KR20150045611A - OsGASD gene involved in gibellelin mechanism in plant and uses thereof - Google Patents

Abstract

The present invention relates to an Oryza sativa gibberellic acid sensitive dwarf (OsGASD) gene involved in the gibberellin mechanism of a plant and to uses thereof. The gene that codes rice-derived OsGASD proteins involved in the gibberellin mechanism boosts the growth of a plant and increases the content of the gibberellin in the plant, thus fostering growth of the plant by controlling the expression of OsGASD gene and increasing the content of gibberellin in the plant. Accordingly, the gene can be used to develop plants with enhanced productivity.

Description

OsGASD gene related to gibberellin mechanism of plant and use thereof < RTI ID = 0.0 >

The present invention relates to an OsGASD gene related to a gibberellin mechanism of a plant and a use thereof. More particularly, the present invention relates to a gene encoding gibberellin mechanism related gene OsGASD ( Oryza sativa gibbelellin acid sensitive dwarf) derived from rice, a gene encoding the protein, a recombinant vector, to increase the improvement of plant growth and gibberellin content as compared to wild-type, which was transformed with a recombinant vector into a plant cell comprising a host cell, the OsGASD gene transformed with the recombinant vector comprises the step of overexpressing OsGASD gene , A method for producing a transgenic plant having improved growth and increased gibberellin content compared to a wild type using a recombinant vector comprising the OsGASD gene, a method for producing a transgenic plant having improved gibberellin content, Transgenic plants and their seeds, and O and a composition for increasing the growth and increasing the gibberellin content of a plant containing a recombinant vector containing the sGASD gene.

In traditional cross-breed breeding, it takes a lot of time and effort to isolate and fix the traits, and because there are limited natural genetic resources that can be used, traditional cross-breed breeding is the only way to develop new varieties, It is difficult to go. Therefore, it is necessary to expand the basic research for the development of internationally competitive new varieties of rice using biotechnological techniques. To do this, it is important to find and secure genes that are useful for agriculture.

Dwarf phenotypes are one of the agricultural traits that play an important role in the mechanism of regulating plant growth and development, and they affect crop yields because they are resistant to stress by environmental factors. Gibberellin is one of the plant growth regulators associated with these dwarf phenotypes and plays an important role in promoting the growth of plants, promoting seed germination, promoting flowering, increasing seedling growth and plant growth. The most characteristic of the action of gibberellin is the action of stimulating the growth of the stem, which is effective against the whole plant. In addition, since gibberellin has a strong effect on the flowering phenomenon not greatly affected by auxin, treatment of gibberellin under a single condition induces flowering of the plant. In addition, gibberellin is known to cause dormant seeds and snow to wake up from dormancy and to start growing, and to cause fruit to grow on some plants, or to stimulate fruit growth. Therefore, in the present invention, useful genes related to the gibberellin mechanism are discovered, and the functions of the genes are analyzed and identified to secure genes useful for agriculture.

On the other hand, Korean Patent No. 0550960 discloses 'sd1 gene involved in anti-derealization of plants and its use', Korean Patent Registration No. 1097211 discloses 'Method for regulating the size of a plant and plants therefrom' . However, as in the present invention, the OsGASD gene related to the gibberellin mechanism of plants and its use has not been disclosed.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-described needs, and it is an object of the present invention to provide a mutant having a dysplastic phenotype in a rice Ds insertion mutant, wherein OsGASD ( Oryza sativa gibbelellin acid sensitive dwarf) The mechanism-related protein OsGASD was isolated, and the growth of the rice plant transformed with the recombinant vector containing the gene encoding OsGASD was improved as compared with that of wild type, and the content of gibberellin in the plant was increased, .

In order to solve the above problems, the present invention provides a rice-derived gibberellin mechanism-related protein OsGASD ( Oryza sativa gibbelellin acid sensitive dwarf).

In addition, the present invention provides a gene coding for the gibberellin mechanism-related protein OsGASD.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a host cell transformed with the recombinant vector.

In addition, the present invention provides a method for enhancing plant growth and gibberellin content in comparison with a wild-type strain comprising a step of overexpressing OsGASD gene by transforming a recombinant vector containing a gene encoding OsGASD protein into plant cells.

The present invention also provides a method for producing a transgenic plant having improved growth and increased gibberellin content compared to wild type using a recombinant vector containing a gene encoding OsGASD protein.

In addition, the present invention provides transgenic plants and seeds thereof, wherein the growth produced by the method is improved and the gibberellin content is increased.

The present invention also provides a composition for increasing the growth and increasing the gibberellin content of a plant containing a recombinant vector comprising a gene encoding OsGASD protein derived from rice.

The gene encoding the rice-derived gibberellin mechanism-related protein OsGASD ( Oryza sativa gibbelellin acid sensitive dwarf) of the present invention improves the growth of the plant and increases the gibberellin content in the plant. Therefore, the growth of OsGASD gene is improved by controlling the expression of the OsGASD gene in the plant And can be used to develop plants with increased productivity due to increased gibberellin content.

Figure 1 shows the results of a comparison of the morphological characteristics of the dorsal mutant ( osgasd ) and the wild type control (WT) at the early stage of growth. (A) The length of the wild type control and dwarf mutants. (BC) Comparison of plant length and leaf length of mature plants. (D) Seed comparison results.
Fig. 2 shows the results of the expression analysis of the T-DNA insertion site and the gene related to the gibberellin mechanism ( OsGASD ) in the dendritic mutants . (A) Results of electrophoresis performed by I-PCR for flanking sequencing analysis. (B) Results of homozygous selection using specific primers. (C) Comparison of OsGASD gene expression levels between wild type control (WT) and dwarf mutant ( osgasd ). (D) Analysis of the T-DNA inserted in the promoter region of the LOC_Os04g43290 gene of chromosome 4.
FIG. 3 is a graph showing the effect of a rice-derived gibberellin mechanism-related gene OsGASD ( Oryza sativa gibbelellin acid sensitive dwarf), which is a knock-out gene of a dendritic mutant, Lt; / RTI >
Fig. 4 shows the amino acid sequence deduced from the cDNA of the rice-derived gibberellin mechanism-related gene OsGASD ( Oryza sativa gibbelellin acid sensitive dwarf).
FIG. 5 shows the result of confirming the expression pattern of the OsGASD gene in wild-type Dong Jinbin . OsGASD gene expression levels of young leaves (YL), mature leaves (ML), stem, root, mature booting and Hd (heading flowering)
FIG. 6 shows the results of evaluating recovery of the dendritic mutants according to the gibberellin treatment (0, 1, 10 and 20 ppm). As the concentration of gibberellin treatment increases, It was confirmed that the length increased.
FIG. 7 shows the results of comparing the initial growth state of wild-type rice (WT), dendritic mutants ( osgasd ) and OsGASD-overexpressing transformants (OsGASD OX). (A) Comparison of transplant lengths of early growing plants. (B) the plant length, (C) the interlingual length, and (D) the length of the second leaf.
8 shows the results of HPLC analysis of gibberellin content of wild-type rice (WT), dendritic mutants ( osgasd ) and OsGASD overexpressing transformants (OsGASD OX) (A) and expression pattern of gibberellin biosynthesis- .

In order to achieve the object of the present invention, the present invention provides a rice-derived gibberellin mechanism-related protein OsGASD ( Oryza sativa gibbelellin acid sensitive dwarf) which enhances plant growth and gibberellin content.

The scope of the OsGASD protein according to the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 2 isolated from rice ( Oryza sativa ) and functional equivalents of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous physiological activity" means an improvement in growth and an activity of increasing gibberellin content. The invention also includes fragments, derivatives and analogues of the OsGASD protein. As used herein, the terms "fragments", "derivatives" and "analogs" refer to polypeptides having substantially the same biological function or activity as the OsGASD polypeptides of the present invention.

In addition, the present invention provides a gene coding for the gibberellin mechanism-related protein OsGASD. The gene of the present invention is characterized by enhancing plant growth and increasing gibberellin content in the plant, and includes all genomic DNA, cDNA, and synthetic DNA encoding OsGASD protein. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 can do. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The present invention also provides a recombinant vector comprising the OsGASD gene according to the present invention. In the recombinant vector of the present invention, the OsGASD gene may preferably consist of the nucleotide sequence of SEQ ID NO: 1.

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. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element. Enhancers, ribosome binding sites, termination signals, polyadenylation signals, and promoters, which are available in eukaryotic cells, are well known in the art. The expression vector can be constructed by a method known in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of a recombinant vector is a Ti-plasmid vector which is capable of transferring a so-called T-region to a plant cell when it is 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.

In the recombinant vector of the present invention, the promoter may be a promoter suitable for transformation, preferably a CaMV 35S promoter, an actin promoter, a ubiquitin promoter, a pEMU promoter, a MAS promoter or a histone promoter, preferably a CaMV 35S 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. Since the selection of the transformant can be carried out by various tissues at various stages, a constant promoter may be preferable in the present invention. Therefore, the constant promoter does not limit the selectivity.

In the recombinant vector of the present invention, the terminator can be a conventional terminator, such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens Agrobacterium tumefaciens , Octopine gene terminator of Agrobacterium tumefaciens , and the like. Regarding the need for a terminator, it is generally known that the terminator region increases the certainty and efficiency of gene transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The recombinant vector of the present invention may 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, antibiotic resistant genes such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, However, the present invention is not limited thereto.

Also provided is a host cell transformed with the recombinant vector of the present invention. Host cells capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any host cell known in the art. Examples of prokaryotic cells include E. coli JM109, E. coli BL21, E. coli Bacillus sp. Strains such as RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, and Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcesensis And enterobacteria and strains such as Pseudomonas spp.

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

The recombinant vector of the present invention can be produced by using a CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (Dower, WJ et al., Nucleic Acids Res., 16: 6127-6145 (1988)), etc., And can be delivered into cells. In addition, when the host cell is a eukaryotic cell, microinjection (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52: 456 (Wong, TK et al., Gene, 10: 87 (1980)), DEAE- (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 The vector can be injected into the host cell.

In addition, the present invention provides a method for improving plant growth and gibberellin content in comparison with a wild type strain comprising the step of overexpressing a OsGASD gene by transforming a recombinant vector comprising a gene encoding a rice-derived gibberellin mechanism-related protein OsGASD into a plant cell .

In one embodiment of the present invention, the OsGASD protein may be an amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

When transforming a vector of the present invention into a plant cell, the host cell may preferably be a Poaceae plant cell, and more preferably, it may be a rice cell, but is not limited thereto. The method of transforming the plant cell is as described above.

The present invention also provides a method for producing a plant cell, comprising the steps of: transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived gibberellin mechanism-related protein OsGASD; And

The present invention also provides a method of producing a transgenic plant having improved growth and increased gibberellin content compared to the wild type, which comprises regenerating a transgenic plant from the transgenic plant cell.

In one embodiment of the present invention, the OsGASD protein may be an amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The method of transforming plant cells is as described above. Regeneration of transgenic plants from the transformed plant cells can be carried out by any method known in the art.

In addition, the present invention provides a transgenic plant and its seeds in which the growth produced by the above method is improved and the gibberellin content in the plant is increased.

In one embodiment of the present invention, the plants used in the present invention are selected from the group consisting of Arabidopsis, potato, eggplant, cigarette, pepper, tomato, burdock, ciliaceae, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, Such as rice, barley, wheat, rye, corn, sugar cane, oats, onions, etc., such as Chinese cabbage, cabbage, gangbu, watermelon, melon, cucumber, amber, pak, strawberry, soybean, mung bean, It may be a monocot plant, preferably a monocot plant, more preferably a Poaceae plant, most preferably rice ( Oryza sativa ), but is not limited thereto.

The present invention also provides a composition for increasing the growth and increasing the gibberellin content of a plant comprising a recombinant vector comprising a gene encoding the rice-derived gibberellin mechanism-related protein OsGASD comprising the amino acid sequence of SEQ ID NO: 2. The composition includes a recombinant vector containing the gene encoding the amino acid sequence of SEQ ID NO: 2 as an active ingredient. The gene can be transformed into a plant to improve plant growth and increase gibberellin content.

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

Example 1. Phenotypic analysis of Ds insertion variants

In the present invention, a mutant having a dysplastic phenotype was used as a Ds insertion mutant, which is a rice gene function analysis material. The morphological differences between wild - type Dong Jin - pine and dwarf mutants were investigated. The length of the stem and the length of the stem were significantly shorter in the mutant strains. The maturation stage was similar to the initial stage. No morphological differences were observed in the seeds, but a decrease in fertility was observed (Fig. 1).

Example 2 T-DNA Insertion Site and Gibberellin Mechanism Related Gene of Dendritic Mutants OsGASD ) Expression analysis

Flanking sequencing analysis was performed using I-PCR to analyze the T-DNA insertion site of dendritic mutants, and the method is as follows. Genomic DNA was isolated from dendritic mutants and 2 의 of DNA was treated with Nla III restriction enzyme at a total reaction volume of 100 쨉 l for 2 hours and 30 minutes at 37 째 C. Then, the reaction solution was transferred to 65 ° C for 30 minutes, 300 μl of 100% ethanol and 10 μl of 3 M sodium acetate were added and kept at -20 ° C. for 20 minutes, followed by centrifugation at 13000 rpm for 15 minutes . The supernatant of the centrifuged sample was removed, and after drying, 360 μl of distilled water was added to dissolve the DNA. The DNA was treated at 55 ° C for 10 minutes, and T4 ligase was added thereto, followed by overnight incubation at 16 ° C to self-ligation. Then, the DNA was purified and purified by using Ds3-37 primer (5'-TATGAAAATGAAAACGGTAGAGG-3 ': SEQ ID NO: 3) and Ds3I-105 primer (5'- AAACGAACGGGATAAATACGG- The first PCR was carried out under the conditions of 94 캜 for 15 seconds, 55 캜 for 1 minute, 72 캜 for 2 minutes and 30 seconds, and 72 캜 for 10 minutes. The resulting PCR product was diluted to 1/100 and then PCR was carried out by using 1: 1 of Ds3-4 primer (5'-GTTACCGACCGTTTTCATCC-3 ': SEQ ID NO: 5) and Ds3I-150 primer (5'-GGTTAAAGTCGAAATCGGACG- The secondary PCR was performed under the same conditions as the secondary PCR. The primary and secondary PCR products were electrophoresed to purify the amplified DNA, followed by Flanking sequencing analysis (FIG. 2A). Analysis of the T-DNA insertion site of the dendritic mutant revealed that T-DNA was inserted into the promoter region 397 bp away from the initiation codon of the LOC_Os04g43290 gene of chromosome 4 (FIG. 2D).

(5'-GCCATGGAGCTAGCACTGTT-3 ': SEQ ID NO: 7) and the reverse primer S2 (5'-GGCTGAAGAATTGGCGGCAA-3') at both positions where the T-DNA was inserted in order to select homozygous individuals in the dendritic mutants. : SEQ ID NO: 8) was prepared, and PCR was carried out using the vector primer Ds3-4 primer (5'-GTTACCGACCGTTTTCATCC-3 ': SEQ ID NO: 5) To select a clean 1 copy homo individual (Fig. 2B).

Since gibberellin is involved in the growth of plants, the expression pattern of the OsGASD gene, a gene related to the gibberellin mechanism in dendritic mutants and wild type Dong Jinbun, was examined by Northern blot. (Forward primer: 5'-TGAGGCTTCGCCATCCAGCG-3 '(SEQ ID NO: 9) and reverse primer: 5'-CGATGGCCAGGTCCGTGTCG-3' having a base sequence specific to the gene ( OsGASD ) related to the gibberellin mechanism based on the entire genome sequence of rice, 3 '(SEQ ID NO: 10)) was prepared and used as a probe. The control group, Dongjinbyeong and dwarf mutants, were cultivated for 2 weeks and RNA was isolated using Trizol reagent (Invitrogen). 10 μg of the separated RNA was electrophoresed on 1.2% (w / v) denaturing formaldehyde agarose gel, and RNA was then attached to a nylon membrane (Amersham). The nylon membrane RNA is attached to ^[32 P] 20% with a probe labeled with dCTP (w / v) SDS, 20 × SSPE, 100 g / L PEG (8,000 mwt), 250 mg / L heparin, and 10 ml / L Hybridization was carried out overnight at 65 ° C with a solution containing Herring sperm DNA (10 mg / ml). As a result of confirming the expression pattern of the OsGASD gene by northern blot, it was confirmed that the OsGASD gene was not expressed in the dendritic mutant (Fig. 2C).

Example 3 Genes Associated with Gibberellin Mechanism from Rice ( OsGASD )

Genomic DNA was isolated from the wild type rice leaves and primers specific for the gene ( OsGASD ) related to the gibberellin mechanism (forward primer: 5'-CACCATGCCTGTTGATCATACCTT-3 '(SEQ ID NO: 11) and reverse primer: 5'-TCACTCCAGCTTCTGGTACT-3' The nucleotide sequence of the gene was confirmed by sequencing after the amplified product was cloned into the pEntr vector. The gene associated with the gibberellin mechanism, OsGASD, has been previously identified The cDNA sequence and the amino acid sequence of the OsGASD gene are shown in detail in FIGS. 3 and 4, respectively.

Example 4. OsGASD  Expression pattern of gene

In order to confirm the expression pattern of OsGASD gene in wild type Dong Jinbun , young leaves (YL), mature leaves (ML), stem, root, mature booting (MB) and flowering head (Hd; in OsGASD is most highly expressed in the results of mature leaves examining the genetic level, the roots of heading flowering) it was confirmed that the lower the expression level (Fig. 5).

Example 5 Evaluation of Recovery of Dendritic Mutants by Gibberellin Treatment

In order to confirm the effect of gibberellin treatment on the dendritic mutants, dendritic mutants and wild type Dongjin cotton seeds were seeded and cultured at 28 ° C for 16 hours light and 8 hours dark cycle. After spraying 0, 1, 10, and 20 ppm of gibberellin on the dwarf mutants of the elongation stage, the dwarf recovery was compared with that of the control wild type. As a result, according to the concentration of gibberellin treatment, It was confirmed that the length increased sharply (Fig. 6).

Example 6. OsGASD  Genetic and phenotypic analysis of transgenic plants with gene overexpression

In order to investigate the function of the OsGASD gene isolated from rice in the plant, the OsGASD gene was transformed into DongGin - paddy to produce an OsGASD - overexpressing transformant. To produce a rice transformant, a coding region of the OsGASD gene was cloned into a destination vector (pH7WG2D, 1) whose expression was regulated by the CaMV 35S promoter, which is a strong allosteric promoter, to construct a construct. The cloned construct was introduced into Agrobacterium tumefaciens EHA105 by a triparental mating method and transformed with Agrobacterium tumefaciens EHA105. Transgenic plants with strong expression of OsGASD gene were selected, cultured, and seeds were harvested. The OsGASD gene was transfected into the transformed T0 plants by PCR (data not shown).

The harvested over - expressing transgenic seeds were cultivated for 2 weeks with wild - type Dong Jin - jin and dwarf mutants. In the case of over-expressing transformants, it was confirmed that the initial growth was remarkably improved, and especially the length of the transplant was prolonged (FIG. 7A). Quantitative investigations also revealed that stem, interspecific, and second leaf lengths of over-expressed transformants were increased compared to wild-type Dong Jin pine and dwarf mutants (FIG. 7B-D). These results indicate that the OsGASD gene is a gene related to the gibberellin mechanism.

Example 7. Analysis of Gibberellin Content of Plants

Based on the above results, the content of gibberellin in the control, dendritic mutant and OsGASD overexpressing transformants was measured. The measurement method is as follows. A 10 g leaf sample was taken in an outbreak period, disrupted with liquid nitrogen, and the crushed sample was repeated three times with 80% methanol and the supernatant was recovered. The recovered supernatant was evaporated at 40 ° C, treated with ethyl acetate to dissolve the pellet, and then again ethyl acetate was evaporated at 42 ° C and treated with 100% methanol. The content of gibberellin dissolved in 100% methanol was measured by HPLC. HPLC was carried out on a C18 column (150 x 4.6 mm) with a methanol-2% acetic acid-water (40:40:20) solution at a flow rate of 2 ml / min at 245 nm, Respectively. As a result of the analysis of the content of gibberellin, the content of gibberellin in the OsGASD- overexpressing transformant was the highest (Fig. 8A).

Furthermore, to investigate the expression pattern of the rice plant gibberellin biosynthesis-related gene, OsCPS (Oryza sativa copalyl diphosphate synthase ), OsKS (O. sativa ent-kaurene synthase), OsKO (O. sativa ent-kaurene 19-oxidase), OsKAO ( O. sativa ent-kaurenoic acid hydroxylase), OsGA20 ( O. sativa GA 20 oxidase) and OsGA2 ( O. sativa GA 2 oxidase) biosynthetic genes. As a result, in the dwarf mutant decreases the expression level of gene OsKAO, the OsGASD overexpressing transformant was confirmed that an increase in expression of OsKAO gene (Fig. 8B).

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> OsGASD gene involved in gibellelin mechanism in plant and uses          the <130> PN13329 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 1035 <212> DNA <213> Oryza sativa <400> 1 atgcctgttg atcatacctt gtttgagttt ggatcaatcc gttaccattt acaggcttct 60 attacagatt cagagaacat atacttgtca atatcaacac catctctttc ttatgaggct 120 tcgccatcca gcggcttgcc tgaaatcaca ttacaggaga caaggaaaat gtatcacaag 180 tttgcagaga tcatcgagcc tgcaaaagaa ggatacactt tgacactgag actgaacttc 240 tctggcctta ctcgaccgaa agatcgcacc aaggcgatca accagatatc gttgctgcaa 300 tccgtcatcc ttagctcgca gctcaaggac atgctggcga gcctcggctc gtccggcacg 360 atgaagctcg tgtacaacca aagggaccct ttcttcgtca gcaaaacgcc ggtgaagata 420 agcgccatat tccccatgcg tttcagggac gacacggacc tggccatcgc gtcgtcgttc 480 ttccaggagc tacaggacct cgggagcacg tcgtccttct ccagggcgcc gcggtgcagc 540 tggtcgccga tcccgccacc cgagctgcgc ggggagtacg tgcaccacct caccaccaac 600 ggtggcttcg tgtccttcga catcctggcg cggcacgtga aggggaggag ggcggctagg 660 acggcgtgga tcctgctcaa tttccagtcc tacgtgaaat accatatcaa gtgcacccga 720 agctacatcc agagcaggat gagaaagcgt ctggagatca tgacagaggt gatcgatgac 780 gcgaagttca gaggaaacga tgagagcagc aagaagctgc aagtgaggaa gagaagcaag 840 agaagatcaa tcaaattcgc gagagccaaa aagctccaaa agggtttcaa agctgtcatc 900 gacaagatca agcggctgcg gctgaggatc agagtgaaag gcctggaccg gttcaggagg 960 cactgccagt gcttcccagt gctcaaactg accatggcac agaggaagga gcagaagtac 1020 cagaagctgg agtga 1035 <210> 2 <211> 344 <212> PRT <213> Oryza sativa <400> 2 Met Pro Val Asp His Thr Leu Phe Glu Phe Gly Ser Ile Arg Tyr His   1 5 10 15 Leu Gln Ala Ser Ile Thr Asp Ser Glu Asn Ile Tyr Leu Ser Ile Ser              20 25 30 Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser          35 40 45 Ile Thr Leu Gln Glu Thr Arg Lys Met Tyr His Lys Phe Ala Glu Ile      50 55 60 Ile Glu Pro Ala Lys Glu Gly Tyr Thr Leu Thr Leu Arg Leu Asn Phe  65 70 75 80 Ser Gly Leu Thr Arg Pro Lys Asp Arg Thr Lys Ala Ile Asn Gln Ile                  85 90 95 Ser Leu Leu Gln Ser Val Ile Leu Ser Ser Gln Leu Lys Asp Met Leu             100 105 110 Ala Ser Leu Gly Ser Ser Gly Thr Met Lys Leu Val Tyr Asn Gln Arg         115 120 125 Asp Pro Phe Phe Val Ser Lys Thr Pro Val Lys Ile Ser Ala Ile Phe     130 135 140 Pro Met Arg Phe Arg Asp Asp Thr Asp Leu Ala Ile Ala Ser Ser Phe 145 150 155 160 Phe Gln Glu Leu Gln Asp Leu Gly Ser Thr Ser Ser Phe Ser Arg Ala                 165 170 175 Pro Arg Cys Ser Trp Ser Pro Ile Pro Pro Pro Glu Leu Arg Gly Glu             180 185 190 Tyr Val His His Leu Thr Thr Asn Gly Gly Phe Val Ser Phe Asp Ile         195 200 205 Leu Ala Arg His Val Lys Gly Arg Arg Ala Ala Arg Thr Ala Trp Ile     210 215 220 Leu Leu Asn Phe Gln Ser Tyr Val Lys Tyr His Ile Lys Cys Thr Arg 225 230 235 240 Ser Tyr Ile Gln Ser Arg Met Arg Lys Arg Leu Glu Ile Met Thr Glu                 245 250 255 Val Ile Asp Asp Ala Lys Phe Arg Gly Asn Asp Glu Ser Ser Lys Lys             260 265 270 Leu Gln Val Arg Lys Arg Ser Lys Arg Arg Ser Ile Lys Phe Ala Arg         275 280 285 Ala Lys Lys Leu Gln Lys Gly Phe Lys Ala Val Ile Asp Lys Ile Lys     290 295 300 Arg Leu Arg Leu Arg Ile Arg Val Lys Gly Leu Asp Arg Phe Arg Arg 305 310 315 320 His Cys Gln Cys Phe Pro Val Leu Lys Leu Thr Met Ala Gln Arg Lys                 325 330 335 Glu Gln Lys Tyr Gln Lys Leu Glu             340 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 tatgaaaatg aaaacggtag agg 23 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 aaacgaacgg gataaatacg g 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gttaccgacc gttttcatcc 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 ggttaaagtc gaaatcggac g 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gccatggagc tagcactgtt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 ggctgaagaa ttggcggcaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 tgaggcttcg ccatccagcg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 cgatggccag gtccgtgtcg 20 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 caccatgcct gttgatcata cctt 24 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 tcactccagc ttctggtact 20

Claims (13)

A rice-derived gibberellin mechanism-related protein OsGASD ( Oryza sativa gibbelellin acid sensitive dwarf) consisting of the amino acid sequence of SEQ ID NO: 2. A gene coding for the gibberellin mechanism-related protein OsGASD of claim 1. 3. The gene according to claim 2, wherein the gene consists of the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 4. A method for enhancing plant growth and gibberellin content compared to a wild type strain comprising the step of overexpressing OsGASD gene by transforming a plant cell with a recombinant vector containing a gene coding for rice gibberellin mechanism-related protein OsGASD. 7. The method of claim 6, wherein the OsGASD protein comprises the amino acid sequence of SEQ ID NO: 2. Transforming a plant cell with a recombinant vector comprising a gene encoding rice-derived gibberellin mechanism-related protein OsGASD; And
And regenerating the transgenic plant from the transformed plant cell, wherein the growth is improved and the content of gibberellin is increased. 9. The method according to claim 8, wherein the OsGASD protein comprises the amino acid sequence of SEQ ID NO: 2. A transgenic plant having improved growth and increased gibberellin content by the method of claim 8. 11. The transgenic plant according to claim 10, wherein the plant is a Poaceae plant. 10. A seed of a transgenic plant according to claim 10. A composition for improving the growth and increasing the gibberellin content of a plant comprising a recombinant vector comprising a gene encoding the rice-derived gibberellin mechanism-related protein OsGASD, the amino acid sequence of SEQ ID NO: 2.

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