table 6 below.

  (X) Centrifugation for 30 seconds. The supernatant was carefully transferred to a clean tube with a pipette. This supernatant contained purified DNA.

  (Xi) Steps (ix) and (x) were repeated and the eluates were combined. The purified DNA was stored at -20 ° C.

(Table 4 Volume of Buffer QX1 added to different fragments)

(Table 5 Volume with QIAEX II added for different fragments)

(Table 6 Incubation time for different fragments)

(1.3 Purification of PCR fragment)
After extracting the PCR fragment, it was purified by StrataPrep (registered trademark) PCR purification kit PCR purification kit (Stratagene # 400771) according to the following protocol.

  (I) To a centrifuge tube containing the PCR product, an equal volume of DNA binding solution was added to the aqueous portion of the PCR product, and the two components were mixed.

  (Ii) Using a pipette, the PCR product-DNA binding solution mixture was transferred to a microspin cup placed in a 2 ml receptacle tube. (Take care not to damage the fiber matrix with the pipette tip.) Close the cap of the 2 ml receptacle tube on the microspin cup.

  (Iii) The tube was rotated at maximum speed for 30 seconds in a microcentrifuge.

  (Iv) The cap of the 2 ml receptacle tube was opened, the microspin cup was removed and held, and the DNA binding solution was discarded.

  (V) Wash buffer 1 was prepared by adding 100% ethanol (40 ml). After the ethanol was added, the box on the container label was marked.

  (Vi) The cap of the 2 ml receptacle tube was opened and 750 μl of wash buffer was added to the microspin cup. Furthermore, the cap of the receptacle tube was closed.

  (Vii) Rotated at maximum speed for 30 seconds on microcentrifuge. The cap of the 2 ml receptacle tube was opened, the microspin cup was removed and held, and the wash buffer was discarded.

  (Viii) The 2 ml receptacle tube was returned to the micro spin cup, and the cap of the receptacle tube was closed on the micro spin cup.

  (Ix) The tube in the microcentrifuge was rotated at maximum speed for 30 seconds. Removed from the centrifuge to ensure removal of all wash buffer from the microspin cup.

  (X) The microspin cup was transferred to a clean 1.5 ml microcentrifuge tube and the 2 ml receptacle tube was discarded.

  (Xi) Elution buffer (50 μl) was added directly to the top of the fiber matrix at the bottom of the microspin cup. The pipette tip was kept out of contact with the fiber matrix.

  (Xii) Tubes were incubated for 5 minutes at room temperature.

  (Xiii) On the microspin cup, the cap of the 1.5 ml microcentrifuge tube was closed, and the tube in the microcentrifuge was rotated at maximum speed for 30 seconds.

  (Xiv) The microcentrifuge tube was opened and the microspin cup was discarded. The purified PCR product was stored at -20 ° C.

(1.4 Preparation of cloning vector)
Plasmid pBI221 was digested for cloning vectors with BamHI and SacI as shown in Table 7. The digested vector was dephosphorylated as shown in Table 8.

  A schematic diagram of a plasmid containing the CGMMV methyltransferase domain and the 129k domain and the respective coding regions is shown in FIG. 1 (lower panel).

(1.5 Agarose gel extraction for cloning vectors)
After dephosphorylating the cleaning vector, it was purified by QIAEX II agarose gel extraction kit (Qiagen (Qiagen) # 20021) according to the protocol described above.

(1.6 Cloning of PCR fragments into plasmid vectors)
After purifying the PCR fragment and cloning vector, the PCR fragment was cloned into the plasmid vector to construct the target plasmid according to the following protocol.

  (I) The above plasmid vector DNA and the PCR DNA fragment to be inserted were combined in a total amount of 10 μl (the recommended amount of DNA is vector: insert = 1: 1-10).

  (Ii) 10 μl of ligation high (ToYoBo Co., LTD (LGK-101)) was added to the DNA solution and mixed thoroughly.

  (Iii) Incubated overnight at 4 ° C.

  (Iv) The ligation reaction mixture is prepared as follows: It can be used directly for transformation with E. coli competent cells.

(1.7 Transformation)
When transformation was performed immediately after ligation, the following protocol was implemented.

  (I) For each ligation reaction, one tube containing 100 μl of competent cells (DH5α) was lysed on ice.

(Ii) 10 μl of the ligation reaction mixture was added to a tube of lysed competent cells. Mix gently (do not mix by pipetting)
(Iii) The transformation mixture was incubated for 30-60 minutes on ice. During the incubation, the SOC medium was pre-warmed to 42 ° C.

  (Iv) The transformation mixture was heat shocked at 42 ° C. for 30-90 seconds.

  (V) The transformation mixture was incubated on ice for 5-15 minutes.

  (Vi) Pre-warmed SOC medium (400 μl) was added to the transformation reaction mixture. Competent cells were allowed to recover by agitation at 37 ° C. for 1 hour (cell tubes were placed horizontally in the shaker for better ventilation).

  (Vii) LB-antibiotic plates were prepared for colony screening during the growth period.

  (Viii) 50-100 μl of the transformation mixture was placed on LB-antibiotic plates. The plate was incubated overnight at 37 ° C.

  (Ix) Colonies were selected for plasmid DNA analysis.

  (X) Miniprep DNA was prepared from selected colonies using the following standard procedure.

(2. Plasmid miniprep)
After transformation, colonies were miniprepped with the Bio-Rad Quantum Prep® kit (Catalog No. 732-6100, BioRad) according to the following protocol.

  (I) The overnight medium (1-2 ml) of the cells containing the plasmid was transferred to a microcentrifuge tube. Cells were pelleted by centrifugation for 30 seconds. All supernatants were removed by aspiration or pipetting.

  (Ii) Add 200 μl of cell resuspension solution and vortex or pipette up and down until the cell pellet is completely resuspended.

  (Iii) 250 μl of cell lysis solution was added and mixed by gently inverting the capped tube for about 10 minutes (not vortexed). As cell lysis occurred, the solution became viscous and slightly clear.

  (Iv) 250 μl of neutralization solution was added and mixed by gently inverting the capped tube for about 10 minutes (not vortexed). A visible precipitate was formed.

  (V) The cell debris was pelleted in a microcentrifuge for 5 minutes. Dense white debris pellets formed along the side or bottom of the tube. The supernatant of this step (clear lysate) contained plasmid DNA.

  (Vi) While waiting for the centrifugation step of step (v), a Spin Filter was placed in one of the 2 ml wash tubes provided in the kit. The Quantum Prep matrix was mixed by repeated bottle shaking and inversion and ensured complete suspension.

  (Vii) The clear lysate (supernatant) from step (v) was transferred to the Spin Filter, 200 μl of completely suspended matrix was added, and the solution was mixed by pipetting up and down. If there were multiple samples, the lysate was transferred first, then the matrix was added and mixed. After the matrix was added to all samples and mixing was complete, it was centrifuged for 30 seconds.

  (Viii) Remove the Spin Filter from the 2 ml tube, discard the filtrate at the bottom of the tube, and place the filter in the same tube. The matrix was washed by adding 500 μl wash buffer and centrifuging for 30 seconds.

  (Ix) The Spin Filter was taken out from the 2 ml tube, the filtrate at the bottom of the tube was discarded, and the filter was put in the same tube. 500 μl of wash buffer was added and the matrix was washed by centrifugation for a full 2 minutes to remove the remaining traces of ethanol.

(X) The Spin Filter was removed and the microcentrifuge tube was discarded. The filter was placed in one of the 1.5 ml collection tubes provided with the kit, or any other standard 1.5 ml microcentrifuge tube (compatible with the Spin Filter). 100 μl of deionized H ₂ O or TE (10 mM Tris-HCl buffer and 1 mM ethylenediaminetetraacetic acid, pH 8.0) was added. DNA was eluted by centrifugation at maximum speed for 1 minute.

  (Xi) The Spin Filter was discarded and the eluted DNA was stored at -20 ° C.

(3. Confirmation of constructed plasmid by sequencing)
Plasmid DNA was extracted by miniprep and then confirmed by sequencing according to the following protocol.

  (I) A reaction mixture was prepared as shown in Table 9 below.

(Ii) The PCR cycle procedure was performed on an iCycler Thermal Cycler (BIO-RAD Co., LTD, # 170-8720JA) as shown in Table 10 below.
Table 9 PCR reaction mixtures for sequencing

(Table 10 PCR cycle procedure for sequencing)

  During the sequencing reaction, a Centri Sep column (Princeton Separations, Inc. # CS-901) for purification of extension products was prepared according to the following procedure.

(I) Column hydration (ii) The column was gently tapped to ensure that the dry gel submerged at the bottom of the spin column.

  (Iii) The column was removed and reagent grade water or buffer (0.8 ml) was added to reconstitute the column. The column end stopper was left in place so that the column could stand alone. The gel was hydrated by changing the column cap and shaking and inverting the column or vortexing briefly. It was important to hydrate all of the dried gel.

(Iv) Hydrate for at least 30 minutes at room temperature before using the column. The reconstituted column can be stored refrigerated at 4 ° C. for 7 days. Longer storage can be achieved in 10 mM sodium azide (NaN ₃ ). Prior to continuing this procedure, the refrigerated column was warmed to room temperature.

  (V) The column was inverted and the column was tapped to remove bubbles from the column gel, and the gel was slurried at the opposite end of the column. The column was placed on a rack of microtubes to precipitate the gel.

  (Vi) After the gel was allowed to settle and air bubbles were removed, the top column cap was first removed, and then the bottom column end stopper was removed.

  (Vii) Excess fluid was drained into a wash tube (2 ml). When the fluid did not flow immediately through the end of the column, a 2 ml latex pipette valve was used to gently apply air pressure to the top of the column so that the fluid began to pass through the column filter. The column was discharged as it was. About 200-250 μl was drained from the column. This fluid was discarded.

  (Viii) The column was rotated in a variable speed centrifuge at 750 × g for 2 minutes, the tube was washed, and the intervening fluid was removed.

  (Ix) About 300 μl of fluid was removed. When there were droplets at the end of the column, they were wiped off and dried. The wash tube and intervening fluid were discarded. The gel material was not excessively dried. Samples were processed within minutes.

(X) The column was left standing towards the light. 20 μl of the completed DyeDeoxy ^™ terminator reaction mixture was transferred to the top of the gel. The sample was carefully placed in the middle of the gel bed at the top of the column without disturbing the gel surface. There was no contact between the column side and the reaction mixture or sample pipette tip. This is because the purification efficiency may be reduced, and excess dye may cause the analysis to be messed up.

  (Xi) A column was placed in a sample collection tube (1.5 ml), and both were placed in a rotor. Maintained proper direction. The highest point of the gel solvent in the column was always located at the outside of the rotor. The column and collection tube were spun at 750 × g for 2 minutes. The purified sample was collected at the bottom of the sample collection tube. The spin column was discarded and the ABI sample preparation procedure proceeded.

  (Xii) The sample was dried by vacuum centrifugation. There was no heating. The PCR product to be sequenced was purified on a Centri Sep column and the sequencing sample was resuspended according to the procedure shown in Table 11 below.

(4. Plasmid Midiprep)
In order to conduct a collision experiment according to the following protocol, the constructed plasmid DNA was confirmed by sequencing, and then the confirmed plasmid was extracted with a Bio-Rad Quantum Prep (registered trademark) kit (Catalog No. 732-6120).

  (I) In a 250 ml Erlenmeyer flask, 50 ml of the liquid medium was inoculated with the plasmid-containing bacteria, and incubated overnight (15-18 hours) at 300 rpm and 37 ° C. in a rotary shaker.

  (Ii) 40 ml of the culture solution after one night was transferred to a 50 ml screw cap centrifuge tube. The cells were spun down by centrifuging at 5,000 rpm (about 3,000 × g) for 5 minutes, and the supernatant was discarded.

  (Iii) 5 ml of cell resuspension was added to the cell pellet and the cells were vortexed to resuspend the pellet. It was ensured that the cell pellet was completely resuspended.

  (Iv) 5 ml of cell lysate was added and mixed by inverting the tube 6-8 times. Vortexing was not performed because chromosomal DNA can be cleaved by vortexing, resulting in contamination of plasmid DNA. It was confirmed that the solution was sticky and somewhat transparent.

  (V) 5 ml of neutralized solution was added to cap the tube, and the tube was mixed by inverting it 6-8 times. It was confirmed that the solution became cloudy and produced a cohesive white precipitate.

  (Vi) The mixture was centrifuged at 8,000 rpm (7500 × g) for 10 minutes, and the supernatant was carefully poured into a new tube. Efforts were made not to transfer the precipitate at all, but small amounts of debris did not affect plasmid purification.

  (Vii) The Quantum Prep matrix was resuspended by vigorously shaking. To the clear lysate obtained in step 3.9.6, 1.0 ml of Quantum Prep matrix was added. Shake gently for 15-30 seconds to mix. The matrix was pelleted by centrifuging at 8,000 rpm for 2 minutes.

  (Viii) The supernatant was carefully removed from the pelleted matrix.

  (Ix) 10 ml of wash buffer was added to the matrix. The matrix was resuspended in wash buffer by shaking.

  (X) Centrifuge at 8,000 rpm for 2 minutes and carefully pour wash buffer from the pelleted matrix. 600 μl of wash buffer was added to the matrix and resuspended.

  (Xi) The spin column was removed from the bag and the tip tab was removed. A spin column was placed inside a 2 ml collection tube, and the resuspension matrix obtained in the previous step was transferred to the spin column. A hole was made in the spin column cap with a small sharp object, and the column was capped with the spin column cap. Rotate at 12-14,000 xg for 30 seconds in a microcentrifuge. The spin column was removed from the microcentrifuge tube and the wash buffer at the bottom of the tube was discarded and placed on a filter in the same tube.

  (Xii) 500 μl of washing buffer was added and rotated in a microcentrifuge at 12-14,000 × g for 30 seconds. The spin column was removed and the wash buffer was discarded.

  (Xiii) Place the spin column in the tube again and rotate for an additional 2 minutes at maximum speed to remove any remaining wash buffer.

  (Xiv) The spin column was transferred to a clean 2 ml microcentrifuge tube. Water or 600 μl of TE was added to the matrix. Spin for 2 minutes in a microcentrifuge and discard the spin column containing the matrix. Plasmid DNA was stored at -20 ° C.

(5. Plasmid maxiprep)
Meanwhile, it is necessary that the confirmed plasmid has been extracted by Bio-Rad Quantum (registered trademark) kit (Catalog No. 732-6130) as in the following protocol.

  (I) Centrifugation at 5,000 × g for 5 minutes (in a 250-500 ml centrifuge bottle) yielded 100-500 ml of cells depending on the density of the culture (see above). The supernatant was discarded.

  (Ii) 15 ml of cell resuspension was added to the cell pellet. Cells were vortexed or pipetted up and down to resuspend the pellet. It was confirmed that the cells were completely resuspended.

  (Iii) 23 μl of cell lysate was added and mixed by shaking the centrifuge bottle. Vortexing was not performed because chromosomal DNA can be cleaved by vortexing, resulting in contamination of plasmid DNA. After a few minutes, it was confirmed that the solution was sticky and somewhat transparent. It was not allowed to incubate for more than 5 minutes before moving on to the next step.

  (Iv) 15 ml of neutralized solution was added and mixed by shaking the centrifuge bottle. It was confirmed that the solution became cloudy and produced a cohesive white precipitate. Immediately proceeded to step 5. It was not allowed to stand for more than 10 minutes before moving on to the next step.

  (V) Centrifugation was performed at 8 to 10,000 × g for 20 minutes, and the supernatant (clarified cell lysate) containing plasmid DNA was poured into a new centrifuge bottle. If the pellet did not adhere well to the wall of the centrifuge bottle, it was filtered through cheesecloth or miracloth. Efforts were made not to transfer the precipitate at all, but even a small amount of debris would not affect the subsequent purification process.

  (Vi) The matrix was resuspended by actively shaking the Quantum Prep matrix. 10 ml of fully resuspended Quantum Prep matrix was added to the clarified cell lysate obtained in step (v).

  (Vii) Mix gently by shaking for 15-30 seconds. The matrix was pelleted by centrifuging at 3,000 × g for 5 minutes.

(Viii) The supernatant was removed from the pelleted matrix.
To the pelleted matrix, 25 ml of wash buffer was added. The matrix was resuspended in wash buffer by shaking.

  (Ix) 25 ml of wash buffer was added to the pelleted matrix and the matrix was resuspended in wash buffer by shaking. Centrifugation was performed at 3,000 × g for 3 to 5 minutes.

  (X) The spin basket was inserted into a 50 ml screw cap centrifuge tube.

  (Xi) The supernatant was removed from the pelleted matrix. 15 ml of wash buffer was added to the pellet. The matrix was resuspended and then the suspension was transferred to the spin basket obtained in step 10. The tube lid was not changed. The spin basket / tube was centrifuged in a swinging bucket rotor at 4,000 rpm (3,000 × g) for 5 minutes.

  (Xii) The basket was removed and the filtrate was poured out from the tube. The basket was again placed in the tube and 10 ml of wash buffer was added to the matrix in the basket. It was centrifuged again at 4,000 rpm (3,000 × g) for 5 minutes.

  (Xiii) The basket was removed and placed in a new sterile 50 ml centrifuge tube (provided). Sterile water or 5 ml of TE was added. Centrifugation was performed at 4,000 rpm (3,000 × g) for 5 minutes. The DNA at this stage is ready for use. If additional purity or concentration is desired, the sample may be precipitated with ethanol as described below.

  (Xiv) Precipitation by adding 1/18 volume of 5M NaCl and 2 volumes of chilled 95-100% ethanol.

  (Xv) It was made into the pellet form by centrifuging at 4,000 xg or 10-20 minutes or more.

  (Xvi) The pellet was washed by adding 10 ml of 70% ethanol and then centrifuged at 4,000 × g or more than 10 minutes. The pellet was dried (air dried or vacuum) and then resuspended in an appropriate amount of TE or sterile water. If the pellet was dried too much, it could be difficult to resuspend the DNA, so it was not allowed to dry too much.

(Example 2. Particle collision procedure)
(1. Preparation of gold particles coated with plasmid DNA)
Initially, gold particles coated with plasmid DNA for the GFP silencing system were made according to the work in Table 12 below.

  Specifically, the 129K protein gene region of the virulent strain (CGMMMV-SH) strain and the attenuated strain (CGMMV-SH33b) and the N-terminal methyltransferase region (MT) thereof are respectively represented by GUS (β-glucuronidase) of the expression vector pBI221. Replaced with gene. The obtained plasmid was subjected to an RNA silencing induction experiment using a particle gun of a reporter gene such as GFP.

(2 Collision conditions)
The mixture plasmids described above, particle inflation Logan by (IDERA GIE-III, TANAKA Company ( Japan)), a pressure 3.57kg / cm ² of the helium gas (the case of rice 6.6 kg / ^cm 2), pressure 460 mmHg (Rice Case 235 mmHG) (20 μl / shot) was bombarded into the onion epidermis (or young leaf).

(3 Detection of cells expressing fluorescence)
For analysis, fluorescence-expressing cells were calculated for each shot 36 hours after collision. Fluorescence images of GFP and DsRed expressing cells were captured with a digital camera C7070 (Olympus, Tokyo, Japan) under the same illumination intensity and exposure time under an IX70 fluorescence microscope (Olympus, Tokyo, Japan). GFP fluorescence was visualized using a filter set consisting of a 475 nm excitation filter, a 505 nm dichroic mirror, and a 535 nm emission filter. DsRed fluorescence was visualized using a filter set consisting of a 540 nm excitation filter, a 570 nm dichroic mirror, and a 575 nm emission filter.

  As a result, the plasmid expressing the full-length, N-terminal methyltransferase domain portion of the 129K protein of the virulent strain SH suppressed silencing seen with the GFP gene. However, it was not suppressed by only a part of the N-terminal side of the methyltransferase domain (see Tables 13 and 14, FIGS. 2 and 3).

  A plasmid expressing the full length of the 129K protein of the attenuated strain SH33b, the methyltransferase domain portion on the N-terminal side and a portion on the N-terminal side of the methyltransferase domain did not suppress silencing. The amino acids in the methyltransferase domain portion on the N-terminal side of SH and SH33b differ at only one place. Therefore, this substitution was thought to affect silencing suppression (see Tables 13 and 14, FIGS. 2 and 3).

  These results showed that the silencing suppression function was similar to the onion and rice experiments.

(Example 3. Production and transformation of nucleic acid constructs using other methyltransferase domains)
A plasmid is prepared by the same method as described in Example 1 using a nucleic acid encoding the amino acid sequence of methyltransferase shown in FIG.

The prepared plasmid is subjected to an RNA silencing induction experiment using a method similar to the particle collision procedure described in Example 2. The resulting plasmid, particles inflation Logan using (IDERA GIE-III, TANAKA Company ( Japan)), the pressure of the helium gas 3.57kg / cm ² (the case of rice 6.6 kg / ^cm 2), pressure 460 mmHg ( In the case of rice, 235 mmHG (20 μl / shot) is applied to the onion epidermis (or young leaf).

  Next, it analyzes by IX70 fluorescence microscope (Olympus, Tokyo, Japan). As a result, it was confirmed that silencing was suppressed in rice and onion even in the plasmid expressing the methyltransferase moiety shown in FIG.

(Example 4. Silencing suppression effect in plants, animals and fungi)
Using the same method as in Examples 2 and 3, a plasmid expressing the methyltransferase moiety shown in SEQ ID NO: 3 and FIG. 5 is constructed. The resulting plasmid is introduced into various organisms. The organisms used are introduced into plants (tobacco, tomatoes, melons, watermelons), animals (ticks, nematodes, Drosophila, mice), and fungi (red bread mold, yeast). The result is observed with a fluorescence microscope to confirm that silencing is suppressed.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  By using the present invention for silencing suppression technology, it becomes possible to produce a large amount of useful proteins or useful substances in living organisms by gene transfer, and applications in various fields such as food, pharmaceuticals, and agrochemical fields are expected.

FIG. 1 (top) shows the PCR products of different fragments of CGMMV. MT Domain: PCR product of the CGMMV methyltransferase domain. 129K Domain: PCR product of the 129k protein domain of CGMMV. FIG. 1 (bottom) shows a schematic diagram of a plasmid containing a CGMMV methyltransferase domain and a 129k domain, each coding region. FIG. 2 is a fluorescence photograph showing the results of GFP and DsRed (control) in onion epidermis after 36 hours of simultaneous particle bombardment with different mixtures of plasmids. DeRed was all expressed in the plasmid, but GFP was expressed only in (i), (iii) and (v). In (ii), (iv) and (vi), silence was generated, and no expression was observed. FIG. 3 is a fluorescence photograph showing the results of GFP and DsRed (control) in rice epidermis after 36 hours of simultaneous particle bombardment with different mixtures of plasmids. DeRed was all expressed in the plasmid, but GFP was expressed only in (i), (iii) and (v). In (ii), (iv) and (vi), silence was generated, and no expression was observed. FIG. 4 shows a CGMMV gene map and a simplified diagram of the gene region used in the experiment. FIG. 5 is an alignment of methyltransferases of TMV-KR, TMV-RAK, TMV, TOMV, PMMV, TMGMV, TMV-OB, ORSV, TVCV, CR-TMV, CRMV, TMV-CG, CGMMV, and SHMV. FIG. 5 is an alignment of methyltransferases of TMV-KR, TMV-RAK, TMV, TOMV, PMMV, TMGMV, TMV-OB, ORSV, TVCV, CR-TMV, CRMV, TMV-CG, CGMMV, and SHMV. FIG. 5 is an alignment of methyltransferases of TMV-KR, TMV-RAK, TMV, TOMV, PMMV, TMGMV, TMV-OB, ORSV, TVCV, CR-TMV, CRMV, TMV-CG, CGMMV, and SHMV. FIG. 5 is an alignment of methyltransferases of TMV-KR, TMV-RAK, TMV, TOMV, PMMV, TMGMV, TMV-OB, ORSV, TVCV, CR-TMV, CRMV, TMV-CG, CGMMV, and SHMV. FIG. 5 is an alignment of methyltransferases of TMV-KR, TMV-RAK, TMV, TOMV, PMMV, TMGMV, TMV-OB, ORSV, TVCV, CR-TMV, CRMV, TMV-CG, CGMMV, and SHMV. FIG. 5 is an alignment of methyltransferases of TMV-KR, TMV-RAK, TMV, TOMV, PMMV, TMGMV, TMV-OB, ORSV, TVCV, CR-TMV, CRMV, TMV-CG, CGMMV, and SHMV. FIG. 5 is an alignment of methyltransferases of TMV-KR, TMV-RAK, TMV, TOMV, PMMV, TMGMV, TMV-OB, ORSV, TVCV, CR-TMV, CRMV, TMV-CG, CGMMV, and SHMV.

  SEQ ID NO: 1 is an example of the full length nucleic acid sequence of the 129K protein of watermelon green spot mosaic virus (CGMMV).

  SEQ ID NO: 2 is an example of the full-length amino acid sequence of the 129K protein of watermelon green spot mosaic virus (CGMMV).

  SEQ ID NO: 3 is an example of the nucleic acid sequence of the methyltransferase domain of watermelon green spot mosaic virus (CGMMV).

  SEQ ID NO: 4 is an example of the amino acid sequence of the methyltransferase domain of watermelon green spot mosaic virus (CGMMV).

  SEQ ID NO: 5 shows the PCR amplification sequence (SH-MT-5'S) for the CGMMV fragment used in Example 1.

  SEQ ID NO: 6 shows the PCR amplified sequence (SH-MT-3 ′S) for the CGMMV fragment used in Example 1.

  SEQ ID NO: 7 shows the PCR amplified sequence (SH-5 ') for the CGMMV fragment used in Example 1.

SEQ ID NO: 8 shows the PCR amplified sequence (SH-MT-3 ′) for the CGMMV fragment used in Example 1 SEQ ID NO: 9 shows the PCR amplified sequence (SH-129K for the CGMMV fragment used in Example 1 -3 ′).

  SEQ ID NO: 10 is an example of an amino acid sequence of TMV-KR methyltransferase.

  SEQ ID NO: 11 is an example of an amino acid sequence of TMV-RAK methyltransferase.

  SEQ ID NO: 12 is an example of an amino acid sequence of TMV methyltransferase.

  SEQ ID NO: 13 is an example of an amino acid sequence of a TOMV methyltransferase.

  SEQ ID NO: 14 is an example of an amino acid sequence of PMMV methyltransferase.

  SEQ ID NO: 15 is an example of an amino acid sequence of TMGMV methyltransferase.

  SEQ ID NO: 16 is an example of an amino acid sequence of TMV-OB methyltransferase.

  SEQ ID NO: 17 is an example of an amino acid sequence of ORSV methyltransferase.

  SEQ ID NO: 18 is an example of an amino acid sequence of a TVCV methyltransferase.

  SEQ ID NO: 19 is an example of an amino acid sequence of a methyltransferase of CR-TMV.

  SEQ ID NO: 20 is an example of an amino acid sequence of CRMV methyltransferase.

  SEQ ID NO: 21 is an example of an amino acid sequence of TMV-CG methyltransferase.

  SEQ ID NO: 22 is an example of an amino acid sequence of CGMMV methyltransferase.

  SEQ ID NO: 23 is an example of an amino acid sequence of SHMV methyltransferase.

Claims (14)

Scan squid A green mottle mosaic virus 129K protein fragments of the nucleic acid sequence or its shown in SEQ ID NO: 1 encoding, the fragment comprises a sequence or sequences corresponding to it are shown in at least SEQ ID NO: 3, a nucleic acid sequence or fragment < br /> the including, a composition for inhibiting gene silencing. The composition of claim 1, wherein the nucleic acid sequence or fragment consists of the nucleic acid sequence shown in SEQ ID NO: 3. It said nucleic acid sequence or fragment is shorter than the total length of SEQ ID NO 1, composition according to claim 1. Furthermore, the composition of any one of Claims 1-3 containing a promoter sequence. 5. The composition according to claim 4 , wherein the promoter sequence is derived from a promoter selected from the group consisting of a 35S promoter and a ubiquitin promoter. Furthermore, the composition of any one of Claims 1-5 containing a terminator arrangement | sequence. The composition according to claim 6 , wherein the terminator sequence is derived from a terminator selected from the group consisting of a Nos terminator and a 35S terminator. The composition according to any one of claims 1 to 3 , further comprising a 35S promoter sequence and a Nos terminator sequence. A gene comprising a nucleic acid sequence or fragment thereof as set forth in SEQ ID NO: 1 encoding a watermelon green spot mosaic virus 129K protein, wherein the fragment comprises at least the sequence set forth in SEQ ID NO: 3 or a sequence corresponding thereto. A vector to suppress silencing . The vector of claim 9 further comprising a promoter sequence. Further comprising a terminator sequence, a vector according to claim 9 or 1 0. A method for suppressing gene silencing caused by gene transfer, which comprises the following:
a) providing a composition according to any one of claims 1 to 8 or a vector according to any one of claims 9 to 11 ; and b) using the composition or vector to produce the gene. Transforming a plant in which silencing has occurred. A kit for suppressing gene silencing caused by gene transfer, the kit comprising:
The composition according to any one of claims 1 to 8, or the vector according to any one of claims 9 to 11 ;
Means for introducing the composition or vector into the plant in which the gene silencing has occurred;
A kit comprising: A method for producing a polypeptide of a foreign gene in plants, gene silencing occurs in the plant during the production, the method comprising,
introducing into the plant a nucleic acid molecule encoding a) the foreign gene;
b) providing the composition according to any one of claims 1 to 8 or the vector according to any one of claims 9 to 11 ;
step transforming the plants with c) the composition or vector; and d) the step of placing the plant under conditions that said polypeptide is expressed,
Including the method.