KR20100053804A - Method for producing reactivation plants of transcriptionally silenced gene using hc/pro gene - Google Patents

Abstract

PURPOSE: A recombinant plant expression vector containing SMV(soybean mosaic virus)-derived S-Hc/Pro(helper component protein) gene is provided to reactivate gene function deleted by TGS(transcriptional gene silencing) by transforming a plant. CONSTITUTION: A recombinant plant expression vector contains SMV(soybean mosaic virus)-derived S-Hc/Pro(helper component protein) gene. The S-Hc/Pro gene comprises a nucleotide of sequence number 1. The gene function that deleted by TGS in a plant is reactivated by transforming with the recombinant plant expression vector. The plant is dicotyledonous plant. A composition for reactivating gene function deleted by TGS in a plant contains SMV-derived S-Hc/Pro gene.

Description

Method for producing reactivation plants of transcriptionally silenced gene using Hc / Pro gene}

The present invention relates to a method for producing a plant that inhibits reactivation and silencing of the TGS gene using an Hc / Pro suppressor gene.

Gene silencing is caused by transcription of 21-27 nt RNA in the form of RISC to degrade target genes (PTGS, Post Transcriptional Gene Silencing), inhibit translation, or enter the nucleus and modify the target DNA. It is a mechanism for regulating gene expression by mechanisms such as TGS (transcriptional gene silencing). This phenomenon is found in most eukaryotes, from single-celled organisms, such as chlamydomonas and yeast to higher plants and humans, primarily to protect their genome from attacks by viruses, bacteria and transposons. It is also known to function to regulate the expression of one's genes in response to developmental processes or stress (Paddison PJ. (2008) Curr Top Microbiol Immunol. 2008; 320: 1-19; Meins et al. (2005) Annu) Rev Cell Dev Biol 21: 297-318; Phillips et al. (2007) FEBS Letters, 581 (19), 3592-3597).

This gene silencing mechanism is also associated with diseases of living organisms, including humans, so the study of this mechanism and its use (RNAi technology) have played an important role in the study and treatment of diseases as well as in basic research (Tan and Yin, 2004, Cell Res. Dec; 14 (6): 460-6; Huang, 2008, Curr HIV / AIDS Rep. 2008 Feb; 5 (1): 33-9). In addition, the gene silencing mechanism is an obstacle to lowering the production efficiency and productivity of transformants in the transformation technology used in the production of useful substances, proteins, and new varieties by transgene silencing expression of transformed foreign genes in eukaryotes. It is becoming.

Therefore, there is a need for a technique that can understand and regulate these gene silencing mechanisms. Several proteins have been found in viruses that inhibit gene silencing. By using such suppressor protein, it is possible to control the expression of genes including foreign genes, and thus can be usefully used for basic research and disease-related research, so that gene expression using various gene silencing inhibitors in animals and plants Research on regulation is ongoing.

Inhibitors such as HC-pro, CMV2b, TBP19, and Tcv-cp (gene silencing inhibitors) not only suppress silence progression but also reactivate silenced genes established by the PTGS mechanism. However, there is no known case of reactivating the silenced gene established by TGS mechanism. In the case of PTGS, studies on the mechanism of PTGS and its use have been developed through studies using various inhibitors as well as mutants. On the other hand, when the silence is in progress, it is not known whether it is via the TGS mechanism or the PTGS mechanism.

In general, when silence is established by the IR structure, when methylation is newly introduced into the promoter region, it is known as silence by the TGS mechanism. However, until now only mutant research has been possible in TGS, there is a limit to the development of understanding and use of the mechanism.

Korean Patent Publication No. 2003-0004305 discloses a target nucleic acid comprising a target nucleotide sequence operably linked to a promoter together with a post-transcriptional gene silencing (PTGS) inhibitor protein as a method for increasing expression from a target nucleotide sequence in an organism. A method comprising temporarily introducing a structure into a cell of an organism is disclosed, but is different from the TGS reactivation method using the Hc / Pro gene of the present invention.

The present invention has been made in accordance with the above-mentioned requirements. In the present invention, the inhibitory effect of PTGS is relatively weak when using HC-pro derived from a virus causing mild disease in soybean, but silenced by suppressing the silence established in TGS. The present invention was completed by showing the results of re-expressing the traits that had been used.

In order to solve the above problems, the present invention provides a recombinant plant expression vector comprising a soybean mosaic virus (SMV) -derived helper component protein (S-Hc / Pro) gene.

In addition, the present invention provides a plant and its seed that have been transformed with the vector with the vector and the gene function that has been lost by transcriptional gene silencing (TGS) has been reactivated.

In addition, the present invention provides a method for reactivating gene function lost by TGS by transforming a plant with the vector.

The present invention also provides a composition for reactivating gene function lost by TGS in a plant, including the S-Hc / Pro gene.

The method of inducing reactivation of TGS silencing established in the present invention will be useful for developing application technology through understanding TGS mechanism.

In order to achieve the object of the present invention, the present invention provides a recombinant plant expression vector comprising a soybean mosaic virus (SMV) -derived helper component protein (S-Hc / Pro) gene.

The recombinant plant expression vector may be, for example, 13 Hc-pro vector described in FIG. 1, but is not limited thereto.

In recombinant plant expression vectors, the term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer a portion of themselves, the so-called T-region, to plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous, especially when it is difficult to properly transform a plant host.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol It doesn't happen.

In the plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

The terminator may be a conventional terminator, and examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

S-Hc / Pro gene according to the present invention may be preferably composed of a nucleotide sequence represented by SEQ ID NO: 1. In addition, variants of such sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in base sequence but has similar functional properties to that of SEQ ID NO: 1. Specifically, the S-Hc / Pro gene has at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1 It may include a base sequence.

The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a plant in which a gene function which has been transformed with the vector of the present invention and lost by transcriptional gene silencing (TGS) has been reactivated. For example, when the npt gene is silenced by the TGS mechanism and loses kanamycin resistance, the tomato plant is transformed using a recombinant vector containing the S-Hc / Pro gene of the present invention to recover kanamycin resistance lost by TGS. You can.

The plant according to the present invention is a monocotyledonous plant such as rice, barley, corn, wheat, rye, oats, grass, forage, millet, sugar cane, rice, orchardgrass, or Arabidopsis, potato, eggplant, tobacco, pepper, tomato , Burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mustard, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, green bean, kidney bean, or pea It may be a dicotyledonous plant, but preferably a dicotyledonous plant, more preferably a tomato.

The present invention also provides seed of the plant. Preferably, the seed is a seed of a dicotyledonous plant, more preferably a tomato seed.

The present invention also provides a method for reactivating gene function lost by transcriptional gene silencing (TGS) comprising transforming a plant with a vector of the present invention to express the S-Hc / Pro gene. to provide. For example, when the npt gene is silenced by the TGS mechanism and loses kanamycin resistance, the tomato plant is transformed using a recombinant vector containing the S-Hc / Pro gene of the present invention to recover kanamycin resistance lost by TGS. You can.

In the method of the present invention, the plant may be a monocotyledonous plant or a dicotyledonous plant, preferably a dicotyledonous plant, more preferably a tomato.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74), electroporation of protoplasts (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102 ), Microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), particle bombardment methods (DNA or RNA-coated) of various plant elements (Klein TM et al., 1987, Nature 327, 70), infection with (incomplete) virus in agrobacterium tumerfaciens mediated gene transfer by invasion of plants or transformation of mature pollen or vesicles, and the like. have. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell. Preferably, the plant is tomato.

"Plant tissue" refers to the tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.

The present invention also provides a composition for reactivating gene function lost by transcriptional gene silencing (TGS) in a plant, including S-Hc / Pro gene derived from soybean mosaic virus (SMV). Since the S-Hc / Pro gene plays a role of reactivating gene function lost by transcriptional gene silencing (TGS) in plants, the composition can be used for TGS reactivation and silence suppression. The S-Hc / Pro gene may be preferably composed of the nucleotide sequence of SEQ ID NO: 1. The plant is preferably a dicotyledonous plant, more preferably a tomato.

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

Example

Example 1 Transformation Vector Construction with S-Hc / Pro Gene

S-Hc / pro gene isolated from SMV showing mild disease was introduced into pET28a vector (Novagen) to determine sequencing, and 1385bp DNA cut with restriction enzyme NotI was treated with Klenow enzyme to have smooth ends. It was prepared so that the insertion of the 13 35S-NOS vector (strain vector of pCAMBIA1300) SmaI position to produce a higher plant transformation vector 13 Hc / pro (Fig. 1).

Example 2 S-Hc / Pro Gene Transformation and Hc / pro Analysis

Germline of Lycopersicon esculentum Mill cv.Joyful germinated in MS medium of line 11 (Jung et al., 2007, J Plant Biotech 34: 323-329), in which wild type and NPTII genes were silencing by TGS mechanism Transformation was performed by coculture with cotyledon segments and Agorbacterium tumefacience LBA4404 with 13 Hc / Pro vector. Shoots developed with resistance to hygromycin after 1 month of culture in MS medium supplemented with 2 mg / L zeatine, 0.1 mg / L IAA, 350 mg / L carbenicillin and 50 mg / L hygromycin The roots were selected by induction of root in MS medium supplemented with 350 mg / L carbenicillin, 100 mg / L kanamycin, 0.5 mg / L indole-butryic acid (IBA). The selected seedlings were transferred to pots and grown to harvest later seeds (FIG. 2).

The leaves of tomato plants grown in greenhouse conditions were collected and analyzed by separating DNA and RNA. Genomic DNA isolation, PCR, electrophoresis, Southern blot analysis, and Northern blot analysis used standard methods (Sambrook and Russell 2001 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press). 60 μg of genomic DNA isolated from transgenic tomatoes was treated with EcoR I restriction enzyme and used for Southern blot analysis. DNA fragments for the preparation of the probe was prepared by the transformation method 13-Hc / Pro as a template and PCR using the primers. 659 bp S-Hc / Pro fragment was PCR method using HC-pro F (5'-GCTCATATGGGCTGTCATGG-3 '; SEQ ID NO: 2) and HC-pro R (5'-TGCGTGACACAACAACAAGG-3'; SEQ ID NO: 3) primers Amplified and separated. Afterwards, it was labeled with [α- ³² P] dCTP and used as a probe, and exposed using Imaging Plate (Fujifilm, Japan) or X-ray film. As a result, a line into which Hc / pro was introduced was selected (FIG. 2).

Since total RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA), 30 ㎍ of total RNA was used for Northern blot analysis. The expression of S-Hc / pro was then examined using the same probe used for Southern blot analysis. As a result, Hc / pro was introduced to select well expressed lines.

Example 3 Recovery of Kanamycin Resistance Lossed by TGS in S-Hc / Pro Transformants

To determine if S-Hc / Pro has a function of restoring TGS in plants, the subsequent seed of the line in which 13-Hc / Pro was introduced into line 11 where the nptII gene was silenced by the TGS mechanism and lost kanamycin resistance was 50 Germination tests were performed in medium supplemented with mg / L kanamycin. As a result, after about three weeks, the seed line (line 11) which did not show resistance to kanamycin, the control, showed a characteristic that the primary root was elongated but the lateral root did not develop (FIG. 3). However, when S-Hc / pro was introduced into line 11, the development of the lateral roots was confirmed, indicating that the resistance to kanamycin was restored (FIG. 3).

The stems of these plants were cut and transferred to a fresh medium to which kanamycin was added. As a result, the control line 11 was sensitive to kanamycin, so that no root was generated. However, when S-Hc / pro was introduced, new roots were generated from 3-4 days, and the recovery of kanamycin resistance was confirmed again. These results suggest that the S-Hc / pro gene used in the present invention is weak in general silence inhibitory effect, but has a new function of reactivating the nptII gene silenced by TGS.

Therefore, in the present invention, although the inhibitory effect of PTGS is relatively weak when using HC / pro derived from a virus causing mild disease in soybean, the results of reexpressing silenced traits by suppressing the silencing established in TGS are shown. Showed. The method of inducing reactivation of TGS silencing established in the present invention will be useful for understanding the mechanism of TGS and developing its application technology (disease research and treatment, increase expression efficiency of foreign genes and increase transformation efficiency).

1 is a schematic diagram of a recombinant plant expression vector comprising the S-Hc / Pro gene of the present invention.

2 is a diagram showing the production of transgenic tomatoes expressing S-Hc / Pro of the present invention.

3 is a diagram showing the recovery of kanamycin resistance by S-Hc / Pro of the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for producing reactivation plants of transcriptionally          silenced gene using Hc / Pro gene <130> PN08231 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1371 <212> DNA <213> Soybean mosaic virus <400> 1 atgcaaaatc ctgaagctca gtttttccgt ggttggaaga aggtgtttga taaaatgccc 60 cctcatgtgg agaatcatga ttgcaccatt gatttcacaa gtgaacaatg tggtgaattg 120 gcagcggcaa taagccaatc aatctttcca gttaagaaac tatcatgtaa gcaatgtcgg 180 cagcacatta agaacctcag ttgggaggag tataagcaat tcctcttagc tcatatgggc 240 tgtcatggga ccgaatggga aactttccag gaaattgatg gcatgaggta tgtgaagaaa 300 gtgattgaga catcaactgc ggagaacgca agtctgcaga catcacttga gattgtgcgt 360 ttaacgcaaa actataagag cactcacatg cttcaaatac aggatattaa taaggccctg 420 atgaagggtc catcggtaac acagagtgag ctggagcaag cttccaagca gctgctcgca 480 atgacacagt ggtggaagaa tcacatggct ttgactgatg aggatgcact taaagtgttc 540 aggaataaga gatcttctaa agcactactc aatccaagtt tactttgtga taaccagttg 600 gacaagaatg gcaattttgt ttggggagag cgtggcaggt attcaaagcg attctttgca 660 aattattttg aagaggtggt tccttctgag gggtacagca agtatgtgat tagaaagaat 720 ccaaacggac aaagggagtt ggcgattggg tcactcattg tgccgttgga ttttgagcgc 780 gctcgaatgg cattacaggg caagagcgta acaagagagc caattacaat gtcatgtatt 840 tcaagacaag acgggaactt tgtgtatcct tgttgttgtg tcacgcatga tgatggaaaa 900 gctttctatt ctgagctcaa gagtcccaca aagcgccact tggttattgg aacatctggc 960 gacccaaaat acattgatct accagccact gatgcagaca ggatgtacat agctaaagaa 1020 ggattttgtt atcttaacat tttcttggca atgttggtta atgtaaatga agatgaggcc 1080 aaagatttca cgaagatggt aagggatgtt attgtaccaa ggttaggaaa gtggccaaca 1140 gtgttagatg tagcaacggc tgcatacatg ctaacagttt ttcatcctga aaccagaaat 1200 gctgagctcc cacgtatttt ggttgaccat gcgtgtcaaa ccatgcacgt gattgactct 1260 tttggatcct tgacagttgg gtaccatgtt cttaaagctg gtacagtgaa tcaattaatt 1320 caatttgctt ctaatgacct tcagagtgag atgaagttct acagagttta a 1371 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HC-pro forward primer <400> 2 gctcatatgg gctgtcatgg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> HC-pro reverse primer <400> 3 tgcgtgacac aacaacaagg 20  

Claims (8)

Recombinant plant expression vector comprising S-Hc / Pro (helper component protein) gene derived from soybean mosaic virus (SMV). The vector of claim 1, wherein the S-Hc / Pro gene consists of a nucleotide sequence of SEQ ID NO: 1. A plant that has been transformed with the vector of claim 1 to the vector and has been deactivated by TGS (transcriptional gene silencing). The plant according to claim 3, which is a dicotyledonous plant. Seeds of plants according to claim 3. A method for reactivating gene function lost by transcriptional gene silencing (TGS) comprising transforming a plant with the vector of claim 1 to express the S-Hc / Pro gene. The method of claim 6, wherein the plant is a dicotyledonous plant. Composition for reactivating gene function lost by transcriptional gene silencing (TGS) in a plant, including S-Hc / Pro gene derived from soybean mosaic virus (SMV).

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