KR101512970B1 - TFC Method for controlling polyploidy of plant using AtTIO TFC mutant and the plant thereof - Google Patents

Abstract

The present invention relates to a method for regulating plant ploidy comprising the step of transforming a plant cell with a recombinant vector comprising a gene encoding a Arabidopsis thaliana-derived protein variant to regulate the expression of the gene, A method for producing a plant having regulated pore size, which comprises transforming a plant cell with a recombinant vector comprising a gene coding for said plant, a method for producing said plant, a method for producing said plant, The present invention relates to a composition for regulating the drainage of a plant containing a recombinant vector comprising a gene coding for a human protein variant as an active ingredient. The present invention relates to a composition for regulating the drainage of a plant having potentially high value by controlling the expression of a mutant gene derived from Arabidopsis thaliana Suggest new ways of breeding.

Description

FIELD OF THE INVENTION The present invention relates to a method for controlling plant ploidy using a transgenic tobacco gene mutant TF △ C and a method for controlling plant polyploidy of plant using AtTIO TF △ C mutant and the plant thereof,

The present invention relates to a method for regulating plant transpiration using the Arabidopsis thaliana gene mutant TF △ C and a plant according to the method, and more particularly, to a method for controlling plant transpiration using a Arabidopsis thaliana (AtTIO, Arabidopsis thaliana TWO-IN-ONE) protein variant and regulating the expression of the gene coding for the transgene protein variant by transforming the plant cell with a recombinant vector containing the gene encoding the thaliana TWO-IN-ONE protein variant , Transforming a plant cell with a recombinant vector comprising a gene encoding the transposon protein variant, a method for producing a transplanted plant having regulated drainage, a method for producing a transplanted plant produced by the method, And a recombinant vector comprising the seed and the gene coding for the transposon protein variant as an active ingredient.

A flowering plant is a plant that has two male spouses (sperm cells) necessary for duplicate fertilization and a vegetative (fertile) organism that carries them to a magnetic spouse (egg cell) cell mature pollen (mature pollen) is made. The germline generation, which is a relatively simple structure composed of three cells, is a crucial process to preserve and breed species, which takes a very short period of plant growth.

Pollen development proceeds by a single meiosis and two mitoses that occur sequentially. Firstly, a diploid mother cell, which is a diploid formed in anther, undergoes meiosis to form four haploid microspore tetrads, The nucleus of the resulting microspores migrates to the generative poles. Polarized microspores become vegetative cells and generative cells through asymmetric division of pollen mitosis I. Then, as the carlos surrounding the germ cells are degraded, the germ cells migrate into the cytoplasm of the malignant cell and become a bicellular pollen of a "cell-within-a-cell" structure. The germ cells undergo pollen mitosis II to form two male spouses, and the malignant cells have no further cell divisor, but instead produce a pollen tube that transfers the male spouse to the magnetic actor itself. One of the two male spouses, who subsequently moved to the magnetic actor through the pollen tube, became a haploid magnetism spouse and an embryo, a diploid embryo that was modified to become the mother of the new plant. The other male spouse was diploid It develops into the central cell and the endosperm of the triploid that fertilizes and nourishes the embryo. Therefore, a triple haploid pollen of three haploid cells is produced in a polymorphic bipolar cell, and a double fertilization with a magnetic spouse is performed to form the same plant of the next generation which is diploid.

The TIO and TWO - IN - ONE genes of the Arabidopsis thaliana gene ( TIO , TWO - IN - ONE ) are responsible for the disruption of cytoplasmic segregation during the first flower polymorphism, (Oh SA et al., 2005, Curr. Biol. 15: 2107-2111). The Arabidopsis thaliana protein consists of a total of 1322 amino acids. In terms of protein structure, there are domains containing four major arm repeat sequences (ARM repeat) at the amino terminus side and at the carboxyl terminus side. Recent studies have shown that modifying the kinase domain or the armadillo repeat domain fails to function normally (Oh SA et al., 2012, Plant J. 72: 308-319). It has already been shown that the phosphorylation gene is required during the first mitosis, somatic cell division and mitotic division of the magnetic actor itself during the development of the pollen. However, what functions in the broader cell division or plant development process including meiosis It is a task to be done. In particular, the effect of overexpression of transgenes on plant development has not been studied.

Korean Patent Publication No. 2012-0053210 discloses a method for producing a mulberry extract, and U.S. Patent No. 8383881 discloses a method for producing an orchid polyploidy plant. However, There is no description about a method for controlling the ploidy of a plant using the Arabidopsis thaliana mutant TFΔC of the present invention and the plant therefrom.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a recombinant vector containing a gene encoding Arabidopsis thaliana-derived protein variant and transforming and growing the plant cell. As a result, . The present inventors have completed the present invention by confirming that the gene coding for the transgene protein variant enables the control of the ploidy of the plant.

In order to solve the above problems, the present invention provides a recombinant vector comprising a recombinant vector comprising a gene coding for AtTIO ( Arabidopsis thaliana TWO-IN-ONE) protein variant, And regulating the expression of the gene encoding the gene encoding the polypeptide.

In addition, the present invention provides a method for producing a transgenic plant having regulated drainage of a plant, which comprises transforming a plant cell with a recombinant vector comprising a gene encoding the transgene protein variant.

The present invention also provides a transplanted plants produced by the above method.

The present invention also provides a seed of the plant.

In addition, the present invention provides a composition for regulating drainage of a plant, which contains, as an active ingredient, a recombinant vector comprising a gene encoding the transgene protein variant.

The present inventors confirmed that the overproduction of Arabidopsis thaliana-derived protein mutants was increased in papermaking ability. Drainage plants are known to have excellent metabolism, good adaptability to various environmental stresses, and high success rate of crossbreeding. Accordingly, the present invention provides a novel method for breeding potentially valuable diploid plants through overexpression of a gene encoding a transgene protein variant.

1 is an intermediate vector containing a gene expression cassette for overexpressing the Arabidopsis thaliana gene.
FIG. 2 shows the result of microscopic observation of nuclei stained with DAPI staining of the maturation pollen of Arabidopsis thaliana transformants overexpressing the transgene gene. (A) is a mature pollen of normal Arabidopsis, and two darkly stained nuclei represent the nuclei of two sperm cells (S). One nucleus that is relatively large and lightly stained is the nucleus of the nutrient cell (V). (BG) is a mature plant of Arabidopsis transgenic plants overexpressing the transgene gene. (HM) is the result of the same observation of the mature pollen of the Arabidopsis tetraspore ( tes ) mutant, which is known to produce pneumatic sperm cells. bar; 10 mu m.
FIG. 3 is a graph showing the results of Western blotting (A), in which expression of the transgene in the transgenic Arabidopsis thaliana plants was confirmed using an antibody against the HA tag, and the results of the abnormal flower polymorphism observed in 5 transgenic Arabidopsis lines Table (B) is a numerical value. con; The E. coli protein containing HA tag was a positive control for HA antibody, 1 to 9; Transgenic Arabidopsis Plants.
FIG. 4 is a microscopic observation of the pollen development process of the normal plant (normal) and the overexpressed plant (abnormal). TET; Immediately after meiosis, microparticle powder, EMS; Lysosomal microspheres initially released after carolysis; LMS; Late sporophyte, EBC; Early two-cell flower pollen immediately after first pollen mitosis, LBC; 2-cell flower pollen cells in which germ cells migrate into the cytoplasm of nutritive cells, TC; 3 pollen after the second pollen mitosis, bar; 10 mu m.
FIG. 5 is a schematic diagram (A) showing a transgenic vector region of transgenic vectors and a plant phenotype of a first-generation transgenic plant using each transformant vector. Then, plant individuals including an abnormal pollen phenotype as shown in FIG. tes-like plant ', and the antibody that detects the HA tag contained in the transformation vector was used as a graph (B) showing a percentage and western blotting confirmed the expression of the translocation protein and its mutant proteins in the transformants The result is (C). K; Lysine, D; Aspartic acid, N; Asparagine, A; Alanine, ARM repeats; Armadillo repeat sequence, proTio-TF-dHA and TF; Normal Tau protein, proTio-KA-dHA and KA; Inositol mutants in which the 35th amino acid lysine (K) is substituted with alanine (A), proTIO-DANA-dHA and DANA; Proline-TFFT-dHA and TF-FT, which are mutants in which the 127th amino acid aspartic acid (D) and the 132st amino acid asparagine (N) are substituted with alanine; A substitution mutant in which the FAX motif (FAIGNAAY) at positions 1175 to 1182 of the amino acid sequence is replaced with a Fax target (YGGAQGGW), proTIO-TFFR-dHA and -FR; Proximal amino acid substitutions of the 11th to 1182nd fax motifs in the amino acid sequence with the Fax random (LTFRDTTH), proTio-TFNT-dHA and -NT; A proximal mutant in which the Nag motif (NAAGALSN) at positions 1213 to 1220 in the amino acid sequence is replaced with the Nag target (QGGAGGDQ), proTIO-TFNR-dHA, and -NR; A proximal mutant in which the Nag motifs at positions 1213 to 1220 in the amino acid sequence are substituted with Nag random (DTTRTMCD), proTio-TF? C-dHA and? C; A proximal amino acid deficient in amino acid 1300 to 1322, proTIO-TFARM4-dHA and -ARM4; A proline-TFARM3-dHA and -ARM3 consisting of a 1 to 1279th amino acid sequence comprising all four armadillo repeat sequences (ARM) at the carboxyl terminus; A proline substitution consisting of a 1 to 1224th amino acid sequence comprising three arthmilar repeat sequences at the carboxyl terminus, proTIO-TFARM2-dHA and -ARM2; A proline variant consisting of the amino acid sequence from amino acid 1 to 1185 comprising two arthermillin repeats at the carboxyl terminus, proTIO-TFARM1-dHA and -ARM1; A proximal amino acid sequence consisting of 1 to 1141 amino acid residues comprising one armadillo repeat sequence at the carboxyl terminus, proTIO-TFARM0-dHA and -ARM0; The proTIO-KIN-dHA and KIN, which consist of the amino acid sequence from amino acid 1 to 1095 in which all four armadillo repeat sequences at the carboxyl terminal are deleted; A pro-amino acid terminal kinase domain, proTIO-CL-dHA and CL; And a kinase domain of the amino terminal is deleted. Wherein said mutants represent mutation positions based on a substitutional protein consisting of the amino acid sequence of SEQ ID NO: 1. (B) The number in parentheses indicates the number of plants used to investigate the phenotype.
FIG. 6 is a result of confirming the somatic cell phenotype common to the pollen plants in addition to the pollen phenotype. A; Normal plant seed pods, B; Drainage plant seed pods, C; Normal trichome, DF; Drainage plants, G; Normal plant shooter, HJ; Drainage flamethrower. An; Anther, Pi; Pistill, Se; Sepal.

In order to accomplish the object of the present invention, the present invention provides a gene encoding the AtTIO ( Arabidopsis thaliana TWO-IN-ONE) protein variant from which the 1300th to 1322nd amino acids of the amino acid sequence of SEQ ID NO: 1 are deleted And transforming the recombinant vector into a plant cell to regulate the expression of the gene coding for the transgene protein variant, thereby controlling the polyploidy of the plant.

The method according to one embodiment of the present invention is preferably, but not limited to, overexpression of the transgene protein mutant coding gene to increase the drainage of the plant.

The present invention relates to a method for producing an Arabidopsis thaliana mutant of Arabidopsis thaliana (AtTIO, Arabidopsis thaliana, transforming a plant cell with a recombinant vector comprising a gene encoding a thaliana TWO-IN-ONE protein variant; And

And regenerating the plant from the transformed plant cell. The present invention also provides a method of producing a transgenic plant having regulated drainage of a plant.

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, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an 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" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

A preferred example of a plant expression vector is a Ti-plasmid vector which is capable of transferring a so-called T-region into a plant cell when present in a suitable host such as Agrobacterium tumefaciens . Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced 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 the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as Kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.

In the plant expression vector 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, constitutive promoters do not limit selectivity.

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) Terminator of the Octopine gene, and the rrnB1 / B2 terminator of E. coli, but the present invention is not limited thereto. Regarding 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.

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae), and the like insect cells, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cell may be used. The host cell is preferably a plant cell.

"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.

The method of delivering the vector of the present invention into a host cell can be carried out by injecting a vector into a host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, can do.

In addition, the present invention provides plants with controlled mobility, preferably increased, and seeds thereof produced by the method.

In a transgenic plant according to an embodiment of the present invention, the plant is selected from the group consisting of Arabidopsis thaliana, eggplant, tobacco, pepper, tomato, burdock, cilia, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, And may be a dicotyledon such as Chinese cabbage, cabbage, gab, watermelon, melon, cucumber, amber, pak, strawberry, soybean, mung bean, kidney bean and pea, and preferably, radish, Chinese cabbage, oilseed rape, It may be a Brassicaceae plant such as cabbage, mustard, horseradish, freshly picked, freshly picked, pollen, flower, flower mackerel, rice cob, rutabaga, forked flower, cauliflower, broccoli, cabbage, cauliflower, But it is not limited thereto.

The present invention also relates to a method for producing an Arabidopsis thaliana mutant (AtTIO, Arabidopsis thaliana) having a 1300 to 1322th amino acid deletion in the amino acid sequence of SEQ ID NO: thaliana TWO-IN-ONE) protein variant as an active ingredient. The present invention also provides a composition for regulating the drainage of a plant, comprising a recombinant vector comprising a gene encoding a thaliana TWO-IN-ONE protein variant.

In the composition of the present invention, the above-mentioned source protein variant is as described above. The composition of the present invention contains, as an active ingredient, a recombinant vector comprising a gene encoding a Arabidopsis thaliana-derived protein variant in which amino acids 1300 to 1322 of the amino acid sequence of SEQ ID NO: 1 are deleted, and the Arabidopsis thaliana- It is possible to increase plant drainage by transforming a plant with a recombinant vector containing a gene encoding a mutant. The plants are as described above.

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

Example  One. Two people  Generation of gene expression vectors

The plant expression vector was prepared based on the previously prepared proMSP1-TF-3XMyc vector (Honys et al., 2006, BMC Plant Biol. 6: 31-39). This vector is based on the commercially available pBluescript and contains a promoter (proTIO) between the restriction enzymes AscI and NotI , a translocation cDNA (TIO cDNA) between NotI and SpeI , and a duplex containing the Nos terminator between SpeI and PacI (double HA) HA (dHA-Noster) was inserted (Fig. 1). AscI and PacI sites were separated from the intermediate vector prepared above, and inserted between AscI and PacI of pER10 , which is a binary vector, to complete a plant transformation vector.

Example  2. Transgenic plant transformation using plant expression vectors

The transforming vector was amplified in E. coli, and these vectors were transferred to Agrobacterium strain GV3101. The Agrobacterium strain was cultured at 28 DEG C in 400 mL of LB medium, and then transferred to Arabidopsis thaliana ). Transformation into Arabidopsis was performed according to the pedicle dipping method (Clough and Bent, 1998, Plant J. 16: 735-743). The seedlings were harvested (T1 seeds) after growing the corresponding plants (T0 plants) transformed by the pedicle dipping method in the growth chamber conditions and subjected to seed sterilization to obtain antibiotic resistance in MS plant medium containing 50 μg / ml of kanamycin antibiotics T1 stems were selected. The selected plants were transferred to soil and cultured under growth conditions. Genomic DNA was extracted from the plant 3-4 weeks after germination, before transplantation, to confirm that the transformant was a transformed T-DNA-inserted transformant.

Example  3. Pollen phenotype observation of transgenic plants

When the T1 plant bloomed, three fresh flowers per plant were collected and placed in a 1.5 ml centrifuge tube containing 250 μl of DAPI reagent. Shake well and extract the pollen from the anther into the solution and submerge it on the bottom of the tube using a small centrifuge. About 2.5 화 of potted pellets were taken with a pipette, and a slide for microscope observation was prepared. DAPI stained pollen cells were observed by UV fluorescence microscopy and pollen counts of approximately 300 lips or more were counted by phenotype.

As a result, the number of sperm cells (yellow S) in the mature pollen of overexpressed Arabidopsis plants was 2 or 3 times as large as in the normal mature pollen (Fig. 2A) 2B), 4 or 6 sperm cells were observed (Fig. 2C-D). There is also a low frequency, but as shown in FIG. 2E-G, two blurred nuclei, which appear to be abnormal vaginal cells (yellow V) without a sperm cell, or one nucleus that appears to be fused, have been observed. The pollen of the tetraspore ( tes ) mutant used as a control (Fig. 2H-M) showed an abnormal flower pollen phenotype very similar to the mature pollen of the transgenic overgrown plant.

Example  4. Abnormal flowerpot phenotype and Two people  Correlation of gene expression

The correlation between the abnormal flower pollen expression level and the expression level of the responder gene shown in Fig. 2 was confirmed. First, the seeds of nine independent T2 generation lines were disinfected and cultivated in a plant medium containing kanamycin, and then plants resistant to the antibiotic were selected. Five plants in each line were transferred to soil to observe the phenotype of the mature pollen After planting, the plants were grown in the growth chamber and protein was extracted from the remaining plants. Western blotting was performed using an antibody that detects the HA tag contained in the transformed vector and the extracted protein to confirm the expression level of the phosphorylated protein. The phenotype of mature pollen was observed for each line.

As a result, all three of the five plants exhibited a high level of abnormal pollen phenotype in line 3, where the phosphorylation protein was expressed at the highest level, and five in line 1, 2, 6 and 7, No abnormal pollen phenotype was observed in all of the plants (Figure 3). On the other hand, in four lines of 4, 5, 8 and 9, where medium level of protein expression was detected, abnormal flower pollen phenotype was observed in only one or two plants (Fig. 3). These results indicate that the overexpression of the transgene protein affects the abnormal phenotype phenotype.

Example  5. Identification of pollen development process of transgenic Arabidopsis thaliana

The pollen development process was compared in transgenic Arabidopsis plants overexpressing normal and responder genes. The other flowers were treated with fixative (Ethanol: Acetic acid 3: 1) at different stages of development, and then separated by a microscope under a microscope. DAPI samples were stained with nuclei and observed under a UV fluorescence microscope.

The arrowheads I and J in FIG. 4 indicate the germ pore observed at the late sporulation (LMS) period, and the transformant shows the appearance of germination balls regularly appearing three in the normal plant (FIG. 4I) (Fig. 4J) or irregularly abnormal (Fig. 4K and L). On the other hand, the arrowhead symbol of MP in FIG. 4 shows a cell wall appearing as a curved shape leaning to one side during the asymmetric first pollen mitosis, which is common to both the normal plant and the pollen of the transformant. It was also found that the second pollen-like mitosis also appeared normally (Fig. 4U-X). In other words, in the case of normal plants, four haploid quartz grains were produced after meiosis, whereas in the transgenic grains overexpressing transformants, two or more multiparticulates were formed after meiosis, Which is the result of disturbance.

Example  6. Two people  gene Mutant  Transgenic Plants Using Transgenic Plants

Mutants in which some of the transgenic cDNA gene sites were substituted or deleted were used to confirm the increase in the drainage of the above-mentioned plants. The mutant used in the present invention is a mutant having amino acid terminal amino acid asparagine (D) and amino acid asparagine (D) having the amino acid sequence of KA (amino acid lysine (K) substituted with alanine (FAIGNAAY) is substituted with Fax Target (YGGAQGGW)), TFFR (Fax Motif is replaced by Fax Motif), TFFT in which a part of amino acids in the carboxyl terminal site or the armadillo repeat domain is substituted (Nag motif (substitution with Nag target (QGGAGGDQ)) and TFNR (Nag motif replaced with Nag random (DTTRTMCD)), a carboxyl terminal site or armadillo repeat (Deletion of 1300 to 1322 amino acid residues), TFARM4 (a variant consisting of amino acid sequences of 1 to 1279 amino acids including all four carboxyl terminal repeat sequences), TFARM3 (Mutant consisting of amino acid sequence 1 to 1224 amino acid sequence containing three terminal arthmylo repeat sequences), TFARM2 (variant consisting of amino acid sequence from 1 to 1185th amino acid sequence containing two carboxyl terminal repeat sequences), TFARM1 5A) which contains or excludes only the amino terminal region kinase domain, and TFARM0 (a mutation consisting of the 1 to 1141th amino acid sequence including an armadillo repeat sequence) and TFARM0 (all four armadillo repeat sequences at the carboxyl terminus are deleted) .

As a result, the transformant of all species except the transgenic vector (KIN) containing only the kinase domain among the transgene cDNA gene region and the empty vector control gene containing the transgene gene contained abnormal phenotype expression at a similar level (Fig. 5B). In addition, western blotting was performed using an antibody that detects the HA tag contained in the vector, and as a result, a mutant protein of the transgene was confirmed in the transgenic plant (Fig. 5C). The results showed that the transplanted plants using the transgenic mutants were also increased in ploidy.

Example  7. Somatic phenotype observation of transgenic plants

In addition to pollen phenotypes, somatic phenotypes appearing in polyploid plants were examined. As a result, when the seed pod is shorter than the normal plant (Fig. 6A) (Fig. 6B), and when the trichome on the surface of the leaf is normal, there are mainly three branches and some four branches ), And a large number of mothers were found in three or more branches in the draining plants (Fig. 6D-F). Also, when the size of the firearm is very large (Fig. 6H) compared with the normal (Fig. 6G) or when the structure is deformed (Figs. (C-F), the red dot represents 4 branches, and the yellow dot represents the trichome with 5 branches.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Method for controlling polyploidy of plant using AtTIO TF delta C          mutant and the plant thereof <130> PN13224 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1322 <212> PRT <213> Arabidopsis thaliana <400> 1 Met Gly Val Glu Asp Tyr His Val Ile Glu Leu Val Gly Glu Gly Ser   1 5 10 15 Phe Gly Arg Val Tyr Lys Gly Arg Arg Lys Tyr Thr Gly Gln Thr Val              20 25 30 Ala Met Lys Phe Ile Met Lys Gln Gly Lys Thr Asp Lys Asp Ile His          35 40 45 Ser Leu Arg Gln Glu Ile Glu Ile Leu Arg Lys Leu Lys His Glu Asn      50 55 60 Ile Ile Glu Met Leu Asp Ser Phe Glu Asn Ala Arg Glu Phe Cys Val  65 70 75 80 Val Thr Glu Phe Ala Gln Gly Glu Leu Phe Glu Ile Leu Glu Asp Asp                  85 90 95 Lys Cys Leu Pro Glu Glu Gln Val Gln Ala Ile Ala Lys Gln Leu Val             100 105 110 Lys Ala Leu Asp Tyr Leu His Ser Asn Arg Ile Ile His Arg Asp Met         115 120 125 Lys Pro Gln Asn Ile Leu Ile Gly Ala Gly Ser Val Val Lys Leu Cys     130 135 140 Asp Phe Gly Phe Ala Arg Ala Met Ser Thr Asn Thr Val Val Leu Arg 145 150 155 160 Ser Ile Lys Gly Thr Pro Leu Tyr Met Ala Pro Glu Leu Val Lys Glu                 165 170 175 Gln Pro Tyr Asp Arg Thr Val Asp Leu Trp Ser Leu Gly Val Ile Leu             180 185 190 Tyr Glu Leu Tyr Val Gly Gln Pro Pro Phe Tyr Thr Asn Ser Val Tyr         195 200 205 Ala Leu Ile Arg His Ile Val Lys Asp Pro Val Lys Tyr Pro Asp Glu     210 215 220 Met Ser Thr Tyr Phe Lys Ser Phe Leu Lys Gly Leu Leu Asn Lys Glu 225 230 235 240 Pro His Ser Arg Leu Thr Trp Pro Ala Leu Arg Glu His Pro Phe Val                 245 250 255 Lys Glu Thr Gln Glu Glu Val Glu Ala Arg Glu Ile His Thr Ala Val             260 265 270 Val Asp Asn Lys Ala Ala Trp Met Leu Lys Gly Asn Gly Gly Gln Gln         275 280 285 Arg Asn Glu Lys Cys Asp Ser Val Thr Leu Val Glu Asp Met Ser Ala     290 295 300 Thr Lys Gly Leu Ala Asp Val Gln Ser Asp Met Lys Ser Ala Val Lys 305 310 315 320 Val Asn Ser Pro Pro Thr Glu Asp Phe Val Gly Phe Pro Thr Gln Glu                 325 330 335 Glu Ile Lys Ser Ser Gly Asn Pro Thr Leu Asp Lys Leu Glu Asn Thr             340 345 350 Ser Arg Thr Val Lys Gly Ala Gln Val Ile Gly Glu Asn Asp Lys Ala         355 360 365 Leu Asp Leu Val Leu Leu Ser Leu Glu Arg Phe Ser Lys Ser Pro Asp     370 375 380 Ser Lys Arg Asp Lys Asp Val Ala Cys Ser Val Gln Ser Leu Arg Ile 385 390 395 400 Ile Ser Asn Leu Val Ala Thr Arg Ala Ile Val Ser Val Gly Leu Ile                 405 410 415 Glu Lys Ile Thr Cys Ala Leu Leu Asp Phe Thr Asp Ala Leu Val Gly             420 425 430 Met Lys Ser Pro Glu Phe Asn Asn Ile Pro Lys Ser Leu Ser Val         435 440 445 Thr Lys Asn Leu Val Gly His Val Glu Gly Asn Asn Ile His Ser Ser     450 455 460 Tyr Ile Arg His Trp Thr Lys Val Val Glu Ile Phe Ile Gln Val Val 465 470 475 480 Arg Trp Glu Glu Glu Gly Thr Gly Arg Ile Ile Tyr Glu Ala Cys Ser                 485 490 495 Cys Ile Thr Thr Met Leu Ser Arg Val Ala Gln Asp Leu Lys Ser Ser             500 505 510 Thr Pro Asp Ser Val Ser Lys Gln Ile Leu Glu His Ala Asn Met Ser         515 520 525 Arg Ile Val Asp His Leu Cys Leu Cys Leu Ala Ser Ser Gly Ser Ser     530 535 540 Leu Thr Ser Gly Ser Ser Gln Met Leu Ala Ala Ala Cys Glu Ala Cys 545 550 555 560 Arg Ala Ile Trp Ile Leu Ile Asp Thr Ser Glu Thr Phe Phe Lys Asn                 565 570 575 Asp Asp Val Asn Ile Leu Pro Leu Asp Ala Leu Gln Asn Arg Leu Ser             580 585 590 Gln His Asp Ile Gly Asn Ser Glu Trp Gly Pro Leu Ser Glu Lys Leu         595 600 605 Val Asp Thr Val Thr Arg Ala Tyr Leu Arg Ser Lys His Val Gln Val     610 615 620 Ala Val Gly His Cys Leu His Gln Arg Val Glu Ala Pro Leu Val Ser 625 630 635 640 Ala Ile Gln Leu Leu Ser Arg Cys Cys Leu His Asn Gly Ile Leu Pro                 645 650 655 Ser Met Leu Cys Gly Leu Pro Ser Ser Leu Pro Ile Thr Thr Val Val             660 665 670 Ser Gly Gly Glu Asp Gly Thr Val Ile Ser Glu Ile Phe Ser Ile Leu         675 680 685 Ser Tyr Ala Thr Leu Ser Ser Lys Asp Gln Gln Thr Gly Glu Lys Asp     690 695 700 Asn Phe Glu Gly Arg Leu Asn Asn Leu Val Phe His Ser Cys Leu Met 705 710 715 720 Leu Ala Thr Val Ala Gln Cys Leu Lys Leu Thr Gly Arg Asn Ser Val                 725 730 735 Leu Leu Met Leu Thr Thr Ser Pro Lys Lys His Gln His Arg Leu Ser             740 745 750 Ala Ile Ala Asn His Ile Ala Ser Asp Asp Lys Ile Glu Ala Ser Leu         755 760 765 Gln Asn His Ser Ala Ser Ala Met Leu Ala Leu Ala Ser Ile Leu Ala     770 775 780 Leu Glu Lys Gly Ser Ser Ala Gly Ser Ser Val Ser Glu Leu Val Val 785 790 795 800 Ser Leu Ile Pro Arg Ala Thr Lys Leu Cys Tyr His Leu Arg Pro Met                 805 810 815 Pro Ser Asn Glu Gly Glu Val Ile Ser His Ser Ala Asn Tyr Ala Lys             820 825 830 Trp His Gly Leu Leu Asp Gly Cys Ile Gly Leu Leu Glu Ser Arg Leu         835 840 845 Lys Trp Gly Gly Pro Leu Ala Val Gln Gln Leu Ile Ala Ser Gly Thr     850 855 860 Pro Leu Leu Leu Ile Asn Leu Leu Ala Gly Lys Leu Ser Asn Ala Ser 865 870 875 880 Pro Glu Asp Ile Lys Lys Thr Ser Asn Arg Ile Gly Leu Ser Pro Ile                 885 890 895 Gly Val Val Trp Thr Ile Ser Ser Ile Cys His Cys Leu Ser Gly Gly             900 905 910 Thr Thr Phe Arg Gln Val Leu Val Lys Ile Glu Thr Met Lys Leu Ile         915 920 925 Thr Cys Leu Leu Ser Asp Ala His Ile Lys Leu Val Lys Ser Trp Gly     930 935 940 Gly Pro Gly Gly Gly Lys Asp Gly Val Arg Glu Thr Ile Asn Val Ile 945 950 955 960 Ile Asp Leu Leu Ala Phe Pro Phe Val Ala Leu Gln Ser Gln Pro Gly                 965 970 975 Ser Leu Ser Ala Thr Ala Ser Val Asn Ser Gly Phe Ile Leu Asn Ile             980 985 990 Gly Ser Pro Gly Val Arg Val Cys Met Glu Asp Arg Asp Leu Leu Lys         995 1000 1005 Ala Ile Glu Asp Met Asp Lys Tyr Ile Val Leu Leu Glu Val    1010 1015 1020 Gly Val Pro Ser Leu Ile Leu Arg Cys Leu Asp His Leu Glu Leu Lys 1025 1030 1035 1040 Asp Leu Val Val Pro Val Ala Phe Leu Ala Lys Met Val Gly Arg Pro                1045 1050 1055 Arg Leu Ala Val Asp Leu Val Ser Lys Gly Leu Leu Asp Pro Asn Arg            1060 1065 1070 Met Lys Lys Leu Leu Asn Gln Ser Ser Pro Arg Glu Val Ile Leu Asp        1075 1080 1085 Ile Leu Met Ile Ile Ser Asp Leu Ser Arg Met Asp Lys Ala Phe Tyr    1090 1095 1100 Lys Tyr Ile Gly Glu Ala Ser Val Leu Gln Pro Leu Lys Glu Tyr Leu 1105 1110 1115 1120 Thr His Val Asp Pro Asn Ile Arg Ala Lys Ala Cys Ser Ala Leu Gly                1125 1130 1135 Asn Met Cys Arg His Asn Gly Tyr Phe Tyr Ser Ala Leu Ala Glu His            1140 1145 1150 Gln Ile Gly Leu Leu Ile Asp Arg Cys Ala Asp Pro Asp Lys Arg        1155 1160 1165 Thr Gln Lys Phe Ala Cys Phe Ala Ile Gly Asn Ala Ala Tyr His Asn    1170 1175 1180 Asp Thr Leu Tyr Glu Glu Leu Arg Arg Ser Ile Thr Gln Leu Ala Asn 1185 1190 1195 1200 Val Leu Thr Thr Ala Glu Glu Asp Lys Thr Lys Ala Asn Ala Ala Gly                1205 1210 1215 Ala Leu Ser Asn Leu Val Arg Asn Ser Asn Lys Leu Cys Glu Asp Ile            1220 1225 1230 Val Ser Lys Gly Ala Leu Gln Thr Leu Leu Arg Leu Val Ala Asp Cys        1235 1240 1245 Ser Thr Leu Ala Leu Asn Pro Ser Lys Lys Glu Thr Ala Ser Glu Ser    1250 1255 1260 Pro Leu Lys Ile Ala Leu Phe Ser Leu Ala Lys Met Cys Ser Asn His 1265 1270 1275 1280 Gln Ile Cys Arg Gln Phe Val Lys Ser Ser Glu Leu Phe Pro Val Ile                1285 1290 1295 Ala Arg Leu Lys Gln Ser Pro Glu Ala Asn Ile Ala His Tyr Ala Ser            1300 1305 1310 Val Ile Val Ala Lys Val Ser Gly Glu Ser        1315 1320

Claims (12)

A recombinant vector comprising a gene coding for a protein mutant of AtiTo ( Arabidopsis thaliana TWO-IN-ONE) having a 1300 to 1322th amino acid deletion in the amino acid sequence of SEQ ID NO: 1 is transformed into plant cells, A method of increasing polyploidy of a plant comprising overexpressing a gene encoding a transgene protein variant. delete A plant cell is transformed with a recombinant vector comprising a gene encoding a protein variant of AtTIO ( Arabidopsis thaliana TWO-IN-ONE) lacking 1300 to 1322 amino acids of the amino acid sequence of SEQ ID NO: 1, Overexpressing a gene encoding a transgene protein variant; And
And regenerating the plant from the transformed plant cell. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; delete An improved transplanted plant produced by the method of claim 3. delete 6. The plant according to claim 5, wherein the plant is a dicotyledonous plant. The method of claim 7, wherein the plant is a plant ssangjayeop characterized in that the Brassicaceae (Brassicaceae) plants. A plant seed according to claim 5. A recombinant vector comprising, as an active ingredient, a recombinant vector comprising a gene coding for a protein variant (AtTIO, Arabidopsis thaliana TWO-IN-ONE) lacking the 1300th to 1322nd amino acids of the amino acid sequence of SEQ ID NO: / RTI &gt; 11. The composition according to claim 10, wherein the plant is a dicotyledonous plant. 12. The method of claim 11, wherein the plant is a cruciferous ssangjayeop (Brassicaceae) increase drainage of the plant, characterized in that the plant composition.

Images