Method for inhibiting methylation of target DNA in plants

Abstract

Plants having desired traits can be prepared simply and rapidly by suppressing methylation of target DNA in plants without using recombinant technology. In the RNA-commanded DNA methylation mechanism, scaffold RNA produced by transcription of the target DNA is cleaved.

Description

Technical Field

[0001] The present invention relates to a method for inhibiting methylation of a target DNA in a plant. Background Art

[0002] Gene expression in plants is regulated by DNA methylation and chemical modification of histones, which are called epigenetic control. This is a phenomenon caused by RNA silencing, which is roughly divided into post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). Plants rely on this epigenetic control to regulate the accumulation level of functional components. In order to make plants accumulate these useful components at a high level, a technology that can freely manipulate this epigenetic is needed. However, it is generally believed that epigenetic control is too complicated and it is impossible to specifically remove the methylation of target DNA.

[0003] Up to now, there is almost no technology for inducing DNA sequence-specific demethylation, and only in recent years has a technology for inducing demethylation using dCAS fused to TET1 been reported (Non-patent Document 1). However, when using dCAS fused to TET1 to induce demethylation, recombinant technology is required, so there is a problem that demethylation cannot be induced simply and quickly.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Patent Publication No. WO2014 / 129560

[0007] Non-patent literature

[0008] Non-patent document 1: Gallego-Bartolome J et al., Proceedings of the National Academy of Sciences of the United States of America 55, E2125-E2134 (2018)

[0009] Non-patent document 2: Gallusci P et al., Trends in Plant Science 22, 610-623 (2017)

[0010] Non-patent document 3: Matzke MA and Mosher RA, Nature Reviews Genetics 15, 394-408 (2014)

[0011] Non-patent document 4: Puerta-Fernandez E et al., FEMS Microbiology Reviews 27, 75-97 (2003)

[0012] Non-patent document 5: Liu G et al., Organic & Biomolecular Chemistry 15, 4681-4685 (2017)

[0013] Non-patent document 6: Bussiere F et al., Plant Biotechnology Journal 1, 423-435 (2003)

[0014] Non-patent document 7: Otagaki S et al., Plant Biotechnology 23, 259-265 (2006)

[0015] Non-patent literature 8: Matsunaga W et al., BMC Plant Biology 19, 24 (2019)

[0016] Non-patent literature 9: Philips JG et al., PloS one, 12, e0171311 (2017) Summary of the invention

[0017] Problems to be solved by the invention

[0018] An object of the present invention is to produce plants having desired traits by simply and rapidly inhibiting methylation of target DNA in plants using or without using recombinant technology.

[0019] Means for solving problems

[0020] In view of the above-mentioned problems, the inventors have conducted in-depth research and obtained the following surprising discovery: in the RNA-instructed DNA methylation mechanism, by cutting off the scaffold RNA produced by the transcription of the above-mentioned target DNA, and in particular by expressing one or more ribozymes specific for the above-mentioned scaffold RNA in the above-mentioned plant cells to cut off the scaffold RNA, the methylation of the target DNA in the plant can be specifically inhibited.

[0021] That is, the gist of the present invention is as follows.

[0022] (1) A method for inhibiting methylation of a target DNA in a plant cell, comprising: in an RNA-commanded DNA methylation mechanism, cleaving a scaffold RNA produced by transcription of the target DNA.

[0023] (2) The method according to item 1, wherein the cleavage of the scaffold RNA is performed by expressing one or more ribozymes specific for the scaffold RNA in the plant cell.

[0024] (3) The method according to 2, wherein the expression of the one or more ribozymes is transient.

[0025] (4) The method according to any one of 1 to 3, wherein the target DNA is a promoter of a gene that controls the expression of a desired trait in plant cells.

[0026] (5) The method according to 4, wherein the gene expressing the desired trait is a gene encoding an amino acid sequence of an enzyme that controls the synthesis or accumulation of a plant-derived functional component.

[0027] (6) The method according to any one of 1 to 5, wherein the expression of the one or more ribozymes in plant cells is carried out by a plant virus vector method, an Agrobacterium infiltration method, a magnICON (registered trademark) system, or a particle gun method.

[0028] (7) The method according to any one of 1 to 6, wherein the plant cell is a non-dividing cell of a plant.

[0029] (8) The method according to any one of 1 to 6, wherein the plant cell is a cultured cell.

[0030] (9) A method for producing a plant having a desired trait, comprising: suppressing methylation of DNA involved in expression of the desired trait in the plant using the method according to any one of claims 1 to 8.

[0031] (10) A method for producing a plant-derived functional ingredient, comprising: using the method described in any one of claims 1 to 8 to inhibit the methylation of DNA involved in the synthesis or accumulation of the plant-derived functional ingredient, thereby accumulating the functional ingredient in the above-mentioned plant cells, and recovering the above-mentioned functional ingredient from the above-mentioned plant cells.

[0032] (11) An expression system for accumulating a functional ingredient derived from a plant, comprising:

[0033] (A) a plant body or plant cell that produces a scaffold RNA, wherein the scaffold RNA is produced by transcription of a DNA involved in the synthesis or accumulation of a plant-derived functional component in an RNA-instructed DNA methylation mechanism, and

[0034] (B) One or more ribozymes specific for the above scaffold RNA.

[0035] (12) A ribozyme expression vector comprising a nucleotide sequence encoding one or more ribozymes, wherein the one or more ribozymes are one or more ribozymes specific for scaffold RNA produced by transcription of target DNA in an RNA-commanded DNA methylation mechanism.

[0036] (13) The ribozyme expression vector according to item 12, wherein a nucleotide sequence encoding another self-cleaving ribozyme is adjacent to the 3' and 5' ends of the nucleotide sequence encoding the ribozyme specific for the scaffold RNA.

[0037] (14) The ribozyme expression vector according to item 13, wherein the nucleotide sequence encoding the ribozyme specific to the scaffold RNA is in trans-form, and the nucleotide sequence encoding the other ribozyme capable of self-cleavage is in cis-form.

[0038] (15) A use of the ribozyme expression vector according to any one of claims 12 to 14 for producing a plant having a desired trait.

[0039] (16) Use of the ribozyme expression vector according to any one of claims 12 to 14 for producing a plant-derived functional ingredient.

[0040] Effects of the Invention

[0041] According to the present invention, TGS of target DNA can be specifically controlled simply and quickly by inhibiting methylation of target DNA in plants. Thus, plants with desired traits can be obtained, for example, greatly contributing to the production of useful proteins in plants and the improvement of the accumulation level of functional components. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 A model of DNA methylation in the RdDM pathway is shown.

[0043] Figure 2 A model of DNA methylation relief mediated by ribozyme cleavage of scaffold RNA is shown.

[0044] Figure 3 The diagram shows a method for constructing a CMV-A1 vector containing a 35S promoter sequence and the DNA methylation rate of the 35S promoter in the offspring (208S1) obtained by inoculating the vector into a tobacco ( benthamiana ) GFP gene-introduced line (16C).

[0045] Figure 4 The detection results of the 35S promoter sequence and the scaffold RNA produced by the 35S promoter are shown. The band indicated by the arrow represents the detected scaffold RNA. It should be noted that in the 35S promoter sequence, the sequences indicated by the single line and the double line respectively represent the regions corresponding to the positive strand and the negative strand of the detected scaffold RNA.

[0046] Figure 5 The sequence of the 35S promoter (minus chain) that is a target of the ribozyme and the sequence of the ribozyme are shown.

[0047] Figure 6 The black arrow indicates the cleavage position of the 5' end of the scaffold RNA cleaved by the ribozyme. The gray arrow indicates the cleavage position of the control (208S1).

[0048] Figure 7 Represents GFP fluorescence and methylation of the 35S promoter region in tobacco progeny expressing the ribozyme using a CMV vector. Figure 7 In C, regions near transcription start sites where DNA methylation is particularly reduced are indicated by rectangles.

[0049] Figure 8 The target sequence of the ribozyme for Arabidopsis thaliana introduction is shown. The three GUCs present in the positive strand of the scaffold RNA of the 35S promoter are designated as A, B, and C, respectively.

[0050] Fig. 9 The sequence of the ribozyme 35S(+)A2BC cassette for introduction into Arabidopsis thaliana is shown.

[0051] Fig.10 The structure of the cis-trans-cis ribozyme and the self-cleavage site of the cis-type ribozyme are shown.

[0052] Fig.11 The structure of the expression vector pBGGN used for transformation of Arabidopsis thaliana is shown.

[0053] Fig.12 The figures show the results of investigating the in vitro cleavage activity of the Arabidopsis thaliana introduced ribozyme.

[0054] Fig.13 The results of PCR detection of the ribozyme A2BC box introduced into the Arabidopsis Col-0 GFP-TGS strain are shown. In the electrophoresis photograph, the upper section shows the position of the PCR primers used to confirm whether the ribozyme is integrated in the transformed plant. The lanes in the lower section represent the individual recombinants. The + mark indicates that the ribozyme is confirmed.

[0055] Fig.14 The graph shows the expression changes of the GFP gene in the Arabidopsis thaliana Col-0 GFP-TGS line into which the ribozyme was introduced. DETAILED DESCRIPTION

[0056] [1. Preface]

[0057] Gene expression in plants is controlled by epigenetics. "Epigenetics" refers to changes in gene function that do not cause changes in the DNA sequence and are transmitted via cell division, and their mechanisms. As one of epigenetics, DNA methylation is known (Non-patent Document 2). It has always been believed that there is a reaction in which a methyl group is added to the 5-carbon atom of the pyrimidine ring of cytosine or the 6-nitrogen atom of the purine ring of adenine, and gene expression in plants is mainly controlled by cytosine methylation. That is, by methylation and demethylation of cytosine in DNA, switching between the opening / closing of gene expression is performed without changing the base sequence information itself. This method of inhibiting gene expression by DNA methylation is called transcriptional gene silencing (TGS).

[0058] The present invention is based on the following basic idea: by artificially regulating epigenetic control in plants, it is possible to change the desired plant phenotype without genetic recombination.

[0059] The understanding of DNA methylation in Arabidopsis thaliana, a model plant, is progressing rapidly. In plants, CpG, CpHpG, and CpHpH sites (H represents a nucleotide other than guanine) are methylated, and DRM2, MET1, CMT3, and CMT2 are known as the main DNA methyltransferases that transfer methyl groups to DNA and make them covalently bonded. Currently, DNA methyltransferases are divided into two types: de novo enzymes that form new methyl marks on DNA, and maintenance enzymes that recognize the methylation sites of the DNA parent chain and pass the new methylation to the daughter chains after DNA replication. Among them, only DRM2 is a de novo DNA methyltransferase. It is still unclear how cells determine the sites of de novo DNA methylation, but current evidence shows that the RNA-directed DNA methylation (RdDM) mechanism is involved at multiple sites.

[0060] As described above, the present invention is based on the following discovery: in the RNA-directed DNA methylation mechanism, methylation of the target DNA in the plant can be specifically inhibited by cutting off the scaffold RNA produced by transcription of the target DNA, and in particular, by expressing one or more ribozymes specific to the scaffold RNA in the plant cells to cut off the scaffold RNA. Therefore, the RNA-directed DNA methylation mechanism and ribozymes, which are the premise of the present invention, are first described.

[0061] [1-1.RdDM mechanism]

[0062] like Figure 1As shown, in the RdDM mechanism, PolIV is first recruited to the methylated target genomic region and transcribes the RNA of the target region. The transcription product of PolIV is converted into double-stranded RNA by RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) on the spot, and is cut into 24-nucleotide siRNA by DICER-LIKE3 (DCL3), an enzyme like RNaseIII (non-patent document 3). After the 3' end of the siRNA is methylated by HUA ENHANCER1 (HEN1), it is taken into ARGONAUT 4 (AGO4) to form a silencing effector complex. The complementary siRNA taken into the silencing effector complex forms a base pair with the RNA transcribed by DNA-dependent RNA polymerase V (PolV) (hereinafter referred to as "scaffold RNA") to recruit the complex. Then, through the silencing effector complex, DOMAINS REARRANGEDMETHYLTRANSFERASE (DRM2), which is a de novo methyltransferase, is recruited in the DNA region corresponding to the siRNA to methylate the DNA. It is believed that RNA-DIRECTED DNA METHYLATION 1 (RDM1), DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3), which have the ability to bind to AGO4, DRM2, and methylated DNA, play an important role in the recruitment.

[0063] RNA transcribed from target DNA by PolIV and PolV (these are transcribed from the same region, so the sequences are generally the same, but RNA transcribed by PolV is specifically called scaffold RNA) plays a role in generating siRNA and guiding the methyltransferase DRM2 to the target DNA in the RdDM pathway. Figure 2 As shown, the target DNA is specified by the sequence of the scaffold RNA and the siRNA bound thereto, so if the specific scaffold RNA is cut off and removed from the pathway, only the methylation of the DNA corresponding to the scaffold RNA is inhibited, and then it enters the direction of demethylation. In contrast, the method of inhibiting the function of other proteins involved in the RdDM pathway will destroy the entire RdDM pathway, which is harmful to plants and is not preferred.

[0064] It should be noted that the cleavage position of the scaffold RNA is limited depending on the sequence, and it is necessary to contain sequences such as GUC, GUA, GUU, etc. with high ribozyme cleavage efficiency (Non-Patent Document 1), but as long as this condition is met, the cleavage position can be selected arbitrarily.

[0065] [1-2. Ribozyme]

[0066] As a means of removing the above-mentioned scaffold RNA, a method of sending a large amount of antisense RNA into the nucleus to capture the scaffold RNA is considered. However, antisense RNA may form a double strand with the target and generate siRNA, which is likely to promote TGS instead. In view of such a problem, the inventors have conducted in-depth research and found that the ribozyme produced in large quantities in cells in recent years and used for the cutting of RNA viruses, etc., cuts off the scaffold RNA, thereby successfully inhibiting the methylation of the target DNA in the plant. The ribozyme is complementary to only 8 bases of the scaffold RNA, so it will not form a double strand and will not generate siRNA.

[0067] Ribozymes are special RNA molecules with enzyme activity that cuts nucleic acid sequences site-specifically. Since their discovery in 1981, their structure and function have been extensively studied. As a result, the structure (shape and nucleic acid sequence) of the enzyme active site has been elucidated. Ribozymes that can specifically cut any sequence site have been obtained by chemical synthesis and have been used in a variety of fields. The ribozymes used in the present invention are not particularly limited as long as they can sequence-specifically cut and degrade the scaffold RNA produced by transcription of the target DNA. Such ribozymes can be designed by methods well known to those skilled in the art. As ribozymes that cut target RNA in vivo, hammerhead ribozymes, hairpin ribozymes and pseudoknot ribozymes are known, among which functional RNA based on hammerhead ribozymes has been developed in particular. Hammerhead ribozymes can cut RNA phosphodiester bonds at specific sites. The smallest hammerhead ribozyme of the trans type is made based on natural hammerhead ribozymes and is used to inhibit target gene expression in vivo through RNA-mediated gene control. Hammerhead ribozyme is made up of the following regions: the active region (helix II) constituted by the conservative core sequence with catalytic activity of the central part, the recognition region (helix III and helix I) constituted by 2 heterozygote formation arm sequences of the recognition target sequence present in the 3' side and 5' side of the active region (non-patent literature 4). By transforming the above-mentioned heterozygote formation arm sequence for target RNA according to meeting the simple Watson-Crick base pairing rule, it is possible to make the hammerhead ribozyme of target specificity. In addition, the hammerhead ribozyme with different core sequences can be designed to target RNA, using the sequence of the specific triplet of the target RNA to form a complementary relationship with the heterozygote sequence to be bound to the target RNA, the phosphodiester bond present in the 3' side of the triplet is cut off.

[0068] The sequence required as a substrate for the ribozyme is a triplet with a U at the second nucleotide, and DWH (D=A / U / G, W=A / U, H=A / U / C) is particularly easy to cut (Non-patent Document 5). Therefore, if a part of the sequence corresponding to DWH is synthesized on the target RNA and is complementary to the sequences on both sides, the part becomes an RNA cutting enzyme that cuts the target RNA at the 3' side of DWH. Thus, those skilled in the art can freely design a ribozyme that can sequence-specifically cut and degrade it based on the homology with the target sequence of the scaffold RNA.

[0069] Therefore, in the present invention, a ribozyme is designed that selects a DNA sequence that is involved in the expression of a desired trait in a plant and is suppressed by methylation as a cleavage site and targets the DNA sequence.

[0070] [2. Method for inhibiting methylation of target DNA in plant cells]

[0071] According to a first aspect of the present invention, there is provided a method for inhibiting methylation of a target DNA in a plant cell, comprising: cleaving a scaffold RNA produced by transcription of the target DNA in an RNA-commanded DNA methylation mechanism.

[0072] The scaffold RNA is an inducer of DNA methylation that inhibits the expression of desired traits in plants. By specifically cutting the scaffold RNA, the recruitment of DRM2 to the DNA region corresponding to the siRNA is inhibited, thereby specifically inhibiting the methylation of the target DNA in the plant. For example, by introducing one or more ribozymes specific to the scaffold RNA into the plant cells and expressing the one or more ribozymes in the plant cells, the scaffold RNA can be specifically cut.

[0073] The target DNA is not particularly limited as long as it is a DNA that participates in the expression of the desired trait in the plant and is inhibited by methylation, and examples thereof include promoters of genes that control the expression of the desired trait in plant cells. The desired traits include, for example, properties observed as appearance (morphology) such as shape, color, size, physiological properties observable such as flowering period, and physiological properties such as resistance to pathogens and temperature, and are preferably properties related to the accumulation of functional components (metabolic components) in the plant. Therefore, the gene expressing the desired trait is preferably a gene encoding the amino acid sequence of an enzyme that controls the synthesis or accumulation of plant-derived functional components.

[0074] Examples of plant-derived functional ingredients include acetylene, thiophene, glycoside, thioglycoside, purine, pyrimidine, alkaloid, phenols (e.g., quinones), essential oils, vitamins, terpenes (e.g., iridoids, sesquiterpenes, diterpenes, and triterpenes), lignin, and flavonoids.

[0075] The expression of ribozymes in plant cells is preferably carried out using recombinant techniques or transient expression systems for simplicity and rapidity, for example, by plant virus vector method, Agrobacterium infiltration method, magnICON (registered trademark) system, particle gun method, or a combination thereof.

[0076] The plant virus vector method is a method in which the cDNA of the plant virus genome into which the target DNA is inserted is transcribed in a test tube, the resulting RNA is inoculated into plants as a vector for infection, and the plant is expressed with the target gene by utilizing the virus's own proliferation ability and overall migration ability. Since this method utilizes the virus's self-replication ability to express the target gene, if a vector based on CMV or TMV, which has a particularly high proliferation ability, is used, the expression amount of the target gene per plant cell can be increased.

[0077] Agrobacterium infiltration is a method in which the culture solution of Agrobacterium transformed with a T-DNA vector inserted with a target gene is introduced into plant tissues by physical methods (injection with a syringe, etc., penetration by decompression, etc.) to infect the plant, thereby causing the target gene to be transiently expressed in the plant. According to this method, Agrobacterium with strong infectivity is generally migrated to the entire plant body by physical methods (injection or penetration) to infect it, and the target gene can be expressed equally in all tissues of the plant. Agrobacterium is a general term for fungi that are pathogenic to plants in the genus Rhizobium in soil bacteria belonging to Gram-negative bacteria. As an example of Agrobacterium, Agrobacterium tumefaciens related to root nodule disease can be cited. When using an Agrobacterium-derived vector to make a plant transiently express an exogenous gene, the vector is usually introduced into plant tissues by physical methods (injection with a syringe, etc., penetration by decompression, etc.) to infect the plant. Agrobacterium-derived vectors have strong infectivity due to the T-DNA region, so if they are physically transferred to the entire plant body for infection (injection, infiltration, etc.), all tissues of the plant can be uniformly expressed with foreign genes.

[0078] The magnICON (registered trademark) system is a method in which the cDNA of the TMV or PVX genome into which the target gene is inserted is introduced into a T-DNA vector, the obtained T-DNA vector is used to transform Agrobacterium, and the obtained culture solution of Agrobacterium is introduced into plant tissues by a physical method (injection by a syringe, etc., penetration by decompression, etc.) to infect the plant, thereby causing the target gene to be transiently expressed in the plant. That is, the entire plant body can be generally infected with the vector by a physical method (injection or penetration), and the target gene can be expressed in all tissues of the plant. In addition, since the target gene is expressed by utilizing the self-replication ability of the virus (TMV or PVX), the expression amount of the target gene in each plant cell can be increased. Thus, this method is a method having the advantages of both the above-mentioned plant virus vector method and the Agrobacterium infiltration method.

[0079] In addition, the sequence of the RNA2 genome of cucumber mosaic virus (CMV) equivalent to replacing part or all of the gene encoding 2b protein with an exogenous gene is functionally combined with the T-DNA sequence of Agrobacterium, and the resulting nucleic acid molecule is introduced into a host plant that functionally expresses the RNA1 genome and RNA3 genome of CMV and protein 2b, and the plant is cultivated to express the exogenous gene, so that the exogenous gene can be universally migrated to the cells of the entire plant body and expressed, and the expression efficiency of the exogenous gene in each cell can be improved, achieving high expression of the entire plant body (Patent Document 1). By using this method, it can become a technology comparable to the above-mentioned magnICON (registered trademark) system using TMV and PVX, and can increase the freedom of host plant species and the size of exogenous genes that can be introduced.

[0080] The particle bombardment method is a method in which microparticles of a metal such as gold or tungsten coated with DNA or a vector are ejected at high speed to introduce target DNA into cells.

[0081] The ribozyme expression vector used in the present invention is not particularly limited as long as it contains a nucleotide sequence encoding one or more ribozymes specific to the scaffold RNA produced by transcription of the target DNA and can express the ribozyme in plant cells. Typically, it is a plant virus vector or a T-DNA vector, and a plant virus vector is particularly preferred. The plant virus vector is not particularly limited as long as it can invade the nucleus of the plant cell, and examples thereof include various single-stranded RNA viruses such as tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), potato virus X (PVX), clover vein yellowing virus (ClYVV), bean yellow dwarf virus (BeYDV), beet curly top virus (BCTV), cabbage leaf-curl virus (CaLCuV), wheat dwarf virus (WDV), tomato yellow leaf curl China virus (TYLCCNV), and vectors derived from double-stranded DNA viruses such as cauliflower mosaic virus (CaMV). Among them, cucumber mosaic virus (CMV)-derived vectors, which are RNA viruses, are particularly preferred, and examples thereof include CMV-A1 vectors.

[0082] There is no particular limitation on the plant species to which the present invention can be applied, and typically plants of the Poaceae, Leguminosae, Cruciferae, Asteraceae, Solanaceae, Rosaceae, Cucurbitaceae, Convolvulaceae, etc. can be cited. Preferred plants include, for example, alfalfa, barley, kidney bean, rapeseed rape, cowpea, cotton, corn, clover, lotus, lentil, lupine, millet, oat, pea, peanut, rice, rye, sweet osmanthus, sunflower, sweet pea flower, soybean, sorghum, triticale, yam, edamame, broad bean, wheat, wisteria, nut plants, etc.; Arabidopsis, bran grass, onion, snapdragon, celery, peanut, asparagus, poppy, oat, bamboo, rapeseed, brome, bush violet), camellia, hemp, chili, chickpea, quinoa, chicory, citrus, coffee tree, kawaguchi, cucumber, pumpkin, dogtooth grass, orchard grass, datura, digitalis, yam, oil palm, goosegrass, fine-leaved grass, strawberry, old cranesbill, soybean, sunflower, yellow alfalfa, rubber tree, henbane, sweet potato, lettuce, flat beans, lilies, flax, ryegrass, lotus, tomatoes, oregano, apples, mangoes, cassava, alfalfa, South American seaside fish fillet, tobacco, red bean grass, geranium, pennisetum, petunia, candle grass, bluegrass, cherry, buttercup, radish, gooseberry, castor, raspberry, sugarcane, purple velvet Panax notoginseng, cinnamon, foxtail grass, whitehead, eggplant, corn, blunt-leaved grass, cocoa tree, clover, tonka bean, grapes, etc.

[0083] Plant cells may be in any form, and may be non-isolated cells existing in the plant body, or cultured cells such as tissue culture isolated from the plant body. In the case of cultured cells, they may be differentiated cells, dedifferentiated cells, or redifferentiated cells.

[0084] [3. Method for producing a plant having desired traits]

[0085] By using the above method to inhibit methylation of target DNA, methylation of DNA involved in the expression of desired traits in plants can be inhibited, thereby producing plants with desired traits. Therefore, the second aspect of the present invention provides a method for producing plants with desired traits, which comprises: using the above method to inhibit methylation of DNA involved in the expression of desired traits in plants.

[0086] [4. Method for producing plant-derived functional ingredients]

[0087] In addition, by using the above-mentioned method for inhibiting the methylation of the target DNA to inhibit the methylation of the DNA involved in the synthesis and accumulation of the functional components of the plant, the functional components of the plant can be simply and quickly produced. Therefore, the third aspect of the present invention provides a method for producing a functional component of the plant, which comprises: using the above-mentioned method to inhibit the methylation of the DNA involved in the synthesis and accumulation of the functional components of the plant, thereby accumulating the functional components in the above-mentioned plant cells, and recovering the above-mentioned functional components from the above-mentioned plant cells; and providing an expression system, which is an expression system for synthesizing and accumulating the functional components of the plant, comprising: (A) a plant body or plant cell that produces a scaffold RNA, wherein the scaffold RNA is produced by the transcription of the DNA involved in the synthesis and accumulation of the functional components of the plant in the RNA-instructed DNA methylation mechanism; and (B) one or more ribozymes specific to the above-mentioned scaffold RNA.

[0088] [5. Ribozyme expression vector]

[0089] According to a fourth aspect of the present invention, there is provided a ribozyme expression vector comprising a nucleotide sequence encoding one or more ribozymes, wherein the one or more ribozymes are one or more ribozymes specific to scaffold RNA produced by transcription of target DNA in an RNA-commanded DNA methylation mechanism. The ribozyme expression vector can be used to produce plants having desired traits and / or to produce functional ingredients of plant origin as described above.

[0090] The ribozyme expression vector used in the present invention may have the following structure: the nucleotide sequence encoding other ribozymes capable of self-cleavage is adjacent to the 3' and 5' ends of the nucleotide sequence encoding the ribozyme specific to the scaffold RNA. The nucleotide sequence encoding the ribozyme specific to the scaffold RNA is set to the trans type, and the nucleotide sequence encoding the other ribozyme capable of self-cleavage is set to the cis type, thereby the target ribozyme monomer can be cut out by the self-cleaving ribozyme (Non-patent Document 6). Thus, the trans-type target ribozyme can be sandwiched between the cis-type ribozymes and multiple ribozymes can be configured, thereby increasing the expression amount of the same target ribozyme and expressing heterogeneous target ribozymes at the same time.

[0091] In addition, the ribozyme expression vector may comprise a promoter and / or terminator operably linked to one or more ribozymes encoding specificity of the scaffold RNA and nucleotide sequences encoding other self-cleaving ribozymes linked to the 3' and 5' ends thereof.

[0092] The above promoter is not particularly limited as long as it can express ribozymes in plant cells, and examples thereof include U6 promoter, 35S promoter of cauliflower mosaic virus (CaMV), cassava mosaic virus promoter, figwort mosaic virus promoter, Badnavirus promoter, strawberry vein banding virus (SVBV) promoter, cauliflower (Mirabilis) mosaic virus promoter (MMV), Rubisco promoter, Actin promoter, ubiquitin promoter, etc. Among them, U6 promoter is preferred.

[0093] The terminator is not particularly limited as long as it can express the ribozyme in plant cells, and examples thereof include U6 terminator, nos terminator and heat shock protein (hsp) terminator derived from Agrobacterium, and 35S terminator of cauliflower mosaic virus (CaMV).

[0094] The present invention will be described in more detail below with reference to the following examples. However, the present invention is not limited to the following examples and can be implemented by appropriately modifying the examples.

[0095] Example

[0096] [Experiment 1] DNA methylation induction using CMV-A1 vector containing a partial sequence of the 35S promoter

[0097] (1) Construction of CMV-A1 vector containing a partial sequence of the 35S promoter

[0098] A sequence with StuI and MluI restriction enzyme sites added to the end of the partial sequence (35S-208) of the 35S promoter from the transcription start point to 208 bp upstream was artificially synthesized and integrated into the cloning site (StuI and MluI restriction enzyme sites) of the infectious cDNA cloning plasmid of the CMV-A1 vector (see Non-Patent Document 7) according to a conventional method ( Figure 3 a). 35S-208 was prepared by PCR amplification and cloning from pBI121 (Clontech). The full length of the 35S promoter in pBI121 is 345 bases, and 208 bases including the as-1 site were amplified and cloned to obtain 35S-208 (see Non-Patent Document 8).

[0099] (2) Inoculation of CMV-A1 with 35S-208 into tobacco 16C strain

[0100] The CMV-A1 vector (RNA2) integrated with 35S-208 was transcribed in vitro using T7 RNA polymerase. The vector was mixed with RNA1 and RNA3 obtained by in vitro transcription from CMV-Y RNA1 infectious clone pCY1 and RNA3 infectious clone pCY3, and mechanically inoculated into the GFP gene-introduced strain 16C of Nicotiana benthamiana (see Non-Patent Document 9) ( Figure 3 b) From the CMV-infected 16C individuals, individuals whose GFP fluorescence disappeared due to TGS were selected, and seeds were collected after flowering. The individuals from the seeds were designated as 208S1.

[0101] (3) DNA methylation in the 35S promoter region of 208S1

[0102] DNA was extracted from leaves of 16C and 208S1 using illustra DNA Extraction Kit Nucleon Phytopure (GE Healthcare), and the extracted DNA was treated with bisulfite using EZ DNA Methylation-Lightning Kit (ZymoResearch) to convert unmethylated cytosine to uracil. The 35S promoter region was amplified by PCR from the bisulfite-treated DNA using TaKaRa EpiTaq HS for bisulfite-treated DNA (TaKaRa). As primers for PCR, 35S-346F-bisuT (5'-ATTGAGAYTTTTYAAYAAAGGGTA-3': sequence number 7) and 35S+1A-bisuA (5'-CTCTCCAAATGAAATGAACTTC-3': sequence number 8) were used. The obtained PCR product was ligated into the pTAC1 vector using Dyna Express TA PCR Cloning Kit (Bio Dynamics Laboratory). The plasmid was transformed into E. coli JM109 Competent Cells (TaKaRa) and the plasmid was extracted after proliferation. The extracted plasmid was sequenced according to the conventional method. Based on the sequencing results, the methylation frequency of cytosine in the 35S promoter region was compared between 16C and 208S1 according to the categories of CG, CHG (H = A / C / T) and CHH sites. The results confirmed that methylation of cytosine was induced in 208S1 at any site ( Figure 3 c).

[0103] [Experiment 2] Detection of scaffold RNA of 35S promoter

[0104] In order to detect scaffold RNA transcribed from the 35S promoter, RNA was extracted from leaves of the 208S1 line using RNAzol RT (Molecular Research Center), and the following RT-PCR was performed on the extracted total RNA using SuperScript III First Strand Synthesis SuperMix (Invitrogen). First, RT reaction was performed using primers T7-35S-5-345 (5'-ATTGAGACTTTTCAACAAAG-3': sequence number 9) for detecting the positive strand of RNA and 35S+1R (5'-GTTCTCTCCAAATGAAATGAAC-3': sequence number 10) for detecting the negative strand. Then, PCR was performed using a primer pair of T7-35S-5-345 and 35S-3-130 (5'-GCAGAGGCATCTTCAACGATG-3': sequence number 11) for detecting the positive strand and a primer pair of 35S-5-160 (5'-CCACCCACGAGGAGCATCGTG-3': sequence number 12) and 35S+1R for detecting the negative strand. As a result of RT-PCR, amplification products were obtained from both the positive strand and the negative strand, and their sequencing was consistent with the sequence of the 35S promoter ( Figure 4 ), thus confirming that transcription was carried out from both the positive and negative strands of the 35S promoter.

[0105] [Experiment 3] Construction of CMV vector expressing ribozyme

[0106] (1) Ribozyme design

[0107] The target sequence of the ribozyme and the sequence of the ribozyme are shown in Figure 5 . According to the results of Experiment 2, it can be confirmed that the positive and negative strands of the scaffold RNA are transcribed from the 35S promoter region, but the GUC that is most easily cut in the triplet that becomes the substrate of the ribozyme is mostly present in the negative strand of the scaffold RNA, so the ribozyme was designed with the negative strand of the scaffold RNA as the target. Considering the GC content and the presence or absence of repetitive sequences in the sequence bound by the ribozyme, the GUC present at 79 bp upstream of the transcription start point was selected as the target GUC, and a ribozyme that cuts this GUC was designed.

[0108] (2) Preparation of CMV vector for ribozyme expression

[0109] In order to synthesize and clone the double-stranded DNA encoding the ribozyme designed in (1), oligoDNAs 35S(-)-5RZ-45 (5'-CGAGGCCTGATCTCCACTCTGATGAGTCCGTGAGGACGA AACGTA-3': SEQ ID NO: 13) and 35S(-)-3RZ-32 (5'-CGCACGCGTCCTTACGTTTCGTCCTCACGGAC-3': SEQ ID NO: 14) were synthesized to which StuI and MluI restriction enzyme sites were added to the 5' end, respectively, and these were made into double strands by PCR. These were then integrated into the cloning site (StuI and MluI restriction enzyme sites) of the infectious cDNA cloning plasmid of the CMV-A1 vector according to a conventional method.

[0110] [Experiment 4] Determination of the cleavage position of the ribozyme scaffold RNA

[0111] (1) Inoculation of 208S1 strain with CMV-A1 containing ribozyme

[0112] The CMV-A1 vector (RNA2) with integrated ribozyme was transcribed in vitro with T7 RNA polymerase. It was mixed with RNA1 and RNA3 transcribed in vitro from CMV-Y RNA1 infectious clone pCY1 and RNA3 infectious clone pCY3, and mechanically inoculated into the 208S1 strain. In the case of 208S1, methylation was induced on the 35S promoter, and TGS of GFP was stably generated. S1 refers to the first generation of self-propagation of TGS individuals.

[0113] (2) Determination of the cleavage position of the negative strand of the scaffold RNA

[0114] RNA was extracted from leaves of healthy 208S1 individuals (208S1) and 208S1 individuals infected with a CMV-A1 vector expressing a ribozyme (208S1Rz(-)) using RNAzol RT (Molecular Research Center), and the extracted total RNA was used for 5'RACE to determine the sequence of the 5' end of the negative strand of the scaffold RNA. 5'RACE was performed using a 5' / 3'RACE Kit, 2nd Generation (Roche). Regarding the 5' end of the negative strand of the scaffold RNA of 208S1 used as a control, 13 of the 17 clones were detected at 8 bp upstream of the transcription start site of the GFP gene, and 4 clones were detected at 151 bp downstream of the transcription start site of the GFP gene ( Figure 6). This result indicates that the transcription of the negative strand of the scaffold RNA containing the 35S promoter region is initiated from the GFP gene. On the other hand, in the 208S1 Rz(-) individual, the 5' end was detected at 80 bp upstream of the GFP transcription start point in 6 of the 15 clones, 83 bp upstream in 6 of the clones, and 240 bp upstream in 3 of the clones ( Figure 6 ). This position is located downstream of the transcription start position of the scaffold RNA negative chain in the control, so it is believed that the scaffold RNA negative chain in the 208S1 Rz(-) individual is cut by the ribozyme. Through the cutting of the scaffold RNA negative chain, it can be expected that the demethylation of the 35S promoter is induced in the 208S1 Rz(-) individual. Then, self-propagated seeds were collected from the 208S1 Rz(-) individual, and the DNA methylation of the 35S promoter region was analyzed using the next generation (208Rz(-)S2) that was not infected with the virus (Experiment 5).

[0115] [Experiment 5] GFP fluorescence and methylation of the 35S promoter region in the offspring of Nicotiana benthamiana 16C strain expressing ribozyme via CMV vector

[0116] The GFP fluorescence of the next generation (208S2) of 208Rz(-)S2 and the control 208S1 was compared. As a result, the restoration of GFP fluorescence was observed in the offspring of N. benthamiana expressing the ribozyme using the CMV vector ( Figure 7 a). Since GFP fluorescence was restored in 208Rz(-)S2 compared to 208S2, the frequency of cytosine methylation in the 35S promoter region was analyzed by the same method as that performed in Experiment 1 (3). The results showed that the frequency of methylation at any of the CG, CHG, and CHH sites in 208Rz(-)S2 decreased compared to 208S2 ( Figure 7 b). In addition, the methylation pattern of the 35S promoter region was observed, and the demethylation near the transcription start point in 208Rz(-)S2 was particularly significant ( Figure 7 c) These results show that the cleavage of the scaffold RNA by the ribozyme expressed by the CMV vector can induce the demethylation of the 35S promoter region and increase the expression of the GFP gene. Furthermore, the demethylation state of the 35S promoter region induced by this method will be stably inherited to the next generation.

[0117] [Experiment 6] Construction of a ribozyme expression plasmid vector for Arabidopsis thaliana transfection

[0118] (1) Ribozyme design

[0119] The sequences of the ribozyme target sequence and the ribozyme cassette are shown in Figure 8 and Fig. 9In order to simultaneously cleave the three GUCs (A, B, and C) on the positive strand of the scaffold RNA of the 35S promoter, a cis-trans-cis ribozyme was designed. Fig.10 , a structure is set in which a ribozyme (trans-type) that cleaves the target sequence in trans is sandwiched by a self-cleaving ribozyme (cis-type), and each trans-type ribozyme becomes a monomer and exhibits activity due to the self-cleavage of the adjacent cis-type ribozyme. Here, a box (ribozyme 35S(+)-A2BC) is designed in which each trans-type ribozyme that cleaves GUC at positions A, B, and C is sandwiched by one cis-type.

[0120] (2) Preparation of ribozyme expression plasmid vector

[0121] A double-stranded DNA sequence of the ribozyme 35S(+)-A2BC designed in (1) was artificially synthesized, with a U6 promoter added to the 5' side, a U6 terminator added to the 3' side, and HindIII and SacI restriction enzyme sites added to both ends, respectively. Fig. 9 ). It was integrated into the cloning site (HindIII and SacI restriction enzyme sites) of the expression vector pBGGN (Inplanta Innovations Inc.) according to conventional methods ( Fig.11 ). The pBGGN vector has a BASTA resistance gene (bar gene) integrated therein as one of the selection markers, and transformed plants can be selected by spraying a 0.01% BASTA solution onto the plants.

[0122] [Experiment 7] In vitro cleavage activity of ribozymes introduced into Arabidopsis

[0123] The 35S promoter sequence and ribozyme 35S(+)-A2BC were amplified by PCR using primers to which T7 promoter sequences were added (forward primer for 35S promoter sequence: 5'-CTAATACGACTCACTATAGGGAGACAGCTATGACCATGATT ACGCCAAGC-3' (sequence number 15); reverse primer for 35S promoter sequence: 5'-ACCATGGATCCTCTAGAGTCGACTG-3' (sequence number 16); forward primer for ribozyme 35S(+)-A2BC: 5'-CTAATACGACTCACTATAGGGAGAAGTAGTGATTGGGCGGA C-3' (sequence number 17); reverse primer for ribozyme 35S(+)-A2BC: 5'-CGCCATTGGGATGAGCTCAA-3' (sequence number 18)), and transcription was performed using T7 RNA polymerase (CUGA7 in vitro transcription kit) using the obtained PCR product as a template. 10 μg of the ribozyme transcription product was denatured at 95°C for 2 minutes in 50 μL of reaction solution (50 mM Tris-HCl), and then cooled suddenly. Then, MgCl2 was added to a final concentration of 50 mM, and then incubated at 37°C for 3 hours to allow the cis-type ribozyme to self-cleave. 2.1 μg of monomerized ribozyme was added to 900 ng of 35S promoter RNA fragment in 10 μL of reaction solution to which Tris-HCl was added to a final concentration of 50 mM, and the mixture was denatured at 95°C for 2 minutes and cooled suddenly. MgCl2 (final concentration 50 mM) was added to start the reaction, and then incubated at 37°C for 3 hours to use the trans-type ribozyme to cleave the 35S promoter RNA fragment ( Fig.12 ).

[0124] [Experiment 8] Preparation of Arabidopsis thaliana with ribozyme introduced

[0125] In order to investigate the effect of ribozyme cutting scaffold RNA when introduced by gene recombination, first, a strain in which the 35S promoter region of the GFP gene is methylated is prepared as follows. A construct with the GFP gene integrated downstream of the 35S promoter of the expression vector pBE2113 is prepared, and it is introduced into the Arabidopsis Col-0 strain by the Agrobacterium method. Transformants are screened on kanamycin (50μg / ml) medium. Next, from the transformed individuals (T1 generation), a strain (Col-0GFP-TGS) is selected in which more than 3 copies of the GFP gene are introduced and the expression level is excessive, resulting in inducing methylation of the 35S promoter and almost completely inhibiting the expression of the GFP gene in the T4 generation. The ribozyme expression vector prepared in Experiment 6 is introduced into the Col-0GFP-TGS strain by the Agrobacterium method. Transformed individuals are screened by spraying 0.01% BASTA about the second week after sowing. Since false positive individuals are easily obtained in screening by spraying BASTA, DNA was extracted from the screened individuals according to conventional methods, and PCR was performed on the extracted DNA using a primer pair of ribo-insert-check-F (5'-CCCCTAAGTGGGATATTAAGTCAAG-3': sequence number 19) and ribo-insert-check-R (5'-TATCCCACTTAGGGGTTTGGAAAC-3': sequence number 20) to confirm the introduction of the ribozyme 35S-A2BC ( Fig.13 ).

[0126] [Experiment 9] Analysis of GFP gene expression in Col-0 GFP-TGS individuals into which ribozymes were introduced

[0127] In the T1 individuals introduced with the ribozyme 35S-A2BC obtained in Experiment 8, the positive strand of the scaffold RNA of the 35S promoter was cut by the ribozyme, thereby inducing demethylation of the 35S promoter region, and it was predicted that the expression of the GFP gene would increase. The expression of the GFP gene was compared between individuals introduced with the ribozyme 35S-A2BC and individuals not introduced. The results confirmed that the GFP expression in individuals introduced with the ribozyme 35S-A2BC increased by up to 2 times compared with individuals not introduced. Fig.14). The expression level of the GFP gene was measured by quantitative RT-PCR (GeXP Expression Analysis System, AB Science) using total RNA extracted from leaves using RNAzol RT (Molecular Research Center). The gene used as the internal standard was the UBQ10 gene, and the primer pairs for GFP and UBQ10 genes used in RT-PCR were GeXP-GFP-F (5'-AGGTGACACTATAGAATATATATCATGGCCGACAAGCA-3': sequence number 21), GeXP-GFP-R (5'-GTACGACTCACTATAGGGATGGGTGCTCAGGTAGTGGTT-3': sequence number 22) and GeXP-Atubq10-F (5'-AGGTGACACTATAGAATATTGGCCGACTACAACATTCA-3': sequence number 23), GeXP-Atubq10-R (5'-GTACGACTCACTATAGGGAAAGTGATGGTCTTTCCGGTG-3': sequence number 24).

Claims

1. A method for inhibiting methylation of 35S promoter in plant cells, wherein the method comprises: In the RNA-commanded DNA methylation mechanism, a step of cutting off the scaffold RNA produced by transcription of the 35S promoter, The step of cutting the scaffold RNA is a step of introducing one or more ribozymes specific to the scaffold RNA into the plant cell and expressing the ribozymes in the plant cell, and The plant is of the family Poaceae, Leguminosae, Cruciferae, Asteraceae, Solanaceae, Rosaceae, Cucurbitaceae or Convolvulaceae.

2. The method according to claim 1, wherein: The plant is selected from the group consisting of barley, kidney beans, cowpeas, cotton, corn, clover, lotus, lentils, lupines, millet, oats, peas, peanuts, rice, rye, sweet osmanthus, sunflowers, sweet peas, soybeans, sorghum, triticale, yam, edamame, broad beans, wheat, wisteria, nut plants, Arabidopsis, bran grass, onions, snapdragons, celery, asparagus, poppy, Penglai bamboo, rapeseed, brome, bulohuali, camellia, hemp, peppers, chickpeas, quinoa, chicory, citrus, coffee trees, chuangu, cucumbers, pumpkins, and bermudagrass. , orchard grass, datura, digitalis, yam, oil palm, goosegrass, fine-leaved grass, strawberry, old crane grass, rubber tree, henbane, sweet potato, lettuce, lily, flax, ryegrass, tomato, oregano, apple, mango, cassava, alfalfa, South American sea cucumber fillet, tobacco, red bean grass, geranium, pennisetum, petunia, candle grass, bluegrass, cherry, buttercup, radish, gooseberry, castor, raspberry, sugarcane, purple velvet Panax notoginseng, cinnamon, foxtail grass, white caltrop, eggplant, corn, blunt-leaved grass, cocoa tree, clover, tonka bean and grape.

3. The method according to claim 1, wherein: The plant is selected from the group consisting of rapeseed rape, alfalfa and yellow alfalfa.

4. The method according to claim 1, wherein: The expression of the one or more ribozymes is transient.

5. The method according to claim 1, wherein: The one or more ribozymes are introduced into the plant cells by plant virus vector method, Agrobacterium infiltration method or particle gun method.

6. The method according to claim 1, wherein: The plant cell is a non-dividing cell of a plant.

7. The method according to claim 1, wherein: The plant cells are cultured cells.

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