KR101316597B1 - Method for controlling plant growth with gene derived from Oryza sativa - Google Patents

Abstract

The present invention relates to a method for controlling plant growth or morphology by using a gene isolated from rice, and more specifically, by overexpressing OsTCP6 protein selected from rice ( Oryza sativa ) mutants and isolated from normal rice cells in plants. It relates to a method of increasing genes that play a central role in division and regulating the growth and / or morphology of plants. By expressing a transformant of the OsTCP6 gene in a plant according to the present invention, it is possible to increase gene expression, which acts as a central regulator of cell division, and to control the growth of the plant. It is possible to increase crop productivity by controlling the shape of the leaves of the plant.

Description

Method for controlling plant growth with gene derived from Oryza sativa}

The present invention relates to a method for regulating the growth or form of a plant using a gene isolated from rice, and more specifically, rice ( Oryza sativa ) relates to a method of increasing genes that play a central role in cell division and regulating plant growth and / or morphology by overexpressing OsTCP6 protein selected from mutants and isolated from normal rice in plants.

Plants have a system to cope with and adapt to various environmental changes by regulating growth and development. Throughout the entire living environment, from seed germination to flowering, environmental stimuli and plant development programs are known to increase plant viability through endless interactions. The developmental control of plants according to such environmental stimuli is known to occur mainly through the interaction of the external environment with the plant's intrinsic development program.

Rice is one of the most important crops in the world for the purpose of seed production. In recent years, the entire gene sequence has been revealed, and a lot of efforts have been made to acquire a large number of useful genes and to preoccupy them. As a technique for improving the size and growth of a conventional seed, a method for regulating plant cell growth by modifying the concentration or catalytic activity of cell cycle control protein in plant cells (US Pat. No. 6,087,175) or synthetic zinc Method of controlling the expression level of the target gene in the plant cell by introducing an expression vector bound to the target gene to enhance the nucleotides encoding a synthetic zinc finger protein (plant patent 7,151,201), etc. Is disclosed. Also, there is a technology (Korean Patent Registration No. 10-0794395) in which the gene of cytochrome P450 protein derived from Arabidopsis thaliana is transformed into plant cells to increase the size of plant seeds.

The shape and size of the organisms such as the stem and leaf of the plant are important factors that increase the biomass and affect the agricultural productivity. Research on stemming mechanism of major grains such as rice, maize, wheat, and millet is in the active stage. Since teosinte branched 1 (tb1), a stem-producing gene that enabled corn breeding, was identified, genes related to stem and fire differentiation such as zfl (FLORICAULA / LEAFY), bif (barren inflorescence2), and pin (pinoid) were accelerated. (Lukens and Doebley 2001, Barazesh and McSteen 2008). First known in maize, tb1 has been shown to be a major gene for suppressing germination in rice and Arabidopsis (Takeda et al. 2003, Finlayson 2007, Aquilar-Martinez et al. 2007).

(Schumacher et al. 1999, Schmitz et al. 2002), and other genes such as sugarcane, bamboo, etc. have been identified in addition to major crops such as rice, maize and corn Research into the stem production regulatory genes to promote biomass is at an early stage (Aiteken et al. 2008). In addition, recent studies have shown that tb1 gene of maize is introduced into wheat to inhibit cleavage (Lewis 2008), indicating that these genes can be used in biotechnology.

Among the plant organs, in particular the leaf shape is an important agricultural feature for controlling the ornamental and productivity of plants. The leaves are formed in the leaf primordia around the shoot apical meristem, and the ultimate leaf size and shape are determined by the balance between cell division and cell extension around the three axes of the leaves. It is known. This process is a complex process caused by the stepwise interaction between various functions of genes, and many genes have been reported around the Arabidopsis.

For example, genes involved in foliar formation, such as Knotted-like homeobox (KNOX), PHANTASTICA (PHAN), ROUGH SHEATH (RS), and ASYMETRIC LEAVES 1 (AS1), as well as HD-ZIP III, PHABULOSA (PHB), and PHAVOLUTA (PHV). In addition to genes involved in ischemia formation, such as REVOLUTA (REV), YABBY, KANADI, genes involved in cell proliferation and cell growth (ICK1 (cyclin-dependent kinase inhibitor 1), KRP2 (Kip-related protein), ROT (ROTUNDIFOLIA) ), AN (ANGUSTIFOLIA), and LNG (LONGIFOLIA) are known to alter leaf morphogenesis. Also, unlike genes such as Aintegumenta (ANT), KLUH (KLU), STRUWWELPETER (SWP), Growth-regulating factor (GRF), and GRF-interacting factor (GIF), act as positive regulators of leaf development. (Anastasiu et al., 2007, Autran et al., 2002, Mizukami and Fisher 2000, Horiguchi et al. 2005, Kim and Kende 2004), BIG BROTHER (BB) or PEAPOD (PPD), which inhibit leaf growth Known as a negative regulator. These genes have various functional categories such as transcription factors, biosynthetic genes, and proteolytic factors.They have different functional mechanisms such as foliar formation, leaf axis formation, cell proliferation, and cell growth, which affect leaf shape and size. This suggests that the spectrum of intracellular mechanisms or genes is very diverse.

On the other hand, the TCP transcription factor is a transcription factor including the bHLH domain, and cycloidea (cyc) and teosinte branched 1 (tb1) play a very important role in the morphological evolution of endodermal organs such as plants' flowers and stems. The molecular biological function of proteins is not well known. PCFs with TCP domains have been reported to promote cell division, and TCP transcription factors have been suggested to play an important regulatory role in the growth and differentiation of meristems. (Cubas et al. 1999).

The TCP transcription factor of the cyclodea family is particularly a central gene that regulates morphogenesis of flowers. The most frequent cis-acting element in the promoter of expression-induced genes in Arabidopsis chicks is similar to the TCP binding site (Tatematsu et al. 2005). Inhibiting the function of Arabidopsis TCP3 results in the formation of leaves and flowers with high surface curvature because TCP inhibits the expression of organ differentiation control genes such as CUC (Koyama et al. 2007). Suppressing the function of AtTCP20 is thought to play a role in regulating the balance between cell division and growth because of the severely altered phenotypes of various organs such as cotyledon, leaf and hypocotyl (Herve et al. 2009).

Rice genome analysis shows that there are about 24 TCP transcription factors, and biological functions are unknown except fc1 (OsTB1), which inhibits division, PCF1, which is involved in cell division, and RFP1, which controls the symmetry of reproductive organs (Takeda et al. 2003, Yuan et al. 2009).

Accordingly, the present inventors conducted a study of plant endodermal mutants to find new transcription factors that are involved in the formation of endodermal organs of plants. The present invention was completed by identifying a method of controlling plant growth and morphology by finding OsTCP6, a rice-derived gene that can contribute to increasing productivity of crops.

In the present invention, the genes related to the growth of plants were searched for, and the characteristics thereof were used to increase the productivity of plants. Therefore, the present invention is to provide a method for controlling the growth of plants using the OsTCP6 gene by transforming the OsTCP6 gene isolated from rice in the Arabidopsis by analyzing the function of the OsTCP6 gene and confirming its utility as a useful gene.

The present invention provides the following problem solving means to achieve the above object.

1. A protein that regulates the growth or form of a plant having the amino acid sequence represented by SEQ ID NO: 2.

2. The gene encoding the protein of above 1.

3. The gene of claim 2, wherein the gene has a nucleotide sequence represented by SEQ ID NO: 1.

4. A method of controlling the growth or morphology of a plant by controlling the intracellular level of a protein having the amino acid sequence represented by SEQ ID NO: 2.

5. The method according to the above 4, wherein the regulating the intracellular level increases or decreases the expression of the gene encoding the protein.

6. In the above 4, the method for increasing or decreasing the expression of the gene is to produce a recombinant expression vector linking the gene to the promoter to enhance the expression, or to recombination of the antisense gene for the gene linked to the promoter Method of controlling the growth or shape of the plant, characterized in that to produce an expression vector to reduce its expression.

7. The method according to 6 above, wherein the regulation of intracellular levels is characterized by introducing a gene encoding the protein into plant cells or transforming a plant with the gene.

8. according to the above 4, the plants are Arabidopsis, Chinese cabbage, cabbage, mustard, rape (rapeseed), radish, radish (Brassica napobrassica), turnip (Brassica rapa / Brassica campestris), tricale, cauliflower, broccoli, wasabi , Stork, Pole Sprout, Flower Pot, Brasica juncea, Brasica napus, Brassica oleracea, Brassica caulorapa, Brassica pimbriata, Brassica Brassica ruvo, Brassica septi-ceps, Black mustard (Brassica nigra), Cochlearia officinalis, Mustard radish (Armoracia lapathifolia), Descurinia pinnata (Descurainia) pinnata), and at least one selected from the group consisting of Aubrieta deltoidea.

9. In the above 4, the plants are rice, corn, onions, leeks, barley, wheat, leek, garlic, tac, zimo, astronomical dong, lily of the valley, pado, mammun-dong, jinhwangjeong, erosion, bamboo and phalaenopsis spp , Cymbidium goeringii, Cymbidium ssp, Denparium phalaenopsis, Neofinetia falcata, Aerides japonicum Cattlea ssp, Oncidium sspras, Brassia sspras To regulate the growth or shape of a plant, characterized in that it is at least one selected from the group consisting of orchid family plants, such as Vanda ssp, Papyoedilum ssp, Miltonia ssp, and Cymbidium Kanran How to.

10. The method according to the above 4, wherein the gene having the nucleotide sequence represented by SEQ ID NO: 1 is a gene separated from the meristem of rice.

11. The method according to the above 4, wherein the plant cell growth or morphological changes of the plant is at least one site selected from the group consisting of leaves, side diseases, and hypocotyls.

12. Plants with increased growth or form prepared according to the above 4 methods.

13. A method of increasing gene expression that acts as a central regulator of cell growth, division or cell cycle by overexpressing a protein having the amino acid sequence represented by SEQ ID NO: 2.

14. The method of claim 13, wherein the gene is any one selected from the group consisting of PCNA, cyclinD3, cyclinB1, cdc2b, and expansin.

15. Recombinant expression vector comprising the above gene.

16. A transformant transformed with the recombinant expression vector of 15 above.

17. Composition for controlling growth or form of a plant comprising the protein of the above 1 or the transformant of the above 16.

It is possible to control the growth of the plant by expressing the transformant produced using the OsTCP6 gene and the recombinant vector including the gene in the plant, and to modify the size of the plant or the shape of the leaf of the plant By doing so, it is possible to increase crop productivity.

1 shows a plant expression vector fused with OsTCP6 and GFP.
FIG. 2 confirms the expression of OsTCP6 in the nucleus of tobacco leaf cells transiently transformed with Agrobacterium (GV3101) containing an OsTCP6-GFP fusion vector.
[Figure 3] confirms whether the expression of OsTCP6 transcripts in the stem apical division (SAM) and root meristem (CR) of the Arabidopsis.
4 shows a recombinant vector for overexpression of OsTCP6.
5 is genomic PCR and RT-PCR results confirming that the overexpression of OsTCP6 vector was introduced.
6 is a result of comparing the size of the leaves of the Arabidopsis overexpressed OsTCP6.
7 is a result of comparing the number of leaflets of the Arabidopsis overexpressed OsTCP6.
8 is a comparison of major gene expressions related to cell division and cell growth in Arabidopsis overexpressed OsTCP6.

The present invention relates to a method for regulating the growth or form of a plant using a gene isolated from rice, and more specifically, rice ( Oryza increased expression of genes that act as central regulators of cell division by overexpressing proteins that regulate the growth or morphology of plants having the amino acid sequence represented by SEQ ID NO: 2, selected from mutants of sativa and isolated from meristem of rice. It is about how to let. According to the present invention, by expressing a transformant of a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 in a plant, it is possible to increase the expression of a gene serving as a central regulator of cell division and to regulate the growth of the plant. Thus, according to the present invention it is possible to increase the productivity of the crop by modifying the size of the plant or by adjusting the shape of the leaves of the plant.

Below. The present invention will be described in detail.

The present invention relates to a method for controlling the growth or shape of a plant by controlling the intracellular level of the protein having the amino acid sequence represented by SEQ ID NO: 2.

The protein having the amino acid sequence represented by SEQ ID NO: 2 is a protein having 410 amino acids, and the like, such as the growth of plants, the number and form of leaves of plants, the form of fat nutrient growth organs including hypocotyls and foliar diseases, and the like. Involved in growth.

In addition, the protein includes a homologous protein having homology with a protein having an amino acid sequence represented by SEQ ID NO: 2.

The 'homologous protein' is preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the amino acid sequence of SEQ ID NO: 2. As a protein, it refers to a protein exhibiting substantially the same function as OsTCP6 of the present invention.

The present invention also relates to a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2. More preferably, the gene has a nucleotide sequence represented by SEQ ID NO: 1.

In addition, the gene includes a homologous gene having homology with a gene having a nucleotide sequence represented by SEQ ID NO: 1.

We cloned the OsTCP6 gene, which was selected for high expression in mutants of rice and isolated from meristem of rice, by RT-PCR method, and registered the base sequence as GQ229483 in GenBank (Gen. 2010). Public).

OsTCP6 gene having a nucleotide sequence represented by SEQ ID NO: 1 or a homologous gene thereof is encoded by a protein. SEQ ID NO: 1 consists of 1233 nucleotides.

The 'homologous gene' is preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the nucleotide sequence of SEQ ID NO: 1. As a gene, it refers to a gene exhibiting substantially the same function as OsTCP6 of the present invention.

By "substantially homogeneous function" is meant to be involved in the determination of the morphology of the plant, in particular in the width to length of the plant tissue. Such functional equivalents include amino acid sequence variants in which some of the amino acids of the amino acid sequence represented by SEQ ID NO: 2 are substituted, deleted or added. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows; (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met).

Deletion of amino acids is preferably located in portions not directly involved in the activity of OsTCP6 of the present invention. In addition, the functional equivalent range includes protein derivatives in which some chemical structures of the protein are modified while maintaining the basic skeleton of OsTCP6 and its physiological activity. For example, fusion proteins made of fusions with other proteins such as GFP, while maintaining structural changes and physiological activities to alter the stability, shelf life, volatility or solubility of the protein of the present invention.

In the present invention, the shape of the plant indicates the external structure or shape of the plant, and the shape or structure of the plant tissues or organs such as leaves, stems, roots, and flowers, particularly the nutrient growth organs of the ground, and combinations thereof. The overall appearance is shown.

In the present invention, since the length and width of a part or the whole of the tissues and organs are mainly changed according to the intracellular level of the protein having the amino acid sequence represented by the amino acid sequence of SEQ ID NO: 2, the change of the shape of the plant is mainly The leaves, stems, etc. mainly represent changes in the length and width of each part or whole of the plant ground tissue.

The 'intracellular level' refers to the amount present in the cell, which can be adjusted by various methods known to those skilled in the art. For example, intracellular levels can be regulated through, but not limited to, regulation at the transcriptional stage or regulation at the post-transcriptional stage. Modulation at the transcriptional stage is a method for enhancing the expression of genes known to those skilled in the art, for example, by preparing a recombinant expression vector linking OsTCP6 of SEQ ID NO: 1 or homologous genes thereof to a promoter to enhance expression of the gene. Or a method of inserting an expression control sequence such that expression of the gene is enhanced around OsTCP6 of SEQ ID NO: 1 or a homologous gene thereof. Modulation at the post-transcriptional stage is a method for enhancing protein expression known to those skilled in the art, for example, to enhance the stability of mRNA transcribed into the OsTCP6 gene of SEQ ID NO: 1 or a functional equivalent thereof, the protein or protein It can be carried out by a method of enhancing the stability or a method of enhancing the activity of the protein or protein.

More specific examples of the method may be transformed into DNA sequences encoding RNAs that act on transcribed mRNA such as group 1 intron type, M1 RNA type, hammerhead type, or hairpin type or microRNA type. In addition, couppression may be induced through transformation of DNA having a sequence identical or similar to a target gene sequence.

Preferably, in the present invention, adjusting the intracellular level of the protein of SEQ ID NO: 2 or an equivalent thereof may be performed by a method of increasing or decreasing the expression of a gene encoding the protein. Such increasing or decreasing methods may be methods known to those skilled in the art, respectively. For example, a recombinant expression vector may be prepared by linking a gene encoding the gene of SEQ ID NO: 2 or its equivalent to a promoter to enhance its expression. Alternatively, a recombinant expression vector in which an antisense gene for the gene is linked to a promoter may be prepared to reduce the expression.

The term 'promoter' refers to a DNA sequence that regulates the expression of a nucleic acid sequence operably linked in a particular host cell. 'Operably linked' means that one nucleic acid fragment is combined with another nucleic acid fragment. Its function or expression is affected by other nucleic acid fragments. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation.

Examples of the promoter include a constitutive promoter that induces expression of a target gene at all times or an inducible promoter that induces expression of a target gene at a specific position and time. Examples of the promoter include an SV40 promoter , The CMV promoter, the CAG promoter (Hitoshi Niwa et al., Gene, 108: 193-199, 1991; Monahan et al., Gene Therapy, 7: 24-30, 2000), the CaMV 35S promoter (Odell et al., Nature 313: 810-812, 1985), the Rsyn7 promoter (US patent application Ser. No. 08 / 991,601), the rice actin promoter (McElroy et al., Plant Cell, 2: 163-171, 1990), the ubiquitin promoter Christensen et al., Plant Mol. Biol. 12: 619-632, 1989) and the ALS promoter (US patent application Ser. No. 08 / 409,297). In addition, U.S. Patent Nos. 5,608,149; 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463 and 5,608,142 can all be used.

In one embodiment of the present invention, the expression level of OsTCP6 was increased by preparing and transforming a recombinant expression vector comprising an OsTCP6 gene having the nucleotide sequence represented by SEQ ID NO: 1.

The present invention relates to a recombinant expression vector comprising a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2.

A "recombinant expression vector" refers to a gene construct that is capable of expressing a protein of interest or RNA of interest in a host cell, and includes a gene construct comprising essential regulatory elements operably linked to express the gene insert. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers (promoter) and can be prepared in various ways depending on the purpose. Can be. The expression vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin.

The recombinant expression vector of the present invention may include various structural genes known in the art, and such structural genes are operably linked with various promoters known in the art.

Also, the structural gene may be selected from the group consisting of an antibiotic resistance gene, a herbicide resistance gene, a virus resistance gene, an insect resistance gene, a metabolism-related gene, a luminescent gene, a GFP (green fluorescence protein, AY598428) gene, a GUS (β-glucuronidase, AF485783) (? -galactosidase, AAB49721) gene, and the like.

In one embodiment of the present invention, the recombinant expression vector was inserted into the downstream of the 35S promoter (CaMV35SP) of the cauliflower mosaic virus (CaMV35SP) of the recombinant protein linked to the fluorescent protein smGFP at the C-terminal of OsTCP6 to produce an OsTCP6-GFP fusion plant expression vector. .

In addition, the present invention relates to a transformant transformed with a recombinant expression vector comprising a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2.

In the present invention, transformation with the recombinant expression vector of the present invention may be performed by transformation techniques known to those skilled in the art. Preferably microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion (PEG) mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method), Agrobacteria spraying method (Agrobacteria spraying method) can be used.

Transformants are also Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Staphylococcus and Agro Bacterium tumefaciens (Agrobacterium tumefaciens) is possible, but is not limited thereto.

In addition, the transformant may be a plant or a plant cell, but is not limited thereto. Plants include whole plants, parts of plants, callus, plant tissues, plant cells and plant seeds.

In one embodiment of the present invention, the recombinant vector was transformed into Agrobacterium tumefaciens (GV3101), and inoculated to Arabidopsis plants to prepare a transformant.

The present invention also relates to a plant in which the growth and / or morphology of the plant is changed by controlling the intracellular level of the polypeptide having the amino acid sequence represented by SEQ ID NO: 2.

The plants are Arabidopsis, Chinese cabbage, cabbage, mustard, rape (rapeseed), radish, radish (Brassica napobrassica), turnip (Brassica rapa / Brassica campestris), tricale, cauliflower, broccoli, wasabi, horseradish, pole sprout, Flower Pot, Brasica juncea, Brasica napus, Brassica oleracea, Brassica caulorapa, Brassica fimbriata, Brassica ruvo, Brassica septi-ceps, Black mustard (Brassica nigra), Cochlearia officinalis, Mustard radish (Armoracia lapathifolia), Descurinia pinnata, Aubrietta It may be a pizza plant such as Aubrieta deltoidea, and the tissues affected by the change of the expression of OSTCP11 protein in the plant may be leaves, flowers, hypocotyls, siliques, seeds.

In addition, the plants are rice, corn, onions, leeks, barley, wheat, leek, garlic, taxa, jimyeo, astronomical dong, lily of the valley, pamo, makmun-dong, jinhwangjeong, erosion, bamboo and phalaenopsis spp, Cymbidium goeringii ), Cymbidium ssp, Denparium phalaenopsis, Neofinetia falcata, Aerides japonicum Cattlea ssp, Oncidium ssp, Brassia ssp, Vanda (Vanda) ), Orchid plants such as Papio pedilum ssp, Miltonia ssp and Cymbidium Kanran.

The present invention also relates to a method of increasing gene expression that acts as a central regulator of cell division by overexpressing a protein having the amino acid sequence represented by SEQ ID NO: 2.

As a result of investigating the characteristics of the overexpressing transformant of OsTCP6 in one embodiment of the present invention, the length and width of the leaves of the plant were greatly increased, and the length and hypocotyl of the leaf disease along with the leaf size were also increased. In addition, in the case of wild type, the leaf generation was stopped during the reproductive growth after the extraction, and in the OsTCP6 transgenic plants, the generation of leaflets increased significantly and the number of leaves was confirmed. This is because the OsTCP6 gene or homolog thereof maintains the meristem of the leaves.

The gene acting as a central regulator of cell division is a gene that regulates cell division activity, but is not limited thereto. Proliferating cell nuclear antigen (PCNA), cyclin dependent protein kinase (CDC 2, etc.), CDK activating kinase It regulates the progress of the cell cycle, including cyclin, E2F transcription factor, retinoblastoma tumor suppressor (RB), and KRP (Kip-related proteins, CDK inhibitor).

In an embodiment of the present invention, the RNA expression of OsTCP6 transgenic plants and wild-type RNA was isolated, and the gene expression related to cell division and cell growth was examined by RT-PCR. As a result, the expression of PCNA genes known as central regulators of cell division was increased. The expression of cdc2b, cyclinB1, and cyclinD3 genes associated with the cell cycle was increased. In addition, it was confirmed that the expression of the expansin gene that regulates cell growth was increased. The results show that OsTCP6 can be used to promote cell division to increase leaf development or to promote cell growth to increase leaf size.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[Example]

These examples are only for illustrating the present invention in detail, and the scope of the present invention is not to be construed as limited by the following examples.

Example  1. derived from rice OsTCP6  gene Cloning  And sequencing thereof

RT-PCR was performed by separating RNA from stem base and meristem of Nakdong rice seedlings grown for 2 weeks. Total RNA was used as a template to synthesize a single cDNA chain using oligo (dT) ₂₀ and reverse transcriptase (Superscript III, Invitrogen), to prepare the following pair of oligonucleotide primers.

Forward primer: 5'-ATGACCATGGACGTCGCCGGAGACGCAGG-3 '(SEQ ID NO: 3)

Reverse primer: 5'-CTACGAGTCGCTGGTGCTTATGCTCTG-3 '(SEQ ID NO: 4)

PCR was performed using the primers and Taq polymerase (Pyrobest, Takara) as a template with a single cDNA chain. The amplified cDNA fragment was cloned into pGEM-T vector (Promega), named OsTCP6 and the total sequencing was determined (SEQ ID NO: 1) and registered in Genbank (GQ229483, published on Jul. 2010).

Example 2 Construction of Vectors for Transformation

A plant expression vector was prepared in which recombinant DNA, in which the fluorescent protein smGFP was connected to the C terminus of OsTCP6 , was inserted downstream of the 35S promoter. First, ORF from which the termination codon of OsTCP6 protein was removed was PCR amplified using the following pair of primers including BglII.

Forward primer: 5'-CAGATCTATGACCATGGACGTCGCCGG-3 '(SEQ ID NO: 5)

Reverse primer: 5'-CAGATCTGCGAGTCGCTGGTGCTTATGC-3 '(SEQ ID NO .: 6)

Amplified DNA fragment was cloned into pGEMT vector, and the BglII-cut gene was inserted into the Bam HI site between the 35S promoter of the pCAMBIA1300-derived plant expression vector having hygromycin resistance and the fluorescent protein gene smGFP. (FIG. 1). The prepared vector was introduced into Agrobacterium (GV3101) and cultured in hygromycin medium.

Example 3. Intracellular and Histological Distribution of TCP6 Protein

In order to confirm the intracellular expression of OsTCP6 protein, the plasmid of the OsTCP6- smGFP plant expression vector prepared in Example 2 was coated on the surface of gold particles, and then, on the back of the tobacco leaf using a gene delivery system (PDS2000, BioRad). After inoculating with a syringe and temporarily expressing the protein, fluorescence microscopy was used to observe the distribution of intracellular fluorescent proteins. As a result, OsTCP6 was expressed in the nucleus (FIG. 2).

In addition, paraffin tissue sections were prepared for shoot / root basal sites including SAMs from rice seedlings and in situ hybridization of OsTCP6 gene was performed as follows. OsTCP6 DNA fragment was used as a template to synthesize antisense RNA labeled with DIG using RNA polymerase. Synthetic antisense RNA was added to the hybridization reaction solution containing PolyA and tRNA, and the hybridization reaction was performed by dropping onto a slide glass containing tissue sections. After removing the unbound RNA by RNase treatment, the antibody was reacted with the nucleic acid Anti-DIG-AP solution, and then the color reaction was induced by NBT / BCIP. Tissue sections were dehydrated with ethanol and observed under a microscope. As a result, it was confirmed that OsTCP6 transcript is expressed in stem apical meristem (SAM) and root meristem (CR) (Fig. 3).

Example 4 Transformation Vector Preparation and Arabidopsis Transformation for TCP6 Plant Overexpression

The OsTCP6 gene was linked downstream of the 35S promoter of the Cauliflower mosaic virus to produce a constant plant expression vector. To this end, the ORF of OsTCP6 was PCR amplified using the following pair of primers including BglII, cloned into the pGEMT vector, and the entire nucleotide sequence was determined.

Forward primer: 5'-CAGATCTATGACCATGGACGTCGCCGG-3 '(SEQ ID NO .: 7)

Reverse primer: 5'-CAGATCTACGAGTCGCTGGTGCTTATGC-3 '(SEQ ID NO .: 8)

OsTCP6 gene digested with Bgl II was constructed with a plant expression vector inserted into the BamHI site of a plant expression vector derived from pCAMBIA1300 having hygromycin resistance (FIG. 4). Vectors prepared for plant transformation were introduced into Agrobacterium (GV3101), and transformants were selected from medium containing 15 g / l of hygromycin and grown in a greenhouse.

RNA was isolated from RNeasy plant kit (QIAGEN) in T2 generation of selected OsTCP6 transgenic Arabidopsis, and whole RNA was transformed into template (dT) ₂₀ and a single cDNA chain using reverse transcriptase (Superscript III, Invitrogen). Synthesized. PCR reactions were performed using the nucleotide sequence specific primers of OsTCP6, wherein PCR cycling parameters were 40 rotations at 95 ° C.-30 sec, 57 ° C.-30 sec, and 72 ° C.-40 sec.

Forward primer: 5'-CGAGTGGCTGCTGCAGCAGG-3 '(SEQ ID NO .: 9)

Reverse primer: 5'-CCTCCTTGCGTGCTGTTGC-3 '(SEQ ID NO .: 10)

It was confirmed that the hygromycin resistance gene and the OsTCP gene were stably introduced by genomic PCR (FIG. 5A).

In addition, it was confirmed that the OsTCP6 transcript, which was introduced through the RT-PCR method as the extracted RNA, was expressed (FIG. 5B).

Example 5 Analysis of Phenotype and Gene Expression of Arabidopsis Transformants

OsTCP6 transgenic Arabidopsis showed about 20-35% and 10-20% increase in leaf length and width, respectively, compared to wild type, showing a new phenotype with very large leaf size. It was confirmed that the lengthening (Fig. 6). The hypocotyl length of the transformant seedlings also increased. Therefore, the overexpression of OsTCP6 changed the length or width of nutrient growth organs. In addition, in the wild type, the generation of leaves stops in the reproductive growth after the extraction, whereas in the OsTCP6 transgenic Arabidopsis, the generation of lobules was greatly increased and the number of leaves increased (FIG. 7). These results indicate that the transformant according to the present invention has a function of maintaining the meristem of the leaves.

In order to identify the molecular biological mechanisms associated with the growth of nutrient growth organs such as leaves and long-lived phenotypes, the following pair of primers were prepared according to the nucleotide sequence of genes related to cell division or cell extension. Gene expression was examined. RNA isolation and cDNA synthesis were performed in the same manner as in Example 4, and PCR rotational conditions (cycling parameters) were 32 rotations (expansin) or 40 rotations (95 ° C-30 sec, 57 ° C-30 sec, 72 ° C-40 sec). PCNA, cdc2b, CYCD3, CYCB1).

Cdc2b forward primer 5'-GAGCAGCAATGGCCGG- 3 '(SEQ ID NO .: 11)

Cdc2b reverse primer 5'-CATTGCTACCACACCATTGTG- 3 '(SEQ ID NO .: 12)

CYCD3 forward primer 5'-TCGGGTACCTCCCATCAG- 3 '(SEQ ID NO .: 13)

CYCD3 reverse primer 5 '-GAGTGGCTACGATTGCCC- 3' (SEQ ID NO .: 14)

CYCB1 forward primer 5'-CGTCCTCGTACACGATCTCA- 3 '(SEQ ID NO .: 15)

CYCB1 Reverse Primer 5 '-CCACAGCTGCGAGGTCATTC- 3' (SEQ ID NO .: 16)

PCNA forward primer 5'-GGGTTCTCACTCCAAGCTAT-3 '(SEQ ID NO .: 17)

PCNA Reverse Primer 5'-GAGCACAATGTTAGCGGTTC- 3 '(SEQ ID NO .: 18)

Expansin Forward Primer: 5'-CGT GAG AGT AGA AGA AGC AAG C-3 '(SEQ ID NO: 19)

Expansin Reverse Primer: 5'-GTG AGC ATC GCA AGG ATC ACA AC-3 '(SEQ ID NO .: 20)

RT-PCR results showed increased expression of the expansin gene, which is known to promote cell growth in Arabidopsis transformants, and the proliferating cell nuclear antigen (PCNA) gene, which is a central regulator of cell division, compared to wild type. , cyclinB1, cyclinD3 gene expression was increased (Fig. 8). In conclusion, it can be seen that gene expression related to cell division and kidney is induced when OsTCP6 is overexpressed, and expression regulation of central regulatory genes of cell division such as PCNA occurs.

GFP: Green fluorescent protein

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Claims (18)

A protein that controls the growth or form of a plant having an amino acid sequence represented by SEQ ID NO: 2. Gene encoding the protein of claim 1. The gene according to claim 2, wherein the gene has a nucleotide sequence represented by SEQ ID NO: 1. A method for regulating the growth or shape of a plant, characterized in that for producing a recombinant expression vector linking a gene encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 to a promoter to enhance the expression of the gene. delete delete delete The method of claim 4, wherein the plant is a Arabidopsis vulgaris. delete The method according to claim 4, wherein the gene has a nucleotide sequence represented by SEQ ID NO: 1 and is a gene isolated from meristem of rice. The method of claim 4, wherein the growth or morphology of the plant is at least one region selected from the group consisting of leaves, side diseases, and hypocotyls. Arabidopsis with increased growth or form prepared according to the method of claim 4. A method of increasing gene expression that acts as a central regulator of cell growth, division or cell cycle by overexpressing a protein having an amino acid sequence represented by SEQ ID NO: 2. The method of claim 13, wherein the gene is any one selected from the group consisting of PCNA, cyclinD3, cyclinB1, cdc2b, and expansin. Recombinant expression vector comprising the gene of claim 3. A Arabidopsis transformant transformed with the recombinant expression vector of claim 15. A composition for controlling growth or form of a plant comprising the protein of claim 1 or the transformant of claim 16. Agrobacterium tumefaciens transformants transformed with the recombinant expression vector of claim 15.

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