WO2011108794A2 - Gene regulating cytokinesis, plants transformed with the gene, and method for regulating growth of plants using same - Google Patents

Gene regulating cytokinesis, plants transformed with the gene, and method for regulating growth of plants using same Download PDF

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WO2011108794A2
WO2011108794A2 PCT/KR2010/007322 KR2010007322W WO2011108794A2 WO 2011108794 A2 WO2011108794 A2 WO 2011108794A2 KR 2010007322 W KR2010007322 W KR 2010007322W WO 2011108794 A2 WO2011108794 A2 WO 2011108794A2
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gene
atsh3p
plant
represented
recombinant vector
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PCT/KR2010/007322
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French (fr)
Korean (ko)
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WO2011108794A3 (en
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황인환
김혜란
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포항공과대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield

Definitions

  • the present invention relates to a gene for controlling cytoplasmic division, a plant transformed with the gene, and a method for controlling growth of a plant using the same.
  • the AtSH3P-P2 gene is able to regulate the growth of a plant when the gene is involved in cellular division and regulates its expression. Revealed to complete the present invention.
  • one example of the present invention provides an AISH3P-P2 protein represented by the amino acid sequence of SEQ ID NO: 3.
  • Another example provides an AtSH3P-P2 gene encoding the AtSH3P-P2 protein.
  • Another example provides a recombinant vector comprising the AtSH3P-P2 gene.
  • Another example provides a plant transformed with the recombinant vector. Another example provides seeds of the transformed plant.
  • Another example provides a plant growth promoter containing at least one AtSH3P-P2 protein, an AtSH3P-P2 gene encoding the same, and a recombinant vector comprising the gene as an active ingredient.
  • AtSH3P-P2 protein Another example provides the use of the AtSH3P-P2 protein, the AtSH3P-P2 gene encoding the same, and at least one plant growth promoter selected from the group consisting of a recombinant vector comprising the gene.
  • AtSH3P-P2 protein of the present invention includes a protein having the amino acid sequence of SEQ ID NO: 3 and a functional equivalent of the protein.
  • the functional equivalent is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 3.
  • substantially homogeneous physiological activity refers to the growth of plants when overexpressed in the plant. When promoted or when expression is suppressed in a plant, it means an activity in which plant growth is inhibited.
  • AtSH3P-P2 gene of the present invention is not limited thereto as long as it can encode the AtSH3P-P2 protein, and includes both genomic DNA and cDNA.
  • the AISH3P-P2 gene represented by the nucleotide sequence of SEQ ID NO: 2 and variants thereof.
  • the AtSH3P-P2 gene variant has a nucleotide sequence having 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: 2. It may include.
  • the '% sequence homology' of the nucleotide sequence is identified by comparing two optimally arranged sequences with a comparison region, wherein a portion 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 AtSH3P-P2 gene of the present invention is not limited thereto. It is preferred to have the nucleotide sequence of the number 2 (see ⁇ Example 1>).
  • the recombinant vector of the present invention comprises the AtSH3P-P2 gene.
  • said recombinant vector is a recombinant plant expression vector.
  • the term "recombinant” refers to a cell replicating, expressing a heterologous nucleic acid or expressing a protein encoded by the heterologous nucleic acid.
  • the 'vector' is used when referring to DNA fragment (s), nucleic acid molecules that are delivered into cells.
  • the vector replicates DNA and can be reproduced independently in a host cell.
  • the expression vector or recombinant vector refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.
  • the 'plant expression vector (recombinant vector expressible in plants)' is not limited thereto, but when present in a suitable host such as Agrobacterium tumerfaciens, a portion of itself, a so-called T-region that can transfer T-regions to plant cells
  • a plasmid vector and a preferred form may be the so-called binary vector described in EP0120516B1 and US Pat. No. 4,940,838.
  • Other suitable plant expression vectors can also be selected from viral vectors, such as those that may be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc., for example incomplete plant virus vectors. .
  • the use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.
  • the plant expression vector is used frequently in the art pBI101 (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121 and pCAMBIA vector. Most preferably it is a pCAMBIA vector (see ⁇ Example 2>).
  • the recombinant vector of the invention 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 herbicide resistance genes such as glyphosate or phosphinothricin (phosphinothricin), kanamycin, G418, bleomycin, hygromycin, chloramphenicol There are antibiotic resistance genes such as, but not limited to.
  • the promoter that may be included in the recombinant vector of the present invention is a promoter capable of initiating transcription in plant cells, and preferably may be capable of overexpressing a gene inserted into the plant. More preferably, it may be a 'constitutive promoter' which is active under most environmental conditions and developmental conditions or cell differentiation. That is, the constitutive promoter is preferable because the selection of the transformant can be made by various tissues at various stages. Even more preferably the promoters that may be included in the expression vector of the invention are CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoters. May be, but is not limited thereto.
  • 'promoter' refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.
  • the terminator that can be included in the recombinant vector of the present invention may use a conventional terminator, such as nopalin synthase (NOS), rice ⁇ -amylase RAmyl A terminator, phaseoline (phaseoline) terminator, agrobacterium Terminator of the octopine gene of agrobacterium tumefaciens, but is not limited thereto.
  • NOS nopalin synthase
  • rice ⁇ -amylase RAmyl A terminator phaseoline (phaseoline) terminator
  • phaseoline phaseoline terminator
  • agrobacterium Terminator of the octopine gene of agrobacterium tumefaciens but is not limited thereto.
  • such regions are generally known to increase the certainty and efficiency of transcription in plant cells.
  • the recombinant vector in one embodiment of the present invention is prepared by inserting the AtSH3P-P2 gene having the nucleotide sequence of SEQ ID NO: 2 into a pCAMBIA vector using CaMV 35S as a promoter and nopaline synthase (NOS) as a terminator. . More specifically, the recombinant vector was prepared by removing a stop codon (TGA) of the AtSH3P-P2 gene and inserting a construct linking the initiation codon (AUG) of the green fluorescent protein (GFP) into a pCAMBIA vector. It can be displayed on a map, it can be confirmed whether the expression of the AtSH3P-P2 gene through GFP (see ⁇ Example 2>).
  • the transformed plant of the present invention is transformed with the recombinant vector will be.
  • Any transformation method can be used for transferring the nucleic acid to the plant for transforming the plant.
  • the transformation method is not limited thereto but is preferably a calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), 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.
  • Agrobacterium tumerfaciens mediated gene transfer by transformation can be used as appropriately selected from (incomplete) viral infection (EP 0 301 316) and the like.
  • Preferred methods according to the invention may be Agrobacterium mediated transformation methods (see ⁇ Example 3>).
  • Plants that can be transformed by introducing the AtSH3P-P2 gene in the present invention is not limited thereto, but may be food crops, vegetable crops, special crops, fruit trees, flowers and feed crops, preferably rice, wheat, barley, Corn, beans, potatoes, eight, oats, sorghum, baby pole, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot, ginseng, tobacco, cotton, sesame, candy Sorghum, Beet, Perilla, Peanut, Rape, Apple tree, Pear, Jujube, Peach, Yard, Grape, Citrus, Persimmon, Plum, Apricot, Banana, Rose, Gladiolus, Gerbera, Carnation, It may be selected from the group consisting of chrysanthemum, lily, lily, lygras, red clover, orchardgrass, alfalfa, pescue and perennial lice.
  • AtSH3P-P2 gene in the present invention can be effectively expressed in the plant, including the baby pole, to promote plant growth, the transformed plant of the present invention may be much better than the non-transformed plant.
  • the seed of the present invention is a seed produced by culturing the transformed plant.
  • the seed of the transformed plant may be introduced by the AtSH3P-P2 gene, thereby promoting the growth of the seed.
  • the plant growth promoter of the present invention contains at least one selected from the group consisting of the AtSH3P-P2 protein of the present invention, the AtSH3P-P2 gene encoding the same, and a recombinant vector comprising the gene as an active ingredient.
  • the AtSH3P-P2 protein, the AtSH3P-P2 gene encoding the same, and a recombinant vector comprising the gene can effectively promote plant growth by inducing cytoplasmic division of the plant.
  • AtSH3P-P2 gene is not limited thereto but preferably It may be represented by the nucleotide sequence of SEQ ID NO: 2.
  • the recombinant vector is not limited thereto, but may be preferably represented by the cleavage map of FIG. 1.
  • the AtSH3P-P2 gene is expressed at the cell plate position, that is, the AtSH3P-P2 gene is present in the cell plate appearing at the cytoplasmic division after the cell division stage of nuclear division, the AtSH3P-P2 gene
  • the AtSH3P-P2 gene is involved in cytoplasmic division, and furthermore, the AtSH3P-P2 gene maintains the cytoplasmic division since the position where the AtSH3P ⁇ P2 gene is expressed is maintained from the initial cell plate formation until the cell plate growth progresses and the cellular division is completed. There is inducing activity (see ⁇ Example 4>).
  • the growth of the leaves and roots in the transgenic plant is significantly superior to the wild type, the root length is longer than 30% compared to the wild type, the transgenic plant to the wild type Larger leaf sizes and longer root lengths indicate that the AtSH3P-P2 gene effectively induces cellular division and promotes plant growth (see Example 5).
  • the AtSH3P-P2 protein, the AtSH3P-P2 gene encoding the same, and a recombinant vector including the gene can effectively promote plant growth by inducing cytoplasmic division of the plant.
  • the method of controlling the growth of the plant of the present invention is the AtSH3P-P2 It is a way to control the growth of plants by controlling the expression of genes.
  • the plant growth regulation includes not only promoting plant growth by inducing expression of the AtSH3P-P2 gene, but also inhibiting plant growth by inhibiting the expression of the gene.
  • the plant growth may be promoted by introducing the AtSH3P-P2 gene into a recombinant vector and expressing or overexpressing the AtSH3P-P2 gene by transforming the plant with the recombinant vector.
  • the 'overexpression of the gene' means that the AISH3P-P2 gene is expressed above the level expressed in wild-type plants.
  • Recombinant vectors and transformation methods capable of expressing or overexpressing the gene are as described above, and the recombinant vector is not limited thereto but may be represented by the cleavage map of FIG. 1.
  • the method of controlling the growth of the plant of the present invention includes the step of inducing the expression of the AtSH3P-P2 protein coding gene represented by the amino acid sequence of SEQ ID NO: 3, it may be characterized by promoting the growth of the plant have.
  • Transforming the plant with the recombinant vector may be performed by the signed plant transformation method.
  • Plants that may be transformed by introducing the AtSH3P-P2 gene may be food crops, vegetable crops, special crops, fruit trees, flowers and feed crops, preferably rice, wheat, barley, corn, Beans, potatoes, arms, oats, sorghum, baby poles, cabbage, radish, peppers, strawberries, tomatoes, watermelons, cucumbers, cabbage, melons, pumpkins, green onions, onions, carrots, ginseng, tobacco, cotton, sesame seeds, sugar cane, Beet, Perilla, Peanut, Rapeseed, Apple tree, Pear, Jujube, Peach, Yard, Grape, Chisel, Persimmon, Plum, Apricot, Banana, Rose, Gladiolus, Gerbera, Carnation, Chrysanthemum, Lily, Yurilip, Ryegrass It may be selected from the group consisting of, red clover, orchard grass, alpha wave, vesque cue and perennial grass.
  • the AtSH3P-P2 gene in the present invention can be effectively expressed in the plant, including the baby pole, to promote plant growth, the transformed plant was able to grow much better than the untransformed plant.
  • the AtSH3P-P2 gene is expressed at the cell plate position, that is, the AtSH3P-P2 gene is after nuclear division during the cell division stage. Since the AtSH3P-P2 gene is involved in cytoplasmic division, the position where the AtSH3P-P2 gene is expressed is at the initial cell plate formation and cell plate growth proceeds to complete the cell division.
  • the AtSH3P-P2 gene has an activity of inducing cytoplasmic division since it is maintained until it is maintained (see ⁇ Example 4>).
  • the growth of leaves and roots in the transgenic plant is much superior to the wild type, the root length is longer than 30% compared to the wild type, the transgenic plant to the wild type Larger leaf sizes and longer root lengths indicate that the AtSH3P-P2 gene effectively induces cellular division and promotes plant growth (see Example 5).
  • plant growth can be suppressed by suppressing the expression of the AtSH3P-P2 gene.
  • dsRNA can be used to inhibit expression of the AtSH3P-P2 gene.
  • the dsRNA is a high specificity target of the AtSH3P-P2 gene.
  • RNA (primary or processed) can be specifically targeted and the dsRNA can be used to effectively inhibit the expression of these AtSH3P-P2 genes.
  • a vector capable of effectively expressing the dsRNA in plants and Transformation methods using the same are known in the art, and preferably
  • the pTA7002 expression vector represented by the cleavage map of 6 can be used.
  • dsRNA was prepared by expressing 300 bases of the N-terminus in the base sequence of the AtSH3P-P2 gene, and the expression of the AtSH3P-P2 gene was suppressed using the AtSH3P-P2 gene.
  • Expression of the AtSH3P-P2 protein is not able to function as a system, and cytoplasmic division does not occur normally after nuclear division. Furthermore, less differentiated cell walls appear due to the failure of cytoplasmic division, which prevents normal cell layer formation, thereby inhibiting plant growth (see Example 6).
  • the AtSH3P-P2 gene has an activity of inducing cytoplasmic division, and thus the transformed plant can be promoted for growth, and thus can be effectively used for improving the biomass of the plant.
  • FIG. 1 shows a cleavage map of a pCAMBIA expression vector to which a gene of AtSH3P-P2 and a GFP coupled to confirm the expression of the gene.
  • Figure 2 is the result of performing Western blot to confirm whether the gene of AtSH3P-P2 is expressed in plants transformed with AtSH3P-P2.
  • FIG. 3 is a result confirming that the AtSH3P-P2 protein is expressed in the cell plate position in plants transformed with AtSH3P-P2.
  • Figure 4 is a result of comparing the development of plants and wild type leaves and roots transformed with AtSH3P-P2 and wild type.
  • 5 is a result of comparing the root length change of plants transformed with AtSH3P-P2 to wild type after 6 days and 10 days after sowing.
  • 2P # 1 Transgenic Plant Corresponding to Line 1 in Western Blots
  • 2P # 2 Transgenic Plant Corresponding to Line 2 in Western Blots
  • FIG. 6 shows pTA7002 expression inhibiting AtSH3P-P2 gene expression. The cleavage map of the vector is shown.
  • Figure 7 is the result of observing the intracellular nucleus (a) and cell wall formation (b) in plants in which AtSH3P-P2 gene expression is suppressed.
  • the cDNA of AtSH3P-P2 is composed of 18 nucleotide sequences in 5 'ATG and 18 nucleotide sequences in front of 3' TGA as sense and antisense primers, respectively.
  • cDNA of AtSH3P-P2 was extracted by PCR amplification (30 cycles consisting of 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 30 seconds at 72 ° C), and the sequence was determined. It was confirmed by sequencing known in the art. ⁇ 1-2> Experimental Results
  • the gene sequence of AtSH3P-P2 has the nucleotide sequence represented by SEQ ID NO: 1, AtSH3P-P2 It was found that cDNA had a nucleotide sequence represented by SEQ ID NO: 2. In addition, according to SEQ ID NO: 2 it can be seen that the amino acid sequence of AtSH3P-P2 is represented by SEQ ID NO: 3.
  • Example 1> Based on the cDNA of the AtSH3P-P2 gene analyzed in ⁇ Example 1> was prepared an expression vector that can be used for the production of transgenic plants of the gene.
  • the termination codon (TGA) of the AtSH3P-P2 gene was removed, and the initiation codon (AUG) of the green fluorescent protein (GFP) was linked, and inserted between the 35s promoter of pCAMBIA 1300 vector (Cambia Labs) and the nos (nopaline synthease).
  • TGA termination codon
  • AVG initiation codon
  • GFP green fluorescent protein
  • the cDNA of AtSH3P-P2 was cloned into the 326-sGFP vector.
  • a 5 'AtSH3P-2 sGFP Xbal primer of SEQ ID NO: 4 was prepared by adding an Xbal restriction enzyme sequence in front of 5' ATG, and a BamHl restriction enzyme sequence was added to remove the 3 'termination codon (TGA).
  • 5 '3' AtSH3P-2 sGFPBamHl primers were prepared.
  • AtSH3P-P2 cDNA library using the two primers (30 seconds at 94 ° C, 30 seconds at 55 ° C, 30 cycles consisting of 30 seconds at 72 ° C) of AtSH3P-P2 cDNA was extracted and cloned by cutting the 326-sGFP vector with two Xbal, BamHl restriction enzymes (Bioneer, Korea or NEB).
  • the prepared AtSH3P-P2: sGFP sequence was again confirmed through sequencing.
  • the pCAMBIA expression vector to which the gene of AtSH3P-P2 and GFP which can confirm the expression of the gene is bound is represented by the cleavage map described in FIG. 1.
  • the Arabidopsis thaliana plants transformed by the Agrobacterium mage transformation method were prepared using the pCAMBIA vector prepared in ⁇ Example 2>.
  • the transformation method is the Floral dip method (The Plant Journal (1998) 16 (6), 735-743).
  • the hygromycin resistance test was carried out to select first.
  • the Western blot was used for 10% SDS-PAGE and the expression was examined using a GFP specific antibody that recognizes GFP at the end of AtSH3-P2: sGFP.
  • FIG. 2 The results of performing the western blot are shown in FIG. 2, and as shown in FIG. 2, it can be seen that the gene of AtSH3-P2 is effectively expressed in the form of a fusion protein of GFP. That is, it was confirmed that a protein of the correct size of 70 kDa was synthesized. Through this, it was possible to secure a stable transgenic plant line.
  • Each line in FIG. 2 means each plant transformed.
  • AtSH3P-P2 protein is expressed at the cell plate position.
  • the AtSH3P-P2 protein is present in the cell plate appearing at the time of cytoplasmic division after nuclear division and nuclear division. Therefore, the AISH3P-P2 protein is involved in cytoplasmic division, Since the position to be expressed is maintained from the initial cell plate formation until cell plate growth proceeds to complete cell division, AtSH3P-P2 protein can be seen that it can play an essential function in cytoplasmic division.
  • the root length of the transgenic plants was measured on the 6th and 10th day after seeding, respectively, and randomly screened 30 times with wild type. Deviations were obtained and plotted.
  • the transgenic baby pole plant Root length is longer than 30% compared to wild type.
  • the size of the leaves and the length of the roots of the transgenic baby pole plants are larger than those of the wild type in the same time period, and the AtSH3-P2 protein effectively induces cellular division and promotes plant growth.
  • AtSH3P-P2 residues were suppressed using a gene expression suppression system that induced double stranded RNA.
  • pTA7002 expression vector having a cleavage map as shown in FIG. was prepared.
  • the expression vector can inhibit the expression of the AtSH3-P2 gene as needed by dexamethasone (dexamethasone).
  • the baby pole plants were transformed in the same manner as in ⁇ Example 3-1>.
  • the transformed plants were isolated and screened by the hygromycin resistance test, and the isolated and screened plants were germinated in 1/2 MS medium, and 30M dexamethasone was able to act on the gene expression suppression system on the 4th day after germination. Transfer to this containing 1/2 MS medium and incubated for 3 more days.

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Abstract

The present invention relates to a gene regulating cytokinesis, plants transformed with the gene, and a method for regulating growth of plants using the same. As examined above, an AtSH3P-P2 gene has an activity for inducing cytokinesis, thereby promoting the growth of plants transformed with the same, and thus the gene can be effectively used to improve biomass of plants.

Description

【명세서】  【Specification】
【발명의.명칭】  [Invention. Name]
세포질 분열을 조절하는 유전자， 상기 유전자로 형질 전환된 식물 및 이를 이용한 식물의 생장을 조절하는 방법  Genes that control cytoplasmic division, plants transformed with the genes and methods of controlling growth of plants using the same
【기술분야】  Technical Field
본 발명은 세포질 분열을 조절하는 유전자, 상기 유전자로 형질 전환된 식물 및 이를 이용한 식물의 생장을 조절하는 방법에 관한 것이다. 【배경기술】  The present invention relates to a gene for controlling cytoplasmic division, a plant transformed with the gene, and a method for controlling growth of a plant using the same. Background Art
종래에는 식물의 생장을 향상시키기 위하여 다량의 화학비료 및 농약을 살포하였으나, 상기 방법은 각종화학비료의 살포로 인해 토양이 점차 산성화되어 식물의 생장이 오히려 저조해져 갈수록 작황이 나빠질 뿐만 아니라 농약을 과다하게 살포함으로 인하여 식물에 유해한 농약성분이 잔류하게 됨으로써 소비자의 건강에 악영향을 미친다는 중대한 문제점이 발생되게 되었다.  Conventionally, a large amount of chemical fertilizers and pesticides were sprayed to improve plant growth. However, in the above method, the soil is gradually acidified due to the spraying of various chemical fertilizers, and the growth of plants is rather low, and the crops are deteriorated. By spraying the water, harmful pesticides remain in the plant, which is a serious problem that adversely affects the health of consumers.
특히 최근 식물은 단순한 식량으로써의 이용 가치와 함께, 바이오 플랜트로써의 이용 가치가 증가되고 있다. 구체적으로 유용한 물질을 생체 내에서 합성시키기 위해 많은 미생물이나 동물 세포들이 이용되고 있으나, 투자 대비 대량 생산이 용이한 점을 고려하면 식물이 가장 효과적이다. 이에 다양한 바이오 벤처와 다국적 회사에서 식물을 바이오 플랜트로 연구하고자 시도하고 있다. 그러나 바이오 물질의 공급처로써의 식물의 이용은 그 기본 가치인 식량으로써의 이해 관계와 상충되므로， 식물의 바이오 매스 증가를 증가시켜야만 식량 자원으로써의 기본 효용을 안정적으로 층족시키면서, 바이오 물질 공급을 위한 플랜트로써의 이용성을 확보할 수 있게 된다. In particular, in recent years, the value of plants as a food plant, as well as the use of simple food is increasing. Although many microorganisms or animal cells are used to synthesize useful substances in vivo, plants are most effective in consideration of easy mass production for investment. Therefore, various bio ventures and multinational companies are trying to study plants as bio plants. However, the use of plants as a source of biomaterials is in conflict with its basic value of food interests, so increasing the plant's biomass growth should increase plant biomass, while stably striking the basic utility as a food resource. As a result, usability can be secured.
따라서 화학 비료를 사용하지 않으면서 식물의 생장을 조절할 수 있는 방법을 개발하는 것이 절실히 필요한 실정이다.  Therefore, there is an urgent need to develop a method for controlling plant growth without using chemical fertilizers.
【발명의 상세한 설명】  [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
이에 본 발명자들은 화학비료를 사용하지 않고, 식물의 바이오 매스를 증가시킬 수 있는 방법에 관하여 연구하던 중, AtSH3P-P2 유전자가 세포질 분열에 관여하고 이의 발현을 조절하는 경우 식물의 생장을 조절할 수 있음을 밝혀 본 발명을 완성하였다.  Therefore, while the present inventors are studying a method of increasing the biomass of a plant without using a chemical fertilizer, the AtSH3P-P2 gene is able to regulate the growth of a plant when the gene is involved in cellular division and regulates its expression. Revealed to complete the present invention.
【기술적 해결방법】  Technical Solution
따라서, 본 발명의 일례는 서열번호 3의 아미노산 서열로 표시되는 AISH3P-P2 단백질을 제공한다.  Thus, one example of the present invention provides an AISH3P-P2 protein represented by the amino acid sequence of SEQ ID NO: 3.
다른 예는 상기 AtSH3P-P2 단백질을 암호화하는 AtSH3P-P2 유전자 제공한다.  Another example provides an AtSH3P-P2 gene encoding the AtSH3P-P2 protein.
또 다른 예는 상기 AtSH3P-P2 유전자를 포함하는 재조합 백터를 제공한다.  Another example provides a recombinant vector comprising the AtSH3P-P2 gene.
또 다른 예는 상기 재조합 백터로 형질 전환된 식물을 제공한다. 또 다른 예는 상기 형질 전환된 식물의 종자를 제공한다. Another example provides a plant transformed with the recombinant vector. Another example provides seeds of the transformed plant.
또 다른 예는 상기 AtSH3P-P2 단백질 ,·이를 암호화하는 AtSH3P-P2 유전자 및 상기 유전자를 포함하는 재조합 백터로 이루어진 군에서 선택된 1종 이상을 유효성분으로 함유하는 식물 생장 촉진제를 제공한다.  Another example provides a plant growth promoter containing at least one AtSH3P-P2 protein, an AtSH3P-P2 gene encoding the same, and a recombinant vector comprising the gene as an active ingredient.
또 다른 예는 상기 AtSH3P-P2 단백질, 이를 암호화하는 AtSH3P-P2 유전자 및 상기 유전자를 포함하는 재조합 백터로 이루어진 군에서 선택된 1종 이상의 식물 생장 촉진을 위한 용도를 제공한다.  Another example provides the use of the AtSH3P-P2 protein, the AtSH3P-P2 gene encoding the same, and at least one plant growth promoter selected from the group consisting of a recombinant vector comprising the gene.
또 다른 예는 상기 AtSH3P-P2 유전자의 발현을 조절함으로써 식물의 생장을 조절하는 방법을 제공한다. 이하 본 발명을 보다 상세히 설명한다. 본 발명의 AtSH3P-P2 단백질은 서열번호 3의 아미노산 서열을 가지는 단백질 및 상기 단백질의 기능적 동등물을 포함한다.  Another example provides a method of controlling plant growth by regulating the expression of the AtSH3P-P2 gene. Hereinafter, the present invention will be described in more detail. AtSH3P-P2 protein of the present invention includes a protein having the amino acid sequence of SEQ ID NO: 3 and a functional equivalent of the protein.
상기 '기능적 동등물 '이란 아미노산의 부가， 치환 또는 결실의 결과, 상기 서열번호 3으로 표시되는 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상， 더욱 바람직하게는 90%이상, 더 더욱 바람직하게는 As a result of the addition, substitution or deletion of the amino acid, the functional equivalent is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 3. Makes
95% 이상의 서열 상동성을 갖는 것으로, 서열번호 3으로 표시되는 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. '실질적으로 동질의 생리활성'이란 식물체 내에서 과다 발현되는 경우 식물의 생장이 촉진되거나， 식물체 내에서 발현이 억제되는 경우 식물의 생장이 억제되는 활성을 의미한다. It refers to a protein having 95% or more of sequence homology and exhibiting substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 3. 'Substantially homogeneous physiological activity' refers to the growth of plants when overexpressed in the plant. When promoted or when expression is suppressed in a plant, it means an activity in which plant growth is inhibited.
또한 본 발명의 AtSH3P-P2 유전자는 상기 AtSH3P-P2 단백질을 암호화할 수 있는 것이라면 이에 한정되지 않으며 게놈 DNA 및 cDNA를 모두 포함한다. 바람직하게는 서열번호 2의 염기 서열로 표시되는 AISH3P-P2 유전자 및 이의 변이체를 포함한다.  In addition, the AtSH3P-P2 gene of the present invention is not limited thereto as long as it can encode the AtSH3P-P2 protein, and includes both genomic DNA and cDNA. Preferably the AISH3P-P2 gene represented by the nucleotide sequence of SEQ ID NO: 2 and variants thereof.
상기 AtSH3P-P2 유전자 변이체는 서열번호 2의 염기 서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다. 상기 염기서열의 '서열 상동성의 %'는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열 (추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제 (즉, 갭)를 포함할 수 있다. The AtSH3P-P2 gene variant has a nucleotide sequence having 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: 2. It may include. The '% sequence homology' of the nucleotide sequence is identified by comparing two optimally arranged sequences with a comparison region, wherein a portion 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).
본 발명의 일실시예에서 서열번호 1의 전체 AtSH3P-P2 유전자에서 In one embodiment of the invention in the entire AtSH3P-P2 gene of SEQ ID NO: 1
AISH3P-P2 단백질을 코딩할 수 있는 cDNA를 분리하고 이의 염기서열을 분석한 결과, 상기 서열번호 2의 염기서열을 가짐을 알 수 있으며, 따라서 본 발명의 AtSH3P-P2 유전자는 이에 한정되지 않지만 상기 서열번호 2의 염기서열을 가짐이 바람직하다 (<실시예 1> 참조). As a result of isolating the cDNA encoding the AISH3P-P2 protein and analyzing its nucleotide sequence, it can be seen that it has the nucleotide sequence of SEQ ID NO: 2. Therefore, the AtSH3P-P2 gene of the present invention is not limited thereto. It is preferred to have the nucleotide sequence of the number 2 (see <Example 1>).
본 발명의 재조합 백터는 상기 AtSH3P-P2 유전자를 포함한다. 바람직하게는 상기 재조합 백터는 재조합 식물 발현 백터이다. The recombinant vector of the present invention comprises the AtSH3P-P2 gene. Preferably said recombinant vector is a recombinant plant expression vector.
상기 '재조합'은 세포가 이종의 핵산을 복제하거나, 발현하거나 또는 이종의 핵산에 의해 암호된 단백질을 발현하는 것을 지칭하는 것이다.  The term "recombinant" refers to a cell replicating, expressing a heterologous nucleic acid or expressing a protein encoded by the heterologous nucleic acid.
상기 '백터 '는 세포 내로 전달하는 DNA 단편 (들) , 핵산 분자를 지칭할 때 사용된다. 상기 백터는 DNA를 복제시키고， 숙주세포에서 독립적으로 재생산될 수 있다.  The 'vector' is used when referring to DNA fragment (s), nucleic acid molecules that are delivered into cells. The vector replicates DNA and can be reproduced independently in a host cell.
상기 '발현백터 또는 재조합 백터'는 목적한 코딩 서열과, 특정 숙주 생물에서 작동 가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다.  The expression vector or recombinant vector refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.
상기 '식물 발현 백터 (식물에서 발현 가능한 재조합 백터) '는 이에 한정되지 않지만 아그로박테리움 투머파시엔스와 같은 적당한 숙주에 존재할 때 그 자체의 일부, 소위 T-영역을 식물 세포로 전이시킬 수 있는 Ti-플라스미드 백터일 수 있으며, 바람직한 형태는 EP0120516B1호 및 미국 특허 제 4,940,838호에 기재된 소위 바이너리 (binary) 백터일 수 있다. 또한 다른 적합한 식물 발현 백터는 이중 가닥 식물바이러스 (예를 들면, CaMV) 및 단일 가닥 바이러스, 게미니 바이러스 등으로부터 유래될 수 있는 것과 같은 바이러스 백터， 예를 들면 비완전성 식물 바이러스 백터로부터 선택될 수 있다. 그러한 백터의 사용은 특히 식물 숙주를 적당하게 형질 전환하는 것이 어려울 때 유리할 수 있다.  The 'plant expression vector (recombinant vector expressible in plants)' is not limited thereto, but when present in a suitable host such as Agrobacterium tumerfaciens, a portion of itself, a so-called T-region that can transfer T-regions to plant cells A plasmid vector, and a preferred form may be the so-called binary vector described in EP0120516B1 and US Pat. No. 4,940,838. Other suitable plant expression vectors can also be selected from viral vectors, such as those that may be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc., for example incomplete plant virus vectors. . The use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.
보다 바람직하게는 상기 식물 발현 백터는 당업계에서 자주 사용되는 pBI101(Cat#: 6018-1, Clontech, 미국), pBIN19(Genbank 수탁번호 U09365), pBI121 및 pCAMBIA 백터일 수 있다. 가장 바람직하게는 pCAMBIA 백터일 수 있다 (<실시예 2> 참조). More preferably the plant expression vector is used frequently in the art pBI101 (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121 and pCAMBIA vector. Most preferably it is a pCAMBIA vector (see <Example 2>).
본 발명의 재조합 백터는 바람직하게는 하나 이상의 선택성 마커를 포함할 소 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질 전환된 세포를 비형질 전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 그 예로는 글리포세이트 (glyphosate) 또는 포스피노트리신 (포스피노트리신)과 같은 제초제 저항성 유전자， 카나마이신 (Kanamycin), G418, 블레오마이신 (Bleomycin), 하이그로마이신 (hygromycin), 클로람페니콜 (chloramphenicol)과 같은 항생제 내성 유전자가 있으나, 이에 한정되는 것은 아니다.  The recombinant vector of the invention 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 herbicide resistance genes such as glyphosate or phosphinothricin (phosphinothricin), kanamycin, G418, bleomycin, hygromycin, chloramphenicol There are antibiotic resistance genes such as, but not limited to.
본 발명의 재조합 백터에 포함될 수 있는 프로모터는 식물 세포에서 전사를 개시할 수 있는 프로모터로써, 바람직하게는 식물 내에 삽입된 유전자를 과발현시킬 수 있는 것일 수 있다. 보다 바람직하게는 대부분의 환경 조건 및 발달 상태 또는 세포 분화 하에서 활성이 있는 '구성적 (constitutive) 프로모터'일 수 있다. 즉 형질 전환체의 선택이 각종 단계에서 각종 조직에 의해서 이루어질 수 있기 때문에 구성적 프로모터가 바람직하다. 보다 더 바람직하게는 본 발명의 발현 백터에 포함될 수 있는 프로모터는 CaMV 35S, 액틴, 유비퀴틴, pEMU, MAS 또는 히스톤 프로모터일 수 있으나, 이에 제한되지 않는다. The promoter that may be included in the recombinant vector of the present invention is a promoter capable of initiating transcription in plant cells, and preferably may be capable of overexpressing a gene inserted into the plant. More preferably, it may be a 'constitutive promoter' which is active under most environmental conditions and developmental conditions or cell differentiation. That is, the constitutive promoter is preferable because the selection of the transformant can be made by various tissues at various stages. Even more preferably the promoters that may be included in the expression vector of the invention are CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoters. May be, but is not limited thereto.
상기 프로모터'란 용어는 구조 유전자로부터의 DNA 상류 (upstream) 의 영역을 의미하며 전사를 개시하기 위하여 RNA 폴리머라아제가 결합하는 DNA 분자를 말한다.  The term 'promoter' refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.
본 발명의 재조합 백터에 포함될 수 있는 상기 터미네이터는, 통상의 터미네이터를 사용할 수 있으며， 그 예로는 노팔린 신타아제 (NOS), 벼 α-아밀라아제 RAmyl A 터미네이터, 파세올린 (phaseoline) 터미네이터, 아그로박테리움 투메파시엔스 (agrobacterium tumefaciens)의 옥토파인 (Octopine) 유전자의 터미네이터 등이 있으나, 이에 한정되는 것은 아니다. 터미네이터의 필요성에 관하여, 그러한 영역이 식물 세포에서의 전사의 확실성 및 효율을 증가시키는 것으로 일반적으로 알려져 있다. The terminator that can be included in the recombinant vector of the present invention may use a conventional terminator, such as nopalin synthase (NOS), rice α -amylase RAmyl A terminator, phaseoline (phaseoline) terminator, agrobacterium Terminator of the octopine gene of agrobacterium tumefaciens, but is not limited thereto. With regard to the need for terminators, such regions are generally known to increase the certainty and efficiency of transcription in plant cells.
본 발명의 일실시예에서의 재조합 백터는 프로모터로 CaMV 35S를 사용하고 터미네이터로 노팔린 신타아제 (NOS)를 사용한 pCAMBIA 백터에 서열번호 2의 염기서열을 가지는 AtSH3P-P2 유전자를 삽입하여 제조한 것이다. 보다 구체적으로 상기 재조합 백터는 AtSH3P-P2 유전자의 종결 코돈 (TGA)를 제거하고 녹색 형광 단백질 (GFP)의 개시 코돈 (AUG)을 연결한 구조체를 pCAMBIA 백터에 삽입함으로써 제조한 것으로 도 1에 기재된 개열지도로 표시될 수 있으며， GFP를 통해 AtSH3P-P2 유전자의 발현 유무를 확인할 수 있다 (<실시예 2> 참조).  The recombinant vector in one embodiment of the present invention is prepared by inserting the AtSH3P-P2 gene having the nucleotide sequence of SEQ ID NO: 2 into a pCAMBIA vector using CaMV 35S as a promoter and nopaline synthase (NOS) as a terminator. . More specifically, the recombinant vector was prepared by removing a stop codon (TGA) of the AtSH3P-P2 gene and inserting a construct linking the initiation codon (AUG) of the green fluorescent protein (GFP) into a pCAMBIA vector. It can be displayed on a map, it can be confirmed whether the expression of the AtSH3P-P2 gene through GFP (see <Example 2>).
본 발명의 형질 전환된 식물은 상기 재조합 백터로 형질 전환된 것이다. The transformed plant of the present invention is transformed with the recombinant vector will be.
상기 식물을 형질 전환시키기 위하여 핵산을 식물에 전이시키는 임의의 형질 전환 방법을 사용할 수 있다. 상기 형질전환 방법은 이에 한정되지 않지만 바람직하게는 원형질체에 대한 칼슘 /폴리에틸렌 글리콜 방법 (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8， 363-373), 원형질체의 전기천공법 (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), 식물 요소로의 현미주사법 (Crossway A. et al., 1986, Mol. Gen. Genet.202, 179-185), 각종 식물 요소의 (DNA 또는 RNA-코팅된) 입자 층격법 (Klein T.M. etal., 1987, Nature 327, 70)， 식물의 침윤 또는 성숙 화분 또는 소포자의 형질전환에 의한 아그로박테리움 투머파시엔스 매개된 유전자 전이에서 (비완전성) 바이러스에 의한 감염 (EP 0 301 316호) 등으로부터 적당하게 선택하여 사용될 수 있다. 본 발명에 따른 바람직한 방법은 아그로박테리움 매개된 형질 전환 방법일 수 있다 (<실시예 3> 참조).  Any transformation method can be used for transferring the nucleic acid to the plant for transforming the plant. The transformation method is not limited thereto but is preferably a calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), 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), (DNA or RNA-coated) particle stratification of various plant elements (Klein TM etal., 1987, Nature 327, 70), of plant infiltrating or maturing pollen or vesicles. Agrobacterium tumerfaciens mediated gene transfer by transformation can be used as appropriately selected from (incomplete) viral infection (EP 0 301 316) and the like. Preferred methods according to the invention may be Agrobacterium mediated transformation methods (see <Example 3>).
본 발명에서 AtSH3P-P2 유전자가 도입되어 형질 전환될 수 있는 식물은 이에 한정되지 않지만, 식량작물류, 채소작물류, 특용작물류, 과수류， 화훼류 및 사료작물류일 수 있으며， 바람직하게는 벼， 밀， 보리, 옥수수， 콩, 감자, 팔， 귀리, 수수, 애기 장대, 배추, 무, 고추, 딸기, 토마토, 수박, 오이， 양배추， 참외, 호박, 파, 양파, 당근, 인삼, 담배, 목화, 참깨， 사탕수수, 사탕무우, 들깨, 땅콩, 유채， 사과나무, 배나무, 대추나무, 복숭아， 양다래, 포도, 감귤, 감, 자두， 살구, 바나나, 장미， 글라디을러스, 거베라， 카네이션, 국화, 백합, 률립， 라이그라스, 레드클로버， 오차드그라스， 알파파, 를페스큐 및 페레니얼라이그라스로 이루어진 군으로부터 선택된 것일 수 있다. Plants that can be transformed by introducing the AtSH3P-P2 gene in the present invention is not limited thereto, but may be food crops, vegetable crops, special crops, fruit trees, flowers and feed crops, preferably rice, wheat, barley, Corn, beans, potatoes, eight, oats, sorghum, baby pole, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot, ginseng, tobacco, cotton, sesame, candy Sorghum, Beet, Perilla, Peanut, Rape, Apple tree, Pear, Jujube, Peach, Yard, Grape, Citrus, Persimmon, Plum, Apricot, Banana, Rose, Gladiolus, Gerbera, Carnation, It may be selected from the group consisting of chrysanthemum, lily, lily, lygras, red clover, orchardgrass, alfalfa, pescue and perennial lice.
특히 본 발명에서 AtSH3P-P2 유전자는 애기 장대를 포함한 상기 식물에서 효과적으로 발현되어 식물 생장을 촉진할 수 있으므로, 본 발명의 형질 전환된 식물은 형질 전환되지 않은 식물보다 생장이 매우 우수할 수 있다.  In particular, the AtSH3P-P2 gene in the present invention can be effectively expressed in the plant, including the baby pole, to promote plant growth, the transformed plant of the present invention may be much better than the non-transformed plant.
또한 본 발명의 종자는 상기 형질 전환된 식물을 재배하여 생산된 종자이다. 상기 형질 전환된 식물의 종자는 AtSH3P-P2 유전자가 도입되어, 상기 종자의 생장이 보다 촉진될 수 있다.  In addition, the seed of the present invention is a seed produced by culturing the transformed plant. The seed of the transformed plant may be introduced by the AtSH3P-P2 gene, thereby promoting the growth of the seed.
한편 본 발명의 식물 생장 촉진제는 본 발명의 AtSH3P-P2 단백질, 이를 암호화하는 AtSH3P-P2 유전자 및 상기 유전자를 포함하는 재조합 백터로 이루어진 군에서 선택된 일종 이상을 유효성분으로 함유한다.  Meanwhile, the plant growth promoter of the present invention contains at least one selected from the group consisting of the AtSH3P-P2 protein of the present invention, the AtSH3P-P2 gene encoding the same, and a recombinant vector comprising the gene as an active ingredient.
또한 본 발명의 또 다른 예는 상기 AtSH3P-P2 단백질， 이를 암호화하는 AtSH3P-P2 유전자 및 상기 유전자를 포함하는 재조합 백터로 이루어진 군에서 선택된 1종 이상의 식물 생장 촉진, 또는 식물 생장 촉진제 제조를 위한 용도를 제공한다.  In still another embodiment of the present invention, the AtSH3P-P2 protein, an AtSH3P-P2 gene encoding the same, and a recombinant vector comprising the gene, the use of at least one plant growth promoter or plant growth promoter, to provide.
상기 AtSH3P-P2 단백질, 이를 암호화하는 AtSH3P-P2 유전자 및 상기 유전자를 포함하는 재조합 백터는 식물의 세포질 분열을 유도함으로써 식물의 생장을 효과적으로 촉진시킬 수 있다.  The AtSH3P-P2 protein, the AtSH3P-P2 gene encoding the same, and a recombinant vector comprising the gene can effectively promote plant growth by inducing cytoplasmic division of the plant.
상기 AtSH3P-P2 유전자는 이에 한정되지 않지만 바람직하게 서열번호 2의 염기 서열로 표시될 수 있다. The AtSH3P-P2 gene is not limited thereto but preferably It may be represented by the nucleotide sequence of SEQ ID NO: 2.
또한 상기 재조합 백터는 이에 한정되지 않지만 바람직하게는 도 1의 개열지도로 표시될 수 있다.  In addition, the recombinant vector is not limited thereto, but may be preferably represented by the cleavage map of FIG. 1.
본 발명의 일실시예에서는 AtSH3P-P2 유전자가 세포판 위치에서 발현되고, 즉 AtSH3P-P2 유전자가 세포 분열 단계 증 핵 분열이 일어난 뒤 세포질 분열 시에 나타나는 세포판에 존재하므로, 상기 AtSH3P-P2 유전자는 세포질 분열에 관여하며, 나아가 AtSH3Pᅳ P2 유전자가 발현되는 위치가 초기 세포판 형성 시부터 세포판 성장이 진행되어 세포질 분열이 완성이 될 때까지 계속 유지되는 것이므로， AtSH3P-P2 유전자는 세포질 분열을 유도하는 활성이 있다 (<실시예 4〉 참조).  In one embodiment of the present invention, the AtSH3P-P2 gene is expressed at the cell plate position, that is, the AtSH3P-P2 gene is present in the cell plate appearing at the cytoplasmic division after the cell division stage of nuclear division, the AtSH3P-P2 gene The AtSH3P-P2 gene is involved in cytoplasmic division, and furthermore, the AtSH3P-P2 gene maintains the cytoplasmic division since the position where the AtSH3P ᅳ P2 gene is expressed is maintained from the initial cell plate formation until the cell plate growth progresses and the cellular division is completed. There is inducing activity (see <Example 4>).
또한 본 발명의 일실시예에서는 상기 형질 전환 식물에서 잎 및 뿌리의 생장이 야생형에 비하여 월등히 우수함을 알 수 있으며， 야생형에 비하여 30%이상 뿌리 길이가 길어짐을 알 수 있어, 형질 전환 식물이 야생형에 비해 잎의 크기가 커지고 뿌리의 길이가 길어지는 것은, AtSH3P-P2 유전자가 세포질 분열을 효과적으로 유도하여 식물의 생장을 촉진하는 활성이 있음올 알 수 있다 (<실시예 5〉 참조).  In addition, in one embodiment of the present invention it can be seen that the growth of the leaves and roots in the transgenic plant is significantly superior to the wild type, the root length is longer than 30% compared to the wild type, the transgenic plant to the wild type Larger leaf sizes and longer root lengths indicate that the AtSH3P-P2 gene effectively induces cellular division and promotes plant growth (see Example 5).
따라서 상기 AtSH3P-P2 단백질， 이를 암호화하는 AtSH3P-P2 유전자 및 상기 유전자를 포함하는 재조합 백터는 식물의 세포질 분열을 유도함으로써 식물의 생장을 효과적으로 촉진시킬 수 있다.  Therefore, the AtSH3P-P2 protein, the AtSH3P-P2 gene encoding the same, and a recombinant vector including the gene can effectively promote plant growth by inducing cytoplasmic division of the plant.
한편 본 발명의 식물의 생장을 조절하는 방법은 상기 AtSH3P-P2 유전자의 발현을 조절함으로써 식물의 생장을 조절할 수 있는 방법이다. 상기 식물 생장 조절은 AtSH3P-P2 유전자의 발현을 유도함으로써 식물의 생장을 촉진하는 것뿐만 아니라， 상기 유전자의 발현을 억제함으로써 식물의 생장을 억제하는 것을 포함한다. On the other hand, the method of controlling the growth of the plant of the present invention is the AtSH3P-P2 It is a way to control the growth of plants by controlling the expression of genes. The plant growth regulation includes not only promoting plant growth by inducing expression of the AtSH3P-P2 gene, but also inhibiting plant growth by inhibiting the expression of the gene.
먼저 식물 생장을 촉진시키기 위하여, AtSH3P-P2 유전자를 재조합 백터에 도입시켜, 상기 재조합 백터로 식물을 형질 전환함으로써 AtSH3P-P2 유전자를 발현 또는 과발현시킴으로써 식물 생장을 촉진시킬 수 있다.  First, in order to promote plant growth, the plant growth may be promoted by introducing the AtSH3P-P2 gene into a recombinant vector and expressing or overexpressing the AtSH3P-P2 gene by transforming the plant with the recombinant vector.
상기 '유전자의 과발현 '은 야생형 식물에서 발현되는 수준 이상으로 AISH3P-P2 유전자가 발현되도록 하는 것을 의미한다.  The 'overexpression of the gene' means that the AISH3P-P2 gene is expressed above the level expressed in wild-type plants.
상기 유전자를 발현 또는 과발현시킬 수 있는 재조합 백터 및 형질 전환 방법에 대해서는 앞서 기재한 바와 같으며， 상기 재조합 백터는 이에 한정되지 않지만 도 1의 개열지도로 표시될 수 있다.  Recombinant vectors and transformation methods capable of expressing or overexpressing the gene are as described above, and the recombinant vector is not limited thereto but may be represented by the cleavage map of FIG. 1.
따라서, 본 발명의 식물의 생장을 조절하는 방법은 서열번호 3의 아미노산 서열로 표시되는 AtSH3P-P2 단백질 코딩 유전자의 발현을 유도하는 단계를 포함하고， 식물의 생장을 촉진시키는 것을 특징으로 하는 것일 수 있다.  Therefore, the method of controlling the growth of the plant of the present invention includes the step of inducing the expression of the AtSH3P-P2 protein coding gene represented by the amino acid sequence of SEQ ID NO: 3, it may be characterized by promoting the growth of the plant have.
보다 구체적으로, 상기 유전자의 발현을 유도하는 단계는  More specifically, the step of inducing the expression of the gene
서열번호 3의 아미노산 서열을 가지는 AtSH3P-P2 단백질 코딩 유전자를 포함하는 재조합 백터를 준비하는 단계; 및  Preparing a recombinant vector comprising an AtSH3P-P2 protein coding gene having the amino acid sequence of SEQ ID NO: 3; And
재조합 백터로 식물을 형질 전환시키는 단계 를 포함하는 것일 수 있다. Transforming Plants with Recombinant Vectors It may be to include.
상기 재조합 백터로 식물을 형질 전환시키는 단계는 상기 서명한 식물 형질 전환 방법에 의하여 수행할 수 있다.  Transforming the plant with the recombinant vector may be performed by the signed plant transformation method.
이와 같이 제조된 재조합 백터를 도입한 식물을 통상의 재배 방법으로 재배함으로써 식물의 생장을 조절하여 바이오매스 증가 또는 생장 억제 등의 소망하는 목적을 달성할 수 있다.  By cultivating the plant incorporating the recombinant vector thus prepared in a conventional cultivation method, it is possible to achieve the desired purpose of controlling the growth of the plant to increase biomass or inhibit growth.
상기 AtSH3P-P2 유전자가 도입되어 형질 전환될 수 있는 식물은 이에 한정되지 않지만, 식량작물류， 채소작물류, 특용작물류, 과수류, 화훼류 및 사료작물류일 수 있으며, 바람직하게는 벼, 밀, 보리, 옥수수, 콩， 감자, 팔, 귀리, 수수, 애기 장대， 배추, 무， 고추, 딸기, 토마토, 수박， 오이, 양배추， 참외, 호박, 파， 양파, 당근, 인삼， 담배， 목화, 참깨, 사탕수수, 사탕무우, 들깨, 땅콩, 유채, 사과나무, 배나무, 대추나무, 복숭아, 양다래, 포도, 감글， 감, 자두, 살구, 바나나, 장미, 글라디올러스， 거베라, 카네이션, 국화, 백합, 률립, 라이그라스, 레드클로버, 오차드그라스, 알파파, 를페스큐 및 페레니얼라이그라스로 이루어진 군으로부터 선택된 것일 수 있다.  Plants that may be transformed by introducing the AtSH3P-P2 gene may be food crops, vegetable crops, special crops, fruit trees, flowers and feed crops, preferably rice, wheat, barley, corn, Beans, potatoes, arms, oats, sorghum, baby poles, cabbage, radish, peppers, strawberries, tomatoes, watermelons, cucumbers, cabbage, melons, pumpkins, green onions, onions, carrots, ginseng, tobacco, cotton, sesame seeds, sugar cane, Beet, Perilla, Peanut, Rapeseed, Apple tree, Pear, Jujube, Peach, Yard, Grape, Chisel, Persimmon, Plum, Apricot, Banana, Rose, Gladiolus, Gerbera, Carnation, Chrysanthemum, Lily, Yurilip, Ryegrass It may be selected from the group consisting of, red clover, orchard grass, alpha wave, vesque cue and perennial grass.
특히 본 발명에서 AtSH3P-P2 유전자는 애기 장대를 포함한 상기 식물에서 효과적으로 발현되어 식물 생장을 촉진할 수 있으므로, 상기 형질 전환된 식물은 형질 전환되지 않은 식물보다 생장이 매우 우수할 수 았다. 본 발명의 일실시예에서는 AtSH3P-P2 유전자가 세포판 위치에서 발현되고， 즉 AtSH3P-P2 유전자가 세포 분열 단계 중 핵 분열이 일어난 뒤 세포질 분열 시에 나타나는 세포판에 존재하므로， 상기 AtSH3P-P2 유전자는 세포질 분열에 관여하며, 나아가 AtSH3P-P2 유전자가 발현되는 위치가 초기 세포판 형성 시부터 세포판 성장이 진행되어 세포질 분열이 완성이 될 때까지 계속 유지되는 것이므로, AtSH3P-P2 유전자는 세포질 분열을 유도하는 활성이 있다 (<실시예 4> 참조). In particular, the AtSH3P-P2 gene in the present invention can be effectively expressed in the plant, including the baby pole, to promote plant growth, the transformed plant was able to grow much better than the untransformed plant. In one embodiment of the present invention, the AtSH3P-P2 gene is expressed at the cell plate position, that is, the AtSH3P-P2 gene is after nuclear division during the cell division stage. Since the AtSH3P-P2 gene is involved in cytoplasmic division, the position where the AtSH3P-P2 gene is expressed is at the initial cell plate formation and cell plate growth proceeds to complete the cell division. The AtSH3P-P2 gene has an activity of inducing cytoplasmic division since it is maintained until it is maintained (see <Example 4>).
또한 본 발명의 일실시예에서는 상기 형질 전환 식물에서 잎 및 뿌리의 생장이 야생형에 비하여 월등히 우수함을 알 수 있으며, 야생형에 비하여 30%이상 뿌리 길이가 길어짐을 알 수 있어, 형질 전환 식물이 야생형에 비해 잎의 크기가 커지고 뿌리의 길이가 길어지는 것은, AtSH3P-P2 유전자가 세포질 분열을 효과적으로 유도하여 식물의 생장을 촉진하는 활성이 있음을 알 수 있다 (<실시예 5〉 참조).  In addition, in one embodiment of the present invention it can be seen that the growth of leaves and roots in the transgenic plant is much superior to the wild type, the root length is longer than 30% compared to the wild type, the transgenic plant to the wild type Larger leaf sizes and longer root lengths indicate that the AtSH3P-P2 gene effectively induces cellular division and promotes plant growth (see Example 5).
둘째로， 식물 생장을 억제시키기 위하여, AtSH3P-P2 유전자의 발현을 억제함으로써 식물의 생장을 억제할 수 있다.  Secondly, in order to inhibit plant growth, plant growth can be suppressed by suppressing the expression of the AtSH3P-P2 gene.
상기 억제를 위하여 당업계에 공지된 식물 유전자의 억제 방법을 제한 없이 사용할 수 있다.  For the inhibition can be used without limitation methods of inhibition of plant genes known in the art.
예를 들어 , AtSH3P-P2 유전자의 발현을 억제하기 위하여 dsRNA를 사용할 수 있다. 상기 dsRNA는 높은 특이성으로 AtSH3P-P2 유전자의 표적 For example, dsRNA can be used to inhibit expression of the AtSH3P-P2 gene. The dsRNA is a high specificity target of the AtSH3P-P2 gene.
RNA(1차 또는 프로세싱됨)를 특이적으로 표적화할 수 있으며, 상기 dsRNA를 사용하여 이들 AtSH3P-P2 유전자의 발현을 효과적으로 억제할 수 있다. 또한 상기 dsRNA를 식물에 효과적으로 발현시킬 수 있는 백터 및 이를 이용한 형질 전환 방법은 당업계에 공지되어 있으며, 바람직하게는 도RNA (primary or processed) can be specifically targeted and the dsRNA can be used to effectively inhibit the expression of these AtSH3P-P2 genes. In addition, a vector capable of effectively expressing the dsRNA in plants and Transformation methods using the same are known in the art, and preferably
6의 개열지도로 표시되는 pTA7002 발현백터를 사용할 수 있다. The pTA7002 expression vector represented by the cleavage map of 6 can be used.
본 발명의 일실시예에서는 AtSH3P-P2 유전자의 염기서열 중 N-말단의 300개의 염기를 마주보게 하여 발현시킴으로써 dsRNA를 제조하고 이를 이용하여 AtSH3P-P2 유전자의 발현을 억제한 결과， AtSH3P-P2 유전자 발현이 억제되어 AtSH3P-P2 단백질이 계 기능을 다하지 못해, 핵 분열이 일어난 뒤 세포질 분열이 정상적으로 일어나지 못한다. 나아가 세포질 분열 실패에 따른 덜 분화된 세포벽들이 나타나게 되고 이로 인해 정상적인 세포층 형성이 이뤄지지 않아， 식물 성장이 억제된다(<실시예 6> 참조).  In an embodiment of the present invention, dsRNA was prepared by expressing 300 bases of the N-terminus in the base sequence of the AtSH3P-P2 gene, and the expression of the AtSH3P-P2 gene was suppressed using the AtSH3P-P2 gene. Expression of the AtSH3P-P2 protein is not able to function as a system, and cytoplasmic division does not occur normally after nuclear division. Furthermore, less differentiated cell walls appear due to the failure of cytoplasmic division, which prevents normal cell layer formation, thereby inhibiting plant growth (see Example 6).
이상 살펴본 바와 같이, AtSH3P-P2 유전자는 세포질 분열을 유도하는 활성이 있어, 이에 의해 형질 전환된 식물은 생장이 촉진될 수 있으므로， 식물의 바이오 매스 향상에 효과적으로 이용될 수 있다.  As described above, the AtSH3P-P2 gene has an activity of inducing cytoplasmic division, and thus the transformed plant can be promoted for growth, and thus can be effectively used for improving the biomass of the plant.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 AtSH3P-P2의 유전자 및 상기 유전자의 발현 유무를 확인할 수 있는 GFP가 결합된 pCAMBIA 발현백터의 개열지도를 나타낸 것이다. 도 2는 AtSH3P-P2로 형질 전환된 식물에서 AtSH3P-P2의 유전자가 발현되는지 여부를 확인하기 위해 웨스턴 블랏을 수행한 결과이다.  1 shows a cleavage map of a pCAMBIA expression vector to which a gene of AtSH3P-P2 and a GFP coupled to confirm the expression of the gene. Figure 2 is the result of performing Western blot to confirm whether the gene of AtSH3P-P2 is expressed in plants transformed with AtSH3P-P2.
도 3은 AtSH3P-P2로 형질 전환된 식물에서 AtSH3P-P2 단백질이 세포판 위치에서 발현됨을 확인한 결과이다. 도 4는 AtSH3P-P2로 형질 전환된 식물과 야생형의 잎 및 뿌리의 발달 여부를 야생형과 비교한 결과이다. 도 5는 AtSH3P-P2로 형질 전환된 식물의 뿌리 길이 변화를 파종 후 6일 및 10일이 경과한 다음， 야생형과 비교한 결과이다. (2P#1: 웨스턴 블랏에서의 라인 1번에 해당하는 형질 전환 식물, 2P#2: 웨스턴 블랏에서의 라인 2번에 해당하는 형질 전환 식물) 도 6은 AtSH3P-P2 유전자 발현을 억제시킨 pTA7002 발현백터의 개열지도를 나타낸 것이다. 도 7은 AtSH3P-P2 유전자 발현이 억제된 식물에서 세포 내 핵 (a) 및 세포벽 형성 모습 (b)을 관찰한 결과이다. 3 is a result confirming that the AtSH3P-P2 protein is expressed in the cell plate position in plants transformed with AtSH3P-P2. Figure 4 is a result of comparing the development of plants and wild type leaves and roots transformed with AtSH3P-P2 and wild type. 5 is a result of comparing the root length change of plants transformed with AtSH3P-P2 to wild type after 6 days and 10 days after sowing. (2P # 1: Transgenic Plant Corresponding to Line 1 in Western Blots, 2P # 2: Transgenic Plant Corresponding to Line 2 in Western Blots) FIG. 6 shows pTA7002 expression inhibiting AtSH3P-P2 gene expression. The cleavage map of the vector is shown. Figure 7 is the result of observing the intracellular nucleus (a) and cell wall formation (b) in plants in which AtSH3P-P2 gene expression is suppressed.
【발명의 실시를 위한 형태】 [Form for implementation of invention]
이하, 본 발명을 실시예에 의해 상세히 설명한다.  Hereinafter, the present invention will be described in detail by way of examples.
단， 하기 실시예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다. <실시예 1>  However, the following examples are merely to illustrate the present invention, and the content of the present invention is not limited to the following examples. <Example 1>
세포질 분열을 조절하는 유전자 0 tSH3P-P2)의 cDNA 분리 및 서열분석  CDNA isolation and sequencing of gene 0 tSH3P-P2) that regulates cytoplasmic division
<1-1> 실험방법 <1-1> Experimental Method
AtSH3P-P2의 cDNA는 5' ATG에서 18개의 염기서열과 3' TGA 앞 쪽으로 18개의 염기서열을 각각 센스 프라이머와 안티센스 프라이머로 이용하여, cDNA library에서 PCR 증폭 (94 °C에서 30초, 55 °C에서 30초， 72°C에서 30초로 구성된 사이클을 30회 반복)을 통해 AtSH3P-P2의 cDNA를 추출하였고 그 서열은 당업계에 공지된 염기서열 분석을 통해 확인하였다. <1-2> 실험결과 The cDNA of AtSH3P-P2 is composed of 18 nucleotide sequences in 5 'ATG and 18 nucleotide sequences in front of 3' TGA as sense and antisense primers, respectively. Using this method, cDNA of AtSH3P-P2 was extracted by PCR amplification (30 cycles consisting of 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 30 seconds at 72 ° C), and the sequence was determined. It was confirmed by sequencing known in the art. <1-2> Experimental Results
AtSH3P-P2의 유전자 전체 서열에서 단백질 발현에 필요한 cDNA 서열을 애기장대 cDNA 라이브러리에서 PCR 방법을 통해 분리한 결과, AtSH3P-P2의 유전자 전체 서열은 서열번호 1로 표시되는 염기서열을 가지며, AtSH3P-P2의 cDNA는 서열번호 2로 표시되는 염기서열을 가지는 것을 알 수 있었다. 또한 상기 서열번호 2에 따라 AtSH3P-P2의 아미노산 서열은 서열번호 3으로 표시됨을 알 수 있었다.  As a result of separating the cDNA sequence required for protein expression from the gene sequence of AtSH3P-P2 by PCR method in Arabidopsis cDNA library, the gene sequence of AtSH3P-P2 has the nucleotide sequence represented by SEQ ID NO: 1, AtSH3P-P2 It was found that cDNA had a nucleotide sequence represented by SEQ ID NO: 2. In addition, according to SEQ ID NO: 2 it can be seen that the amino acid sequence of AtSH3P-P2 is represented by SEQ ID NO: 3.
<실시예 2> <Example 2>
세포질 분열을 조절하는 유전자 (AtSH3P-P2)의 발현백터 제작  Expression vector production of gene (AtSH3P-P2) that regulates cellular division
<2-1> 실험방법 <2-1> Experimental method
상기 <실시예 1>에서 분석된 AtSH3P-P2의 유전자의 cDNA를 기초로 상기 유전자의 형질 전환 식물 제조에 사용될 수 있는 발현백터를 제조하였다.  Based on the cDNA of the AtSH3P-P2 gene analyzed in <Example 1> was prepared an expression vector that can be used for the production of transgenic plants of the gene.
즉 AtSH3P-P2 유전자의 종결 코돈 (TGA)를 제거하고 녹색 형광 단백질 (GFP)의 개시 코돈 (AUG)을 연결하고， 이를 pCAMBIA 1300백터 (Cambia Labs) 의 35s 프로모터와 nos(nopaline synthease) 사이에 삽입하여, 상기 AtSH3P-P2의 유전자 및 상기 유전자의 발현 유무를 확인할 수 있는 GFP가 결합된 발현백터를 제조하였다.  That is, the termination codon (TGA) of the AtSH3P-P2 gene was removed, and the initiation codon (AUG) of the green fluorescent protein (GFP) was linked, and inserted between the 35s promoter of pCAMBIA 1300 vector (Cambia Labs) and the nos (nopaline synthease). Thus, the expression vector to which the gene of AtSH3P-P2 and GFP which can confirm the expression of the gene was combined was prepared.
구체적으로 AtSH3P-P2의 cDNA는 326-sGFP 백터에 클로닝하기 위해 Specifically, the cDNA of AtSH3P-P2 was cloned into the 326-sGFP vector.
5' ATG앞에 Xbal 제한효소 서열을 추가하여 하기 서열번호 4의 5' AtSH3P-2:sGFP Xbal 프라이머를 제작하였고, 3'의 종결 코돈 (TGA)를 제외하고 BamHl 제한효소 서열을 추가하여 하기 서열번호 5의 3' AtSH3P-2:sGFPBamHl 프라이머를 제작하였다. A 5 'AtSH3P-2: sGFP Xbal primer of SEQ ID NO: 4 was prepared by adding an Xbal restriction enzyme sequence in front of 5' ATG, and a BamHl restriction enzyme sequence was added to remove the 3 'termination codon (TGA). 5 '3' AtSH3P-2: sGFPBamHl primers were prepared.
상기 두 개의 프라이머를 이용하여 AtSH3P-P2의 cDNA library에서 PCR 증폭 (94 °C에서 30초, 55°C에서 30초, 72°C에서 30초로 구성된 사이클을 30회 반복)을 통해 AtSH3P-P2의 cDNA를 추출하였고， 326-sGFP 백터를 Xbal, BamHl 두 제한 효소 (Bioneer, Korea or NEB)로 잘라서 클로닝을 완성하였다. 제작한 AtSH3P-P2:sGFP 서열은 염기서열 분석을 통해 다시금 확인하였다.  PCR of the AtSH3P-P2 cDNA library using the two primers (30 seconds at 94 ° C, 30 seconds at 55 ° C, 30 cycles consisting of 30 seconds at 72 ° C) of AtSH3P-P2 cDNA was extracted and cloned by cutting the 326-sGFP vector with two Xbal, BamHl restriction enzymes (Bioneer, Korea or NEB). The prepared AtSH3P-P2: sGFP sequence was again confirmed through sequencing.
<2-2> 실험결과 <2-2> Experimental Results
상기 AtSH3P-P2의 유전자 및 상기 유전자의 발현 유무를 확인할 수 있는 GFP가 결합된 pCAMBIA 발현백터는 도 1에 기재된 개열지도로 표시된다.  The pCAMBIA expression vector to which the gene of AtSH3P-P2 and GFP which can confirm the expression of the gene is bound is represented by the cleavage map described in FIG. 1.
<실시예 3> <Example 3>
세포질 분열을 조절하는 유전자 (AtSH3-P2)로 형질 전환된 식물의 제조  Preparation of Plants Transformed with Genes That Control Cellular Division (AtSH3-P2)
<3-1> 실험방법 <3-1> Experimental method
상기 <실시예 2>에서 제조된 pCAMBIA 백터를 이용하여 아그로박테리움 매게 형질 전환 방법을 통해 형질 전환된 애기장대 (Arabidopsis thaliana) 식물을 제조하였다. 상기 형질 전환 방법은 Floral dip 방법이다 (The Plant Journal (1998) 16(6), 735-743).  The Arabidopsis thaliana plants transformed by the Agrobacterium mage transformation method were prepared using the pCAMBIA vector prepared in <Example 2>. The transformation method is the Floral dip method (The Plant Journal (1998) 16 (6), 735-743).
상기와 같이 형질 전환된 식물을 선택적으로 선별하기 위하여 하이그로마이신 내성 검사를 실시하여 1차 선별하였다.  In order to selectively select the transformed plants as described above, the hygromycin resistance test was carried out to select first.
구체적으로 상기 선별을 위해 B5 배지에 하이그로마이신 (20units/l)이 첨가된 배지를 이용하였다. 1차 선별된 각 라인 별로 2세대를 확보한 뒤, 선별 마커를 이용하여 죽는 개체와 살아남는 개체가 1:3의 비율로 뚜렷하게 구별이 되는 라인을 2차 선별하였다. 이후 형질 전환된 식물체로 3세대에서 선별 마커에 의해 모두 살아남는 호모라인을 확보한 뒤 웨스턴 블랏으로 과발현체 여부를 확인하고자 하였다. Specifically, a medium in which hygromycin (20 units / l) was added to B5 medium was used for the selection. After securing two generations for each of the first screened lines, the screening markers were secondarily screened to clearly distinguish between the dead and surviving individuals in a ratio of 1: 3. The transformed plants were then transferred to Western blot after securing homolines that survived by the selection marker in the third generation. To determine whether overexpressed.
상기 웨스턴 블랏은 10% SDS-PAGE를 이용하였고 AtSH3-P2:sGFP 말단의 GFP를 인식하는 GFP 특이적 항체를 이용하여 그 발현여부를 살펴보았다.  The Western blot was used for 10% SDS-PAGE and the expression was examined using a GFP specific antibody that recognizes GFP at the end of AtSH3-P2: sGFP.
<3-2> 실험결과 <3-2> Experiment Result
상기 웨스턴 블랏을 수행한 결과를 도 2에 기재하였으며, 상기 도 2에 기재된 바와 같이, AtSH3-P2의 유전자는 GFP의 융합 단백질 형태로 효과적으로 발현되고 있음을 확인할 수 있다. 즉 70 kDa의 정확한 크기의 단백질이 합성되고 있음을 확인하였다. 이를 통하여 안정화된 형질 전환 식물체 라인을 확보할 수 있었다. 상기 도 2에서 각 라인은 형질 전환된 각각의 식물을 의미한다.  The results of performing the western blot are shown in FIG. 2, and as shown in FIG. 2, it can be seen that the gene of AtSH3-P2 is effectively expressed in the form of a fusion protein of GFP. That is, it was confirmed that a protein of the correct size of 70 kDa was synthesized. Through this, it was possible to secure a stable transgenic plant line. Each line in FIG. 2 means each plant transformed.
<실시예 4> <Example 4>
형질 전환된 식물에서 AtSH3-P2 유전자의 발현 위치 확인  Identifying Expression Location of AtSH3-P2 Gene in Transgenic Plants
<4-1> 실험방법 <4-1> Experimental method
상기 <실시예 3〉에서 형질 전환된 애기 장대 식물에서 AtSH3-P2 단백질의 발현 위치를 확인해 보고자, 상기 단백질과 연결된 GFP 형광 단백질을 공초점 현미경 (Carl Zeiss, Meta 510)을 통해 관찰하였다.  In order to confirm the expression position of the AtSH3-P2 protein in the transformed baby pole plants in <Example 3>, the GFP fluorescent protein linked to the protein was observed through confocal microscopy (Carl Zeiss, Meta 510).
<4-2> 실험결과 <4-2> Experimental Results
상기 형질 전환된 애기 장대 식물의 GFP 형광 단백질의 발현되는 부위를 확인한 결과를 도 3에 기재하였다.  The result of confirming the site of expression of the GFP fluorescent protein of the transformed baby pole plants is shown in FIG.
상기 도 3에 기재된 바와 같이， AtSH3P-P2 단백질은 세포판 위치에서 발현됨을 알 수 있다. 즉 AtSH3P-P2 단백질은 세포 분열 단계 증 핵 분열이 일어난 뒤 세포질 분열 시에 나타나는 세포판에 존재하므로, 상기 AISH3P-P2 단백질은 세포질 분열에 관여하며, 나아가 AtSH3P-P2 단백질이 발현되는 위치가 초기 세포판 형성 시부터 세포판 성장이 진행되어 세포질 분열이 완성이 될 때까지 계속 유지되는 것이므로， AtSH3P-P2 단백질은 세포질 분열에 필수적인 기능을 할 수 있음을 알 수 있다. As shown in FIG. 3, it can be seen that the AtSH3P-P2 protein is expressed at the cell plate position. In other words, the AtSH3P-P2 protein is present in the cell plate appearing at the time of cytoplasmic division after nuclear division and nuclear division. Therefore, the AISH3P-P2 protein is involved in cytoplasmic division, Since the position to be expressed is maintained from the initial cell plate formation until cell plate growth proceeds to complete cell division, AtSH3P-P2 protein can be seen that it can play an essential function in cytoplasmic division.
<실시예 5> Example 5
AtSH3P-P2 유전자의 세포질 분열 유도 및 이에 따른 식물의 생장 촉진 활성  Induction of cytoplasmic division of AtSH3P-P2 gene and its growth promoting activity
<5-1> 실험방법 <5-1> Experimental method
상기 <실시예 4>에서 형질 전환된 애기 장대 식물에서 생장이 촉진되는지 여부를 확인하기 위해， AtSH3-P2 유전자가 삽입된 형질 전환 애기 장대와 야생형의 잎 및 뿌리 발달 여부를 1/2 MS 배지 (Duchefa)를 이용하여 일반적인 애기장대 배양 조건에서 세워서 키운 다음 그 표현형 변화를 관찰하였다.  In order to confirm whether growth is promoted in the baby pole plants transformed in <Example 4>, the development of the transgenic baby pole into which the AtSH3-P2 gene is inserted and the leaves and roots of the wild-type half MS medium ( Duchefa) was used to grow in normal Arabidopsis culture conditions and observed for phenotypic changes.
또한 형질 전환 식물의 뿌리 길이 변화를 씨 파종 후 각각 6일째 되는 날과 10일째 되는 날에 뿌리의 길이를 측정하고 야생형과 함께 각기 30개체씩 무작위로 선별하여 3회 반복 실험한 다음， 평균과 표준 편차를 구하고 그래프로 나타내었다.  In addition, the root length of the transgenic plants was measured on the 6th and 10th day after seeding, respectively, and randomly screened 30 times with wild type. Deviations were obtained and plotted.
<5-2> 실험결과 <5-2> Experimental Results
상기 형질 전환 애기 장대 식물과 야생형의 잎 및 뿌리의 발달 여부를 비교한 결과를 도 4에 기재하였다.  The results of comparing the development of the transgenic baby pole plants and wild type leaves and roots are shown in FIG. 4.
상기 도 4에 기재한 바와 같이, 형질 전환 애기 장대 식물에서 잎 및 뿌리의 생장이 야생형에 비하여 월등히 우수함을 알 수 있다.  As shown in FIG. 4, it can be seen that the growth of leaves and roots in the transgenic baby pole plants is superior to the wild type.
또한 형질 전환 식물의 뿌리 길이 변화를 씨 파종 후 각각 6일째 되는 날과 10일째 되는 날에 뿌리의 길이를 측정한 결과를 도 5에 기재하였다.  In addition, the results of measuring the length of the roots of the transgenic plants at the 6th day and the 10th day after seeding, respectively, are shown in FIG. 5.
상기 도 5에 기재한 바와 같이， 상기 형질 전환 애기 장대 식물은 야생형에 비하여 30%이상 뿌리 길이가 길어짐을 알 수 있다. As described in Figure 5, the transgenic baby pole plant Root length is longer than 30% compared to wild type.
상기와 같이 동일한 기간 동안에 형질 전환 애기 장대 식물이 야생형에 비해 잎의 크기가 커지고 뿌리의 길이가 길어지는 것은, AtSH3-P2 단백질이 세포질 분열올 효과적으로 유도하여 식물의 생장올 촉진한 것이다.  As described above, the size of the leaves and the length of the roots of the transgenic baby pole plants are larger than those of the wild type in the same time period, and the AtSH3-P2 protein effectively induces cellular division and promotes plant growth.
<실시예 6> <Example 6>
AtSH3P-P2 유전자의 발현 억제를 통한 세포질 분열 억제 및 식물의 생장 저해 <6-1> 실험방법  Inhibition of cytoplasmic division and inhibition of plant growth by inhibiting expression of AtSH3P-P2 gene
상기 <실시예 5〉에서 AtSH3-P2로 형질 전환된 식물에서 생장이 촉진되는 효과를 다시 한번 검증해 보기 위해, AtSH3-P2 유전자 발현을 억제시킨 새로운 형질 전환체를 제작하였다.  In order to verify the effect of promoting growth in plants transformed with AtSH3-P2 in <Example 5>, a new transformant that suppresses the AtSH3-P2 gene expression was produced.
먼저 이중가닥 RNA를 유도한 유전자 발현 억제 시스템을 이용하여 AtSH3P-P2 유잔자의 발현이 억제되도록 하였다. 구체적으로 AtSH3-P2 유전자의 염기서열 중 N-말단의 300개의 염기를 마주보게 발현될 수 있도록 도 6에 기재된 개열지도를 가지는 pTA7002 발현백터 (The Plant Journal (1997) 11(3), 605-612)를 제조하였다. 상기 발현백터는 덱사메사손 (dexamethasone)에 의해 필요에 따라 AtSH3-P2 유전자의 발현을 억제할 수 있다.  First, the expression of AtSH3P-P2 residues was suppressed using a gene expression suppression system that induced double stranded RNA. Specifically, pTA7002 expression vector having a cleavage map as shown in FIG. ) Was prepared. The expression vector can inhibit the expression of the AtSH3-P2 gene as needed by dexamethasone (dexamethasone).
상기 pTA7002 발현백터를 이용하여 상기 <실시예 3-1>과 동일한 방법으로 애기 장대 식물을 형질 전환하였다. 상기 형질 전환된 식물은 하이그로마이신 내성 검사를 통해 분리 선별하고, 분리 선별한 식물은 1/2 MS 배지에서 발아 시킨 뒤 4일째 되는 날에 유전자 발현 억제 시스팀이 작용할 수 있도톡 하는 30M 덱사메사손이 포함된 1/2 MS배지로 옮기고 3일을 더 배양하였다.  Using the pTA7002 expression vector, the baby pole plants were transformed in the same manner as in <Example 3-1>. The transformed plants were isolated and screened by the hygromycin resistance test, and the isolated and screened plants were germinated in 1/2 MS medium, and 30M dexamethasone was able to act on the gene expression suppression system on the 4th day after germination. Transfer to this containing 1/2 MS medium and incubated for 3 more days.
상기 배양 후, 세포 내 핵을 관찰할 수 있도록 하는 propodium iodide 시약을 이용하여 세포 내 핵을 고르게 염색한 다음, 공초점 현미경을 통해 관찰하였다. <6-2> 실험결과 After the incubation, the intracellular nuclei were evenly stained using a propodium iodide reagent to observe intracellular nuclei, and then observed through confocal microscopy. <6-2> Experimental Results
상기 AtSH3-P2 유전자 발현이 억제된 식물에서 세포 내 핵을 관찰한 결과를 도 7에 기재하였다.  The results of observing intracellular nuclei in plants in which AtSH3-P2 gene expression was suppressed are described in FIG. 7.
상기 도 7에 기재한 바와 같이, 단일 세포 내 2개 이상의 핵이 존재하는 다핵성 세포들이 관찰되었다.  As described in FIG. 7, multinuclear cells with two or more nuclei in a single cell were observed.
상기와 같은 결과는 AtSH3-P2 유전자 발현이 억제되어 AtSH3-P2 단백질이 제 기능을 다하지 못한 결과, 핵 분열이 일어난 뒤 세포질 분열이 정상적으로 일어나지 못한 것이다. 나아가 세포질 분열 실패에 따른 분화된 세포벽들이 나타나게 되고 이로 인해 정상적인 세포층 형성이 이뤄지지 않았다.  The above results indicate that AtSH3-P2 gene expression is inhibited and the AtSH3-P2 protein fails to function properly, and cytoplasmic division does not occur normally after nuclear division. Furthermore, differentiated cell walls appeared due to the failure of cytoplasmic division, which prevented normal cell layer formation.

Claims

【청구의 범위】 [Range of request]
【청구항 1】 [Claim 1]
서열번호 3의 아미노산 서열로 표시되는 AtSH3-P2 단백질.  AtSH3-P2 protein represented by the amino acid sequence of SEQ ID NO.
【청구항 2】 [Claim 2]
제 1항의 AtSH3P-P2 단백질을 암호화하는 AtSH3P-P2 유전자.  AtSH3P-P2 gene encoding the AtSH3P-P2 protein of claim 1.
【청구항 3】 [Claim 3]
저 12항에 있어서, 상기 AtSH3P-P2 유전자는 서열번호 2의 염기 서열로 표시되는 AtSH3P-P2 유전자.  The AtSH3P-P2 gene according to claim 12, wherein the AtSH3P-P2 gene is represented by the nucleotide sequence of SEQ ID NO: 2.
【청구항 4】 [Claim 4]
게 2항의 AtSH3P-P2 유전자를 포함하는 재조합 백터.  A recombinant vector comprising the AtSH3P-P2 gene of Crab 2.
【청구항 5】 [Claim 5]
제 4항에 있어서， 상기 재조합 백터는 도 1의 개열지도로 표시되는 것인 재조합 백터.  The recombinant vector of claim 4, wherein the recombinant vector is represented by a cleavage map of FIG. 1.
【청구항 6】 [Claim 6]
제 4항의 재조합 백터로 형질 전환된 식물.  Plant transformed with the recombinant vector of claim 4.
【청구항 7】 [Claim 7]
제 6항에 있어서, 상기 식물은 벼， 밀, 보리, 옥수수, 콩， 감자, 팔, 귀리, 수수, 애기 장대, 배추, 무, 고추， 딸기， 토마토, 수박, 오이, 양배추, 참외, 호박, 파， 양파, 당근， 인삼, 담배, 목화， 참깨, 사탕수수, 사탕무우, 들깨, 땅콩, 유채, 사과나무, 배나무, 대추나무, 복숭아， 양다래, 포도, 감글, 감, 자두, 살구, 바나나, 장미, 글라디을러스, 거베라, 카네이션, 국화, 백합， 률립, 라이그라스, 레드클로버, 오차드그라스， 알파파, 를페스큐 및 페레니얼라이그라스로 이루어진 군으로부터 선택된 것인， 형질 전환된 식물.The method of claim 6, wherein the plant is rice, wheat, barley, corn, soybeans, potatoes, arms, oats, sorghum, baby pole, Chinese cabbage, radish, red pepper, strawberries, tomatoes, watermelon, cucumber, cabbage, melon, pumpkin, Green onion, onion, carrot, ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed, apple tree, pear tree, jujube, peach, squid, grape, persimmon, persimmon, plum, apricot, banana , Roses, Gladiolus, Gerbera, Carnations, Chrysanthemums, Lilies, Yulips, Rygras, Red Clover, Orchardgrass, Alpha Wave, Fescue and A transformed plant, selected from the group consisting of perennial grass.
【청구항 8】 [Claim 8]
제 6항의 형질 전환된 식물로부터 얻어진 종자.  Seed obtained from the transformed plant of claim 6.
【청구항 9】 [Claim 9]
제 1항의 단백질, 이를 암호화하는 AtSH3P-P2 유전자, 및 상기 유전자를 포함하는 재조합 백터로 이루어진 군에서 선택된 1종 이상을 유효성분으로 함유하는 식물 생장 촉진제.  The plant growth promoter containing at least one selected from the group consisting of the protein of claim 1, the AtSH3P-P2 gene encoding the same, and a recombinant vector comprising the gene as an active ingredient.
【청구항 10】 [Claim 10]
제 9항에 있어서, 상기 AtSH3P-P2 유전자는 서열번호 2의 염기 서열로 표시되는 것인 식물 생장 촉진제.  The plant growth promoter of claim 9, wherein the AtSH3P-P2 gene is represented by the nucleotide sequence of SEQ ID NO: 2. 11.
【청구항 11】 [Claim 11]
제 9항에 있어서, 상기 재조합 백터는 도 1의 개열지도로 표시되는 것인 식물 생장 촉진제.  The plant growth promoter of claim 9, wherein the recombinant vector is represented by a cleavage map of FIG. 1.
【청구항 12]  [Claim 12]
서열번호 3의 아미노산 서열로 표시되는 AtSH3P-P2 단백질 코딩 유전자의 발현을 조절함으로써 식물의 생장을 조절하는 방법.  A method of regulating plant growth by regulating the expression of the AtSH3P-P2 protein coding gene represented by the amino acid sequence of SEQ ID NO.
【청구항 13】  [Claim 13]
제 12항에 있어서, 서열번호 3의 아미노산 서열로 표시되는 AtSH3P-P2 단백질 코딩 유전자의 발현을 유도하는 단계를 포함하고， 식물의 생장을 촉진시키는 것을 특징으로 하는, 식물의 생장을 조절하는 방법.  13. The method of claim 12, comprising inducing the expression of the AtSH3P-P2 protein coding gene represented by the amino acid sequence of SEQ ID NO: 3, and promoting plant growth.
【청구항 14]  [Claim 14]
제 13항에 있어서, 상기 서열번호 3의 아미노산 서열로 표시되는 AISH3P-P2 단백질 코딩 유전자의 발현을 유도하는 단계는 서열번호 3의 아미노산 서열로 표시되는 AtSH3P-P2 단백질 코딩 유전자를 포함하는 재조합 백터를 준비하는 단계; 및 상기 재조합 백터로 식물을 형질 전환시키는 단계 를 포함하는 것인, 식물의 생장을 조절하는 방법. The method of claim 13, wherein the amino acid sequence of SEQ ID NO: 3 Inducing the expression of the AISH3P-P2 protein coding gene comprises preparing a recombinant vector comprising an AtSH3P-P2 protein coding gene represented by the amino acid sequence of SEQ ID NO: 3; And transforming the plant with the recombinant vector.
【청구항 15】 [Claim 15]
제 14항에 있어서, 상기 재조합 백터는 도 1의 개열지도로 표시되는 것인, 식물의 생장을 조절하는 방법.  The method of claim 14, wherein the recombinant vector is represented by the cleavage map of FIG. 1.
【청구항 16]  [Claim 16]
제 12항에 있어서, 상기 식물은 벼， 밀， 보리, 옥수수, 콩, 감자, 팔, 귀리, 수수, 애기 장대, 배추, 무, 고추, 딸기， 토마토, 수박, 오이, 양배추, 참외, 호박, 파， 양파， 당근， 인삼， 담배, 목화, 참깨, 사탕수수, 사탕무우, 들깨 땅콩, 유채， 사과나무, 배나무, 대추나무, 복숭아, 양다래， 포도， 감글, 감, 자두, 살구, 바나나, 장미, 글라디을러스, 거베라, 카네이션, 국화, 백합, 률립, 라이그라스, 레드클로버, 오차드그라스, 알파파, 를페스큐 및 페레니얼라이그라스로 이루어진 군으로부터 선택된 것인 식물의 생장을 조절하는 방법.  The method of claim 12, wherein the plant is rice, wheat, barley, corn, soybeans, potatoes, arms, oats, sorghum, baby pole, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, Green onion, onion, carrot, ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla peanut, rapeseed, apple tree, pear tree, jujube tree, peach, lamb, grape, sweet potato, persimmon, plum, apricot, banana, A method of regulating the growth of a plant selected from the group consisting of roses, gladiolus, gerbera, carnations, chrysanthemums, lilies, yulrip, lygras, red clover, orchardgrass, alfalfa, erfescue and perennialgrass.
【청구항 17]  [Claim 17]
제 12항에 있어서, 서열번호 3의 아미노산 서열로 표시되는 AtSH3P-P2 단백질 코딩 유전자의 발현을 억제함으로써 식물의 생장을 억제시키는, 식물의 생장을 조절하는 방법. The method according to claim 12, wherein the growth of the plant is inhibited by inhibiting the expression of the AtSH3P-P2 protein coding gene represented by the amino acid sequence of SEQ ID NO: 3.
【청구항 18] [Claim 18]
제 1항의 단백질, 이를 암호화하는 AtSH3P-P2 유전자, 및 상기 유전자를 포함하는 재조합 백터로 이루어진 군에서 선택된 1종 이상의 식물 생장 촉진을 위한 용도.  Use of at least one plant selected from the group consisting of the protein of claim 1, the AtSH3P-P2 gene encoding the same, and a recombinant vector comprising the gene.
【청구항 19]  [Claim 19]
제 18항에 있어서, 상기 AtSH3P-P2 유전자는 서열번호 2의 염기 서열로 표시되는 것인 용도. 【청구항 20】  The use according to claim 18, wherein the AtSH3P-P2 gene is represented by the nucleotide sequence of SEQ ID NO. [Claim 20]
제 18항에 있어서, 상기 재조합 백터는 도 1의 개열지도로 표시되는 것인 용도.  Use according to claim 18, wherein said recombinant vector is represented by the cleavage map of FIG.
PCT/KR2010/007322 2010-03-03 2010-10-25 Gene regulating cytokinesis, plants transformed with the gene, and method for regulating growth of plants using same WO2011108794A2 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102851280A (en) * 2012-09-18 2013-01-02 中国科学院遗传与发育生物学研究所 Application of RNA (ribonucleic acid) and gene for generating RNA in regulating development of rice root system

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101334408B1 (en) * 2012-06-08 2013-11-29 포항공과대학교 산학협력단 Genes increasing biomass production and transgenic plants using them

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
'A Predicted interactome for Arabidopsis' BIOINFORMATICS vol. 145, 2007, pages 317 - 329 *
'Expression of auxilin or Ap 180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP 1 or AP2 but not clathrin' JOURNAL OF CELL SCIENCE vol. 114, no. 2, 2000, pages 353 - 365 *
'Multiple roles of Auxilin and Hsc70 in clathrin-mediated endocytosis' TRAFFIC vol. 8, 2007, pages 640 - 646 *
'Role of SH3 doamin-containing proteins in clathrin-mediated vesicle trafficking in Arabidopsis' PLANT CELL vol. 13, 2001, pages 2499 - 2512 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102851280A (en) * 2012-09-18 2013-01-02 中国科学院遗传与发育生物学研究所 Application of RNA (ribonucleic acid) and gene for generating RNA in regulating development of rice root system

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