KR20110029941A - Plant with increased resistance to various stresses transformed with osraf gene - Google Patents

Abstract

PURPOSE: A transgenic plant with enhanced stress resistance and a method for preparing the same are provided to enhance the yield of crops and to detect a novel stress resistant gene. CONSTITUTION: A plant with enhanced stress resistance is prepared by transforming with a recombinant vector containing an Oryza sativa-derived RAF(root abundant factor) gene, OsRAF. The stress is ABA(abscisic acid), drought, bacterial diseases, salicylic acid, or methyl jasmonate. A method for preparing a transgenic plant with enhanced stress resistance comprises: a step of transforming a plant cell with the recombinant vector; and a step of redifferentiating the transgenic plant. A composition for enhancing transcription activity contains a protein having 59 C-terminal amino acids.

Description

Plant with Increased Resistance to Various Stresses Transformed with OsRAF Gene

The present invention relates to a transgenic plant in which the OsRAF gene is introduced to enhance stress resistance.

Plants are often exposed to environmental stresses, including drought and cold, and biological stresses such as attack of pathogens. These stresses adversely affect the growth and development of plants and are important factors in determining crop productivity. In the face of such stress, plants express stress-responsive genes in order to survive and synthesize products (including transcription factors) according to those genes and respond to the stress environment (Shinozaki, K. and Yamaguchi-Shinozaki, K). (1997) Plant Physiol. 115 : 327-34. Variety of transcription factors and cis-acting element (cis -acting elements) will play an important role in the signal derived network for the regulation of gene expression under stress response (Yamaguchi-Shinozaki, K. and Shinozaki , K. (2006) Annu. Rev. Plant Biol. 57 : 781-803).

One representative factor of such environmental stress is drought stress. Drought stress causes osmotic and ionic imbalances in plant cells, inhibiting plant growth and photosynthesis. On the other hand, plants have a defensive mechanism against these stresses, so if they are in an environment unsuitable for growth, they tend to survive by adapting to their environment by controlling their morphology or physiological metabolic processes. The plant's drought resistance mechanisms include ABA (epsis acid) dependent signaling pathways or ABA independent signaling pathways (Finkelstein et. Al ., Plant cell 14, S15-S45, 2002; Himmelbach et. Al., Curr Opin Plant Biol 6, 470-479, 2003; Kuhn and Schroeder, Curr Opin Plant Biol 6: 463-469, 2003).

In addition, one of the phytopathogens, Ralstonia solanacearum , is known to penetrate the roots of plants and block the catheter, inhibiting water supply and killing the leaves of the host plant (Vasse et.al., Mol. Plant). Microbe Interact , 8: 241-251, 1995; Wallis et al., Physiol.Plant Pathol , 13: 307-31, 1978). About 450 major crops are known as tropical host plants including tomatoes, potatoes, tobacco, bananas and olives, subtropical crops and grains (Hayward, Annu. Rev. Phytopatholll , 29: 65). -87, 1991).

Recently, several genes encoding ERF-type transcription factors associated with abiotic stress responses have been identified in various plant species. As an ERF-type transformation factor, barley ( Hordeum) vulgare ) HvRAF ( Hordeum) vulgare Root abundant factor) was isolated from barley (Jung, J., Won, SY, Suh, SC, Kim, H., Wing, R., Jeong, Y., Hwang, I. and Kim, M.). (2007) Planta 225 : 575-88). HvRAF transcripts were more in the roots than the leaves and were induced by salicylic acid, etepon, methyl jasmonic acid, cellulose and methyl biozin. Overexpression of the HvRAF gene in Arabidopsis induces activation of various stress-induced genes including PDF1.2, JR3, PR1, PR5, KIN2 and GSH1 , increases hospital resistance, and possibly salts in an ABA-independent manner Have a property that increases stress tolerance.

Rice is the most important crop in the world, especially in Asia, and has been provided as an important model system for studying monocotyledonous plants. 139 All of the ERF and DREB / CBF-type genes have been identified in rice (Nakano, T., Suzuki, K., Fujimura, T. and Shinshi, H. (2006) Plant Physiol. 140 : 411-432), but only a few Only ERF genes have been isolated and characterized.

In addition, OsRAF gene, which shows 50% amino acid sequence homology (58% similarity) with HvRAF, is induced at low temperature with ethylene, and the transfer of OsRAF :: GFP fusion protein to the nucleus has been reported in protein mobility experiments using onion epidermal cells. (Yibing Hu, Kang Chong, Tai Wang. Plant Growth Regul . (2008) 54: 55-61).

The present invention has been made in accordance with the above requirements, the present invention provides a transformed rice plant prepared by cloning the root abundant factor (RAF) gene OsRAF derived from rice ( Oryza sativa ) by introducing into a vector, Even when exposed to extreme environmental stresses such as bacteria and bacteria, the plant will continue to grow and develop, providing an effective way to improve the productivity of crops.

In order to solve the above problems, the present invention is a rice ( Oryza Provided is a plant with enhanced stress resistance transformed with a recombinant vector comprising a root abundant factor (RAF) gene OsRAF derived from sativa ).

The present invention also provides seed of the plant.

The present invention also provides a method for producing a stress-enhanced transgenic plant comprising transforming a plant cell with a recombinant vector comprising an OsRAF gene.

The invention also, by transforming a plant cell with a recombinant vector comprising a gene OsRAF provides a method for enhancing the stress tolerance of a plant comprising overexpressing OsRAF gene.

The present invention also provides a plant with enhanced stress resistance produced by the above method.

The present invention also provides a composition for enhancing stress resistance of a plant comprising the OsRAF gene.

The present invention also provides a composition for enhancing transcriptional activity comprising a protein consisting of 59 C-terminal amino acids which are acidic regions in a RAF protein derived from rice ( Oryza sativa ) consisting of the amino acid sequence of SEQ ID NO: 2.

The present invention also provides a method of improving plant productivity by transforming plant cells with a recombinant vector comprising the OsRAF gene.

In the present invention, by transferring the OsRAF gene to a plant transformation expression vector in combination, the plant effectively enhances environmental stress resistance such as epsic acid, drought, bacterial disease, salicylic acid and methyljasmonic acid without affecting general growth characteristics. Can be obtained. Therefore, it is expected to greatly contribute to increasing the yield of economic crops. In addition, the present invention can be usefully used to search for new environmental stress resistance genes by revealing a plant resistance mechanism in which expression is regulated by this transcription factor.

In order to achieve the object of the present invention, the present invention provides a plant with enhanced stress resistance transformed with a recombinant vector comprising a root abundant factor (RAF) gene OsRAF derived from rice ( Oryza sativa ). Stress according to an embodiment of the present invention may be epsis acid, drought, bacterial disease, salicylic acid or methyl jasmonate, but is not limited thereto.

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

Terminator may be used a conventional terminator, and examples thereof include fine loam furnace sold synthase (NOS), rice α- amylase RAmy1 A terminator, De Pace (phaseoline) terminator, Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens) ( Octopine) terminators, etc., but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. The use of terminators is therefore highly desirable in the context of the present invention.

Recombinant plant expression vector according to an embodiment of the present invention may be preferably a pBI111L- OsRAF vector described in Figure 3a. The pBI111L- OsRAF vector is a binary vector for plant gene expression prepared by recombining OsRAF cDNA derived from rice into pBI111L. However, the recombinant vector of the present invention is not limited to the vector described in FIG. 3A, and any vector useful for plant transformation can be used.

Plants according to the present invention is rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum food crops selected from the group consisting of; Vegetable crops selected from the group consisting of Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Special crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Fruit trees selected from the group consisting of apple trees, pear trees, jujube trees, peaches, lambs, grapes, citrus fruits, persimmons, plums, apricots and bananas; Flowers selected from the group consisting of roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops selected from the group consisting of rygras, red clover, orchardgrass, alpha alpha, tolsque cue and perennial lysgras. Preferably, the plant is Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, gall, watermelon, Melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, pea, etc. may be dicotyledonous plants, most preferably, the plant is Arabidopsis.

The present invention also provides seed of the plant. Preferably, the seed is a seed of the Arabidopsis.

The present invention also comprises the steps of transforming plant cells with a recombinant vector comprising a root abundant factor (RAF) gene OsRAF derived from rice ( Oryza sativa ); And regenerating the transformed plant from the transformed plant cell.

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

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

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

The method of the present invention comprises the step of transforming plant cells with the recombinant vector according to the present invention, wherein the transformation can be mediated by Agrobacterium tumefaciens . The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

The present invention also provides a method of enhancing stress resistance of a plant, comprising the step of transforming plant cells with the recombinant vector according to the present invention, overexpressing the OsRAF gene. Plant transformation methods, recombinant vectors used for transformation and plants that can be used as host cells are as described above.

The present invention also provides a plant with enhanced stress resistance produced by the above method. Plants that are stress resistant preferably are Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beetroot, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mustard, watermelon , Melon, cucumber, pumpkin, gourd, strawberry, soybean, mung beans, kidney beans, peas, etc. can be dicotyledonous plants, most preferably, the plant is Arabidopsis.

The present invention also provides a composition for enhancing stress resistance of a plant comprising a root abundant factor (RAF) gene OsRAF derived from rice ( Oryza sativa ). The stress may be, but is not limited to, epsic acid, drought, bacterial disease, salicylic acid or methyl jasmonate. In the composition of the present invention, the OsRAF gene may be preferably composed of a nucleotide sequence represented by SEQ ID NO: 1. The composition for enhancing stress resistance of the plant of the present invention includes a root abundant factor (RAF) gene OsRAF derived from rice ( Oryza sativa ) as an active ingredient, and can improve stress resistance of the plant by transforming the gene OsRAF into the plant. It is.

The present invention also provides a composition for enhancing transcriptional activity comprising a protein consisting of 59 C-terminal amino acids which are acidic regions in a RAF protein derived from rice ( Oryza sativa ) consisting of the amino acid sequence of SEQ ID NO: 2.

The range of OsRAF proteins according to the present invention includes proteins having an amino acid sequence represented by SEQ ID NO: 2 isolated from rice and functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" means the function of a transcriptional activator in a plant.

The present invention also provides a method of improving the productivity of a plant comprising the step of transforming plant cells with a recombinant vector comprising a root abundant factor (RAF) gene OsRAF derived from rice ( Oryza sativa ), overexpressing the OsRAF gene. do. Since the expression of the OsRAF gene in the plant of the present invention makes it resistant to stress such as drought stress and bacterial disease stress, the plant transformed with the OsRAF gene can improve the productivity of the plant compared to the control group.

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.

Experimental Method

1.Bacteria and yeast strains, vectors and culture media

Bacterial strain Escherichia coli DH10B [F ^- mcr A Δ (mrr - hsd RMS- mcr BC) φ80d lac ZΔM15 Δ lac X74 rec A1 end A1 deo R Δ (ara, leu) 7697 ara D139 gal U gal K rps L nup G λ ^- ] Was used as a host for plasmid amplification. Agrobacterium tumerfaciens strain C58 was used for transformation of Arabidopsis. Plasmid pGEM T-easy vectors (Promega, Madison, Wis.) Were used for general molecular cloning. pBI111L vector derived from pBI121 (Clontech) was used for transformation with Agrobacterium of OsRAF . Luria-Bartani (LB) medium (1% tryptone, 0.5% yeast extract and 1% NaCl) was used for the culture of E. coli strains. YEP medium ((1% Bakto Peptone, 1% Yeast Extract, 0.5% NaCl) was used for Agrobacterium incubation. LB medium containing antibiotics such as kanamycin (50 μg / ml) and ampicillin (50 μg / ml) Was used for the selection of transformed E. coli YEP medium containing rifampicin (100 μg / ml), gentamycin (50 μg / ml) and kanamycin (50 μg / ml) was used for the conversion of the transformants using Agrobacterium. Used for screening.

2. Plant

Seeds of rice ( Oryza sativa L. spp. Japonica cultivar Hwacheong) were soaked in distilled water for 24 hours at room temperature, and then Hoagland nutrient solution, Hoagland DR, Arnon DI (1950) Calif. Agric.Exp. Stn. Circ, 347: 1-39) was used to incubate at 28 ° C. in long-term conditions (16 hours / 8 hours cancer cycle) in an adjustable growth chamber. Two weeks after germination, the rice plants were subjected to several stress treatments. Arabidopsis (Columbia ecotype) was transformed via Agrobacterium to form OsRAF -overexpressing transgenic plants. During stress treatment, Arabidopsis seeds were sterilized for use in RNA preparation and morphological analysis of plants (Weigel, D., and Glazebrook, J. (2002) Arabidopsis : A Laboratory Manual.Cold Spring Harbor, NY : Cold Spring Harbor Laboratory Press), germination at 0.5 X MS (Murashige T and Skoog F (1962) Physiol Plant 15 (3): 473-497) containing 0.05% MES-KOH (pH 5.7) and 1% phytoagar. I was. After seeding at 4 ° C., dark conditions for 4 days, the seeds were germinated and incubated in a growth chamber at 28 ° C., long day conditions (16 hour light / 8 hour dark cycle).

3. Enzymes, Chemicals and Kits

Taq DNA polymerase and PfuTurb o DNA polymerase were purchased from Coabio Systems (Seoul, South Korea) and Stratazine (La Jolla, CA). SuperScript ^™ III RNase H ^- Reverse Transcriptase was purchased from Invitrogen. dNTP mixtures were purchased from Takara. Restriction enzymes T4 DNA ligase and agarose were purchased from Promega (Madison, USA). Reagents for preparing MS medium (Murashige and Skoog medium) and LB medium were purchased from Duchefa. Most chemicals, including antibiotics, were purchased from Sigma Aldrich (USA). The RNeasy Plant Mini Kit and One-Step RT-PCR Kit were purchased from Qiagen (Valencia, USA). Plasmid extraction kits, gel purification kits, PCR purification kits, and synthetic oligomers were synthesized in Bioneer (Euseong, Korea).

4. Stress Processing and Total RNA Isolation

Rice plants grown in Magenta box for 2 weeks were treated with 200 mM NaCl (salt), 0.1 mM Epsic Acid (ABA), 2 mM Salicylic Acid (SA), 0.1 mM Ethephon (Etephon) and 0.1 mM Methyl Jasmonic Acid ( MeJA) was applied to 0.01% TritonX-100 solution. As a control, nothing was treated at the same time. After treating the stress materials at various times, the plants were harvested, frozen in liquid nitrogen and stored at −70 ° C. for analysis. For mRNA expression analysis over time, total RNA was isolated from shoots and roots of two-week-old rice plants using the RNeasy Plant Mini Kit (Qiagen) as directed by the manufacturer.

5. OsRAF   Cloning of genes

CDNA of OsRAF (Os03g08470) was amplified by two stages of RT-PCR. The first strand of cDNA was synthesized with SuperScript ^™ III RNase H ^- reverse transcriptase (Invitrogen), with minimal alteration of manufacturer's instructions. OsRAF-TGA-R (SEQ ID NO: 3), 1 μg total RNA, 2 pmole reverse primer; 5'- TCA GAA AAG GGC GCC GTC GAT TG-3 'and 10 nmol of each dNTPs were added to a final volume of 13 μl and the mixture was heated at 65 ° C. for 5 minutes and then reacted on ice for 1 minute. 4 μl of 5 × first strand buffer, 1 μl 0.1 M DTT, 1 μl RNase inhibitor and 20 units of SuperScript ^™ III RNase H ⁻ reverse transcriptase were added and the reaction mixture was reacted at 55 ° C. for 1 hour. Reverse transcription was inactivated by heating at 72 ° C. for 15 minutes. PCR reactions were performed with 2 μl first strand reactant solution, 20 pmole forward and reverse primer, OsRAF-mF (SEQ ID NO: 4); 5'-AAA GGC ATT CGC AAC ACA CAC TTG-3 'and OsRAF-TGA-R (SEQ ID NO: 3), 10 nmol dNTPs and 1 unit PfuTurbo DNA polymerase ( stratazine ) were added to a final volume of 50 μl. . The PCR reaction was carried out under the following conditions after performing the initial denaturation reaction at 95 ℃ for 2.5 minutes [94 ℃: 30 seconds, 60 ℃: 30 seconds, 72 ℃: 1 minute, 28 times performed].

6. RNA Gel Blot Analysis

5-20 ug total RNA isolated from each sample was electrophoretically separated on a 1.2% agarose gel containing 2.2 M formaldehyde and transferred to a nylon membrane (Amersham Biosciences, UK). RNA was hybridized with OsRAF and 18S ribosomal RNA specific 32P- labeled DNA probes. For probe generation, 0.3 kb cDNA of the C-terminal coding region of OsRAF was amplified by PCR. Primer sets for PCR include OsAP2-probe-F1 (SEQ ID NO: 5); 5'- GCT ACC TTT CT T CAC CAA CGG CTT -3 'and OsAP2-probe-R1 (SEQ ID NO: 6); 5'- AAG AGG CTG TCG ATG GAC TCG TAG-3 'was used. The membrane was exposed to X-rays for radioactivity detection.

7. Transcriptional Activity Analysis in Yeast Cells

In order to examine the acidic regions of OsRAF proteins with transcriptional activation domains using a yeast assay system, complete and several partial regions of OsRAF coding sequences were obtained by PCR using the following primer sets. OsRAF complete constructs include Nde I-OsRAF-Full-F (SEQ ID NO: 7); 5′-CCCATATGTGTGGAGGCG CCATCCT-3 ′ and RAF-Full-BamHI-R (SEQ ID NO: 8); 5′-CGGGATCCCGAAAAG GGCGCCGTC-3 ′, sRAF ΔC1 constructs include Nde I-OsRAF-Full-F (SEQ ID NO: 7) and OsRAF-ΔC1-BamHI-R (SEQ ID NO: 9); 5′-CGGGATCCCGCTGTTGGCGTCGCC-3 ′, OsRAF ΔC2 constructs include NdeI-OsRAF-Full-F (SEQ ID NO: 7) and OsRAF-ΔC2-BamHI-R (SEQ ID NO: 10); 5′-CGGGATCCCGAAACCCAGCGTGTCG-3 ′, OsRAF ΔC3 constructs include NdeI-OsRAF-Full-F (SEQ ID NO: 7) and OsRAF-ΔC3-BamHI-R (SEQ ID NO: 11); 5′-CGGGATCCCGC TGTCGGTGAAGGAGCT-3 ′, OsRAF ΔN1 constructs include NdeI-OsRAF-ΔN1-F (SEQ ID NO: 12); Primer sets 5′-CCCATATGTCCGACGACGTCGACTC-3 ′ and OsRAF-Full-BamHI-R (SEQ ID NO: 8) were used, respectively. The coding region of the complete polypeptide and several partial polypeptides were linked in frame to the yeast GAL4 DNA binding domain at two restriction enzyme sites, Nde I and Bam HI, of the yeast expression vector pGBKT7 (Clontech), and GAL4 DNA Binding domain-OsRAF fusion proteins were made in transformed yeast cells. Fusion plasmids can be obtained from Saccharomyces cerevisiae with LacZ and HIS3 reporter genes by modified lithium acetate-mediated methods (Gietz et al. 1992). Saccharomyces cerevisiae with AH109 and LacZ Y187 was transformed. Transformed yeast culture was dropped in tryptophan-free synthetic dropout (SD) medium and incubated at 30 ° C. for 3 days. Transformed yeast cells were selected for growth. Transcriptional activity was determined using colony-lift filter assay using X-β-gal, and liquid culture assay was performed using the ο-nitrophenyl β-D-galactopyranoside (ONPG) as substrate as directed by the manufacturer. Analyzes (yeast protocol handbook PT3024-1, Clontech).

8. OsRAF PBI111L- for the production of transgenic Arabidopsis plants that overexpress  OsRAF vector making

The coding region of OsRAF cDNA comprises a forward primer OsRAF-Full-F1 (SEQ ID NO: 13) comprising Xba I site (5'- TCT AGA ACA AGA ACA AAG GCA TTC GCA ACA C-3 ') and Xho I site Amplification was done using reverse primer (SEQ ID NO: 14), 5'-CTC GAG TCA GAA AAG GGC GCC GTC T-3 '. PCR products digested with Xba I and Xho I sites were introduced into the corresponding sites of pBI111L, a binary vector derived from pBI121 (Clontech).

9. Transformation of Agrobacterium by Freeze-thaw Method

2 ml culture (C58) was placed in a 50 ml YEP liquid medium and subcultured at 28 ° C. to make the OD ₆₀₀ approximately 0.6-1.0, followed by centrifugation at 4000 rpm for 10 minutes. The pellet was suspended in 10 ml of 0.15 M NaCl solution, which had been cooled beforehand. After centrifugation for 5 minutes, 1 ml of 20 mM CaCl ₂ , which had been cooled, was added thereto, suspended, and then, 100 µl each of the 0.5 ml microfuse tubes, which had been cooled, was dispensed. After cooling on ice for 20 minutes, 1 μg recombinant plasmid DNA was added and cooled in liquid nitrogen for 2 minutes. Then thawed at 37 ° C. for 5 minutes. Cooling and thawing were repeated one or more times, then cooled on ice for 30 minutes, 1 ml YEP medium was added and plated on YEP-agar medium containing rifampicin, gentamycin and kanamycin.

10. Transformation of Arabidopsis by Agrobacterium-Mediated Transformation

Arabidopsis plants were transformed by the floral dip method (Clough, SJ and Bent, AF (1998). Plant J. 16 : 735-743). Single colonies were inoculated in 5 ml YEP medium containing rifampicin, gentamicin and kanamycin and suspended overnight at 28 ° C. 1 ml of the culture was subcultured in 500 ml YEP medium containing antibiotics so that the OD ₆₀₀ was at least 2.0. The cultures were suspended in 500 ml of infection medium (0.22% MS salt, 5% sucrose, 0.05% MES, 0.044 μM BA and 200 μl Silwet L-77; pH 5.7). The stems of Arabidopsis cultivars grown 4-6 weeks in soil grew to 5-15 cm long and were used as plant material. Plant pots were upside down and soaked in infected medium. After immersing in the infected medium for 10 seconds, the method of immersing in the infected medium for 10 seconds again after 10 seconds was repeated three times. After the pots were drained, the trays were covered with plastic wrap and placed in the growth chamber to maintain humidity. Transformants were selected in MS medium containing 30 μg ml ⁻¹ kanamycin. For subsequent analysis, homozygous transformants were selected as isolation rates for kanamycin resistance.

11. Germination and Root Growth Analysis

At least 80 sterile seeds (wild type, OsRAF overexpressed transgenic Arabidopsis) were seeded in 0.5x MS medium containing 0, 1, 5 μM ABA and 0, 100, 125 and 150 mM NaCl for germination testing. After absorbing and swelling the seeds under dark conditions for 4 days at 4 ° C., the plates were transferred to a growth chamber at 23 ° C., long-term conditions (16h light / 8h cancer) and counted the number of germinated seeds with small roots visible at a given time. It was. Also, for root growth analysis in ABA, sterile seeds (wild type, OsRAF overexpressing transgenic Arabidopsis) were placed perpendicular to 0.5x MS medium containing 0, 1, and 5 μM ABA at 23 ° C., long-term conditions (16 h / Cultures and root lengths were measured in growth chambers, 8h dark).

12. Analysis of drought resistance

The next generation of seeds was harvested by continuing incubating the wild type Arabidopsis and OsRAF overexpressing transformants in the soil. Each seed was sown in 100 soils and grown for two weeks at 23 ° C. under long-term conditions (16 hours light / 8 hours cancer). Then, the water supply was applied for 10 days, and a pair of stresses were applied. Then, the water was resupplied and the growth state was observed from two days later.

 13. Disease Resistance Analysis

Wild type Arabidopsis and OsRAF gene overexpressed transformed Arabidopsis were grown in soil for 4 weeks, and then the root tip was cut about 2cm.

The plant was soaked in 10 mM MgCl ₂ suspended for 10 ⁸ cfu for about 10 hours of Ralstonia solanacearum GMI 1000 and transferred to soil and grown at 23 ° C. Wild type Arabidopsis was used as a control. One day after the pathogen was inoculated, the condition of the disease was examined by the disease index method. The method is a method of marking the degree of infection 1 to 4 depending on the extent of the signs of bedbugs of the entire leaf. In other words, if no signs of allergic leaves are indicated, 0, the symptom of 25% or less of the total leaves is 1, 26 to 50% 2, 51 to 75% 3, 76 to 100% 4 if the method is displayed. Each experiment was performed three times independently.

Example 1 Responsiveness to Abiotic Stress and Phytohormone Treatment OsRAF Gene Expression Patterns>

RNA blot analysis was performed to investigate the induction pattern over time for the expression of the OsRAF gene. Total RNA was extracted from rice plants with various abiotic treatments such as high salt, MeJA and etepon treatments and a drought, stress response phytohormonal treatment. As can be seen from the RNA blot analysis of FIG. 1, the expression level of OsRAF transcript was small after mock treatment. However, transcripts of the OsRAF gene induced expression within 24 hours after ABA, SA and drone treatment. In particular, OsRAF transcripts were induced within one hour at one foot and expressed strongly after 3 and 6 hours, and were strongly expressed 24 hours after ABA and SA treatment. Ribosome RNA was stained with ethidium bromide and used as an internal control for RNA quantification in RNA blot analysis.

Example 2 Functional Role of OsRAF as a Potential Transcription Activator

Several ERF family proteins, including acidic C-terminal domains, are believed to have transcriptional activity in yeast (Jung, J. and Kim, M. (2007) J. Plant Biol. 50 (3) 383-385). OsRAF has an acidic C-terminal region, which is known to be able to act as a transcriptional activity domain (Hahn, S. (1993). Cell 72 : 481 ?? 483). In order to confirm the transcriptional regulator of OsRAF and the functional role of the C-terminal region, transcriptional activity analysis was performed in yeast. The OsRAF coding region and its missing mutations were linked to a GAL4 DNA binding domain expression vector, and each vector was used to analyze the transcriptional activity of the lac Z reporter gene (FIG. 2A). Full-length OsRAF (OsRAF Full) and N-terminal deletion mutants comprising C-terminal acidic regions (OsRAFΔN1) linked to the GAL4 DNA binding domain showed transcriptional activity in liquid culture assays. On the other hand, the mutant (OsRAFΔC1) deficient in 24 C-terminal amino acids, the C-terminal acidic region, was found to be somewhat lower in β-galactosidase activity than OsRAF Full activity. Moreover, the mutant lacking 59 C-terminal amino acids (OsRAFΔC2) and the more mutant (OsRAFΔC3) had no β-galactosidase activity (FIG. 2B). These results indicate that OsRAF can function as a transcriptional activator in yeast, and that the C-terminal acidic region, particularly the downstream site comprising 59 C-terminal amino acids, plays an important role in transcriptional activity.

<Example 3. Examination of drought resistance of transformants overexpressed OsRAF gene>

The next generation of seeds was harvested by continuing incubating the wild type Arabidopsis and OsRAF overexpressing transformants in the soil. Each of the seeds were sown in soil over 100 and grown for two weeks at 23 ° C. under long-term conditions (16 hours light / 8 hours cancer). Then, the water supply was moderated for 10 days, and a pair of stresses were applied. Then, the water was resupplied and the growth state was observed after two days.

As a result, wild type Arabidopsis showed more than 90% death, whereas OsRAF gene overexpressing transformants were mostly survived (FIG. 5).

<Example 4. Disease resistance investigation of the transformant overexpressed OsRAF gene>

Wild-type Arabidopsis and transformed Arabidopsis were grown in soil for 4 weeks, and then the root tip was cut about 2cm.

The plant was soaked in 10 mM MgCl ₂ suspended for 10 ⁸ cfu for about 10 hours of Ralstonia solanacearum GMI 1000 and transferred to soil and grown at 23 ° C. Wild type Arabidopsis was used as a control. Two days after the pathogen was inoculated, the condition of the disease was examined by the disease index method. The method is a method of marking the degree of infection 1 to 4 depending on the extent of the signs of bedbugs of the entire leaf. In other words, if no signs of allergic leaves are indicated, 0, the symptom of 25% or less of the total leaves is 1, 26 to 50% 2, 51 to 75% 3, 76 to 100% 4 if the method is displayed. Each experiment was performed three times independently.

As a result, about 6 days after the inoculation of the disease-free pathogen, wild type Arabidopsis showed more than 60% of symptoms (disease index 2.5 or more), while OsRAF overexpressing transformants showed less than about 25% of symptoms. (Disease index 1) is shown. At 8 days after inoculation of the pathogen, the wild type showed more than 90% of the disease (disease index 3.5 or more), and the transformant showed about 50% of the disease (disease index 2) (FIG. 6b).

Therefore, the symptom rate of the OsRAF overexpressing transformant was slower than that of the wild type. From this, the OsRAF gene isolated from rice, which is a monocotyledonous plant, showed a defense mechanism against pathogens in the Arabidopsis, a dicotyledonous plant.

1 shows the OsRAF gene expressed in response to some exogenous stress. Sterilized water (mock), 200 mM NaCl (salt), 0.1 mM ABA (epsilic acid), a pair, 2 mM SA (salicylic acid), 0.1 mM Ethephon (ethepone) and 0.1 mM MeJA (methyljasmonic acid) respectively After that, the total RNA extracted from the plants grown for 14 days (0, 1, 3, 6, 24 h) was investigated. Each 10 ug total RNA was loaded onto formaldehyde agarose gel. RNA gel blots were hybridized with 0.3 kb specific probes that matched the C-terminal coding region of OsRAF . The same amount of RNA was confirmed by visible rRNA stained with ethidium bromide.

Figure 2 shows the results of performing a transactivation assay (Transactivation assay) of OsRAF in yeast. a . Yeast strain Y187 was transformed by linking the full and missing lengths of OsRAF to the GAL4 DNA binding domain (GAL4 BD) of the yeast expression vector pGBKT7. The transformants were incubated for 30 days at 30 ° C. in tryptophan-free synthetic dropout (SD) medium. Black boxes represent conserved AP2 / ERF DNA-binding domains, and parallel hatched boxes indicate potential acidic transcriptional activity regions, respectively. b. Β-galactosidase activity by liquid culture assay using ο-nitrophenyl β-D-galactopyranoside (ONPG) as substrate to investigate the activity of each translation product as a transcription activator in yeast Was examined (three repeat spheres). One unit of β-galactosidase activity is defined as the amount by which one cell hydrolyzes 1 μM ONPG with ο-nitrophenyl and D-galactose for 1 minute.

3 relates to the preparation of OsRAF -overexpressing transgenic Arabidopsis plants. a . OsRAF cDNA containing 5 'UTR (1105 bp) was inserted into the XbaI and XhoI sites of the pBI111L vector. b . Kanamycin-resistant transgenic plants that continuously express OsRAF , 35S :: OsRAF # 3, # 6, were identified by Northern analysis.

4 is 35S :: OsRAF Resistance to ABA and salt in transgenic Arabidopsis. MS medium containing high levels of salt (0, 100, 125, 150 mM) or ABA (0, 1, 5 μM), a plant hormone, a representative factor that recovers seeds and suppresses germination from Arabidopsis overexpressed OsRAF gene Seeding and germination was measured. At this time, as a control, the seeds recovered from the wild-type Arabidopsis oleifera were sown under the same conditions to measure germination. Germination was expressed as a percentage of the number of seeds from the young root (radicle) penetrating the epidermis 1 mm or more.

Figure 5 compares the growth of OsRAF gene overexpressing transformants and wild type Arabidopsis according to drought stress.

Figure 6 compares the growth of OsRAF gene overexpressing transformants and wild type Arabidopsis according to bacterial diseases. a. OsRAF gene overexpression of the Arabidopsis 6 days after inoculation of the bacterium. b. OsRAF gene overexpressed Arabidopsis inoculated with the disease index over time.

<110> SNU R & DB FOUNDATION <120> Plant with Increased Resistance to Various Stresses Transformed          with OsRAF Gene <130> PN08096 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 1114 <212> DNA <213> Oryza sativa <400> 1 atgtgtggag gcgccatcct cgccgagttc atcccggcgc cgtcgcgcgc cgcggcggcg 60 accaagcggg tgaccgccag ccacctgtgg ccggccggct ccaagaacgc cgcccgcggc 120 aagagcaaga gcaagaggca gcagaggagc ttcgccgacg tcgacgactt cgaggccgcc 180 ttcgagcagt tcgacgatga ctccgacttc gacgacgcgg aggaagaaga cgaaggacac 240 ttcgtgttcg cgtccaaatc tcgtggtaat gttcgtcgcg tgcgatctcg tctaggtgtt 300 cgatcgtgat ggcgatgagt tcttggcttg atctcaccga ggaagattgg tttggtttgg 360 ttttgttcgt gcagtcgtcg ccgggcacga cgggcgcgcg gcggcgaggg cggcgagcaa 420 gaagaagcgg gggcggcact tccgaggcat ccggcagcgg ccatggggga agtgggcggc 480 ggagatccgc gacccgcaca agggcacgcg cgtctggctc ggcacgttca acaccccgga 540 ggaggccgca cgcgcctacg acgtcgaggc gcgccgcctc cgcggcagca aggccaaggt 600 caacttcccc gccacgcccg ccgccgcgcg cccacgccgc ggcaacacga gagccaccgc 660 cgtgccaccg ccggcgacag cacccgccgc cgccccgccg cgcggactga agcgagaatt 720 ctcgccgcct gctgagaccg cgctaccttt cttcaccaac ggcttcgtcg acctgacgac 780 cgccgcggcg ccgccaccgg ccatgatgat gacgagctcc ttcaccgaca gcgtcgccac 840 gtcggagtcc ggcgggagcc ccgccaagaa ggcgaggtcc gacgacgtcg actcgtccga 900 gggcagcgtc ggcggcggca gcgacacgct gggtttcacc gacgagctgg agttcgaccc 960 gttcatgctg ttccagctcc cctactccga cggctacgag tccatcgaca gcctcttcgc 1020 cgccggcgac gccaacagcg cgaacaccga catgaacgcc ggcgtcaacc tgtggagctt 1080 cgacgacttc ccaatcgacg gcgccctttt ctga 1114 <210> 2 <211> 334 <212> PRT <213> Oryza sativa <400> 2 Met Cys Gly Gly Ala Ile Leu Ala Glu Phe Ile Pro Ala Pro Ser Arg   1 5 10 15 Ala Ala Ala Ala Thr Lys Arg Val Thr Ala Ser His Leu Trp Pro Ala              20 25 30 Gly Ser Lys Asn Ala Ala Arg Gly Lys Ser Lys Ser Lys Arg Gln Gln          35 40 45 Arg Ser Phe Ala Asp Val Asp Asp Phe Glu Ala Ala Phe Glu Gln Phe      50 55 60 Asp Asp Asp Ser Asp Phe Asp Asp Ala Glu Glu Glu Asp Glu Gly His  65 70 75 80 Phe Val Phe Ala Ser Lys Ser Arg Val Val Ala Gly His Asp Gly Arg                  85 90 95 Ala Ala Ala Arg Ala Ala Ser Lys Lys Lys Arg Gly Arg His Phe Arg             100 105 110 Gly Ile Arg Gln Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp         115 120 125 Pro His Lys Gly Thr Arg Val Trp Leu Gly Thr Phe Asn Thr Pro Glu     130 135 140 Glu Ala Ala Arg Ala Tyr Asp Val Glu Ala Arg Arg Leu Arg Gly Ser 145 150 155 160 Lys Ala Lys Val Asn Phe Pro Ala Thr Pro Ala Ala Ala Arg Pro Arg                 165 170 175 Arg Gly Asn Thr Arg Ala Thr Ala Val Pro Pro Pro Ala Thr Ala Pro             180 185 190 Ala Ala Ala Pro Pro Arg Gly Leu Lys Arg Glu Phe Ser Pro Pro Ala         195 200 205 Glu Thr Ala Leu Pro Phe Phe Thr Asn Gly Phe Val Asp Leu Thr Thr     210 215 220 Ala Ala Ala Pro Pro Pro Ala Met Met Met Thr Ser Ser Phe Thr Asp 225 230 235 240 Ser Val Ala Thr Ser Glu Ser Gly Gly Ser Pro Ala Lys Lys Ala Arg                 245 250 255 Ser Asp Asp Val Asp Ser Ser Glu Gly Ser Val Gly Gly Gly Ser Asp             260 265 270 Thr Leu Gly Phe Thr Asp Glu Leu Glu Phe Asp Pro Phe Met Leu Phe         275 280 285 Gln Leu Pro Tyr Ser Asp Gly Tyr Glu Ser Ile Asp Ser Leu Phe Ala     290 295 300 Ala Gly Asp Ala Asn Ser Ala Asn Thr Asp Met Asn Ala Gly Val Asn 305 310 315 320 Leu Trp Ser Phe Asp Asp Phe Pro Ile Asp Gly Ala Leu Phe                 325 330 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> OsRAF-TGA-R primer <400> 3 tcagaaaagg gcgccgtcga ttg 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsRAF-m-F primer <400> 4 aaaggcattc gcaacacaca cttg 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsAP2-probe-F1 primer <400> 5 gctacctttc ttcaccaacg gctt 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsAP2-probe-R1 primer <400> 6 aagaggctgt cgatggactc gtag 24 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NdeI-OsRAF-Full-F primer <400> 7 cccatatgtg tggaggcgcc atcct 25 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RAF-Full-BamHI-R primer <400> 8 cgggatcccg aaaagggcgc cgtc 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> OsRAF-C1-BamHI-R primer <400> 9 cgggatcccg ctgttggcgt cgcc 24 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> OsRAF-C2-BamHI-R primer <400> 10 cgggatcccg aaacccagcg tgtcg 25 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> OsRAF-C3-BamHI-R primer <400> 11 cgggatcccg ctgtcggtga aggagct 27 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NdeI-OsRAF-N1-F primer <400> 12 cccatatgtc cgacgacgtc gactc 25 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> OsRAF-Full-F1 primer <400> 13 tctagaacaa gaacaaaggc attcgcaaca c 31 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 14 ctcgagtcag aaaagggcgc cgtct 25  

Claims (10)

Plants having enhanced stress resistance transformed with a recombinant vector comprising a root abundant factor (RAF) gene OsRAF derived from rice ( Oryza sativa ). The plant of claim 1, wherein the stress is ABA (epsis acid), drought, bacterial disease, salicylic acid or methyl jasmonate. Seeds of plants according to claim 1. Transforming plant cells with a recombinant vector comprising a root abundant factor (RAF) gene OsRAF derived from rice ( Oryza sativa ); And Method for producing a transformed plant enhanced stress resistance comprising the step of re-differentiating the transformed plant from the transformed plant cells. Rice ( Oryza sativa) by transforming the plant cell with a recombinant vector comprising a RAF (root abundant factor) gene derived from OsRAF OsRAF A method of enhancing stress resistance of a plant comprising overexpressing a gene. Plants with enhanced stress resistance produced by the method of claim 5. A composition for enhancing stress resistance of a plant comprising a root abundant factor (RAF) gene OsRAF derived from rice ( Oryza sativa ). 8. The composition according to claim 7, wherein the stress is epsis acid (absciscic acid), drought, bacterial disease, salicylic acid or methyl jasmonate. A RAF protein derived from rice ( Oryza sativa ) consisting of the amino acid sequence of SEQ ID NO: 2, comprising a protein consisting of 59 C-terminal amino acids which are acidic regions. Rice ( Oryza sativa) by transforming the plant cell with a recombinant vector comprising a RAF (root abundant factor) gene derived from OsRAF OsRAF A method of improving the productivity of a plant comprising overexpressing a gene.

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