KR20120111715A - Osbhlh148 gene enhancing drought stress tolerance of plant and uses thereof - Google Patents

Abstract

The present invention relates to a transgenic plant prepared by transferring a recombinant DNA comprising the transcription factor OsbHLH148 gene isolated from rice, and more specifically, the transcription factor OsbHLH148 ( Oryza, which regulates expression of genes involved in rice-derived stress resistance. sativa basic helix-loop-helix 148) protein, gene encoding the protein, recombinant vector containing the gene, host cell transformed with the recombinant vector, increased resistance to environmental stress of plants using the recombinant vector The present invention relates to a method for increasing the resistance to environmental stress of a transgenic plant and its seed, and a plant comprising the gene, which is transformed with the recombinant vector to increase resistance to environmental stress.

Description

OsshlH148 gene enhancing drought stress tolerance of plant and uses approximately}

The present invention is a rice (rice, Oryza) The present invention relates to a transgenic plant prepared by transferring a recombinant DNA comprising a transcription factor OsbHLH148 gene isolated from sativa ). More specifically, the genes of OsbHLH148 transcription factor, which are activated by the environmentally-resistant plant hormone abscisic acid (ABA), are introduced into the plant and overexpressed to resist various environmental stresses such as drought, low moisture and drought. It is to provide a transgenic plant.

bHLH (basic / helix-loop-helix) proteins are a group of transcription factors that bind to the DNA promoter and induce the expression of the gene. 167 bHLH genes were found in the rice genome (Li et al., Plant Physiol. 2006, 141: 1167-1184).

ABA is a growth regulator widely present in various plants and plays a role in activating the expression of related genes so that plants have a resistance response under conditions such as drought, high salinity, and low temperature (Xiong et al., Plant Cell 2000: S165-). S183). When plants are subjected to environmental stress, such as drought, cold, and salt, ABA is biosynthesized in large quantities and accumulated in the body. ABA activates relevant transcription factors and induces their expression by binding these activated transcription factors to the promoter region of the resistance gene.

In order to enhance the expression of genes that lead to resistance to environmental stress such as drought, a lot of genetic engineering studies have been conducted to transfer ABA-related transcription factors to crops to enhance resistance. For example, plants overexpressing the CBF / DREB1 transcription factor showed improved resistance to cold, salt and drought. An example has been reported in which the CBF gene of Arabidopsis larvae was added to rapeseed to improve cold resistance and drought resistance (Zhang et al., Plant Physiol. 2004, 135: 615-621). The Arabidopsis overexpressing the gene of rice OsDREB1, a heterologous gene of CBF, also showed improved resistance to cold, salt and drought. Recently, the transfer of the Arabidopsis transcription factor gene AtMYB44 has been reported to improve resistance to various environmental stresses such as cold, salt and drought (Jung et al., Plant Physiol. 2008, 146: 623-635). However, in the transformed crops, the resistance is not greatly improved, or problems such as discoloration and growth disorder have been observed (Liu et al., Plant Cell 1998, 10: 1391-1406).

Korean Patent Registration No. 10-0742193 discloses a novel environmental stress resistant transcription factor and a method of increasing the environmental stress resistance of plants using the same, and Korean Patent Registration No. 10-0571131 describes the coding of the transcription factor of the plant Although the gene is disclosed, it is different from the transcription factor of the present invention.

The present invention has been made in accordance with the above requirements, and while the present inventors studied the effects of ABA on the gene expression of rice, the present inventors searched for the OsbHLH148 gene expressed by this plant hormone and transferred the gene to overexpress it. The present invention has been completed by confirming that the transformed plant exhibits markedly improved resistance to environmental stress such as drought without problems in growth.

In order to solve the above problems, the present invention is a transcription factor OsbHLH148 ( Oryza to regulate the expression of genes involved in rice-derived stress resistance sativa basic helix-loop-helix 148) protein.

The present invention also provides a gene encoding the protein.

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

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

The present invention also provides a method for increasing the resistance to environmental stress of plants using the recombinant vector.

In addition, the present invention provides a transformed plant and its seeds that have been transformed with the recombinant vector and increased resistance to environmental stress.

The present invention also provides a composition for increasing the resistance to environmental stress of plants comprising the gene.

According to the present invention, the rice OsbHLH148 protein of the present invention is a transcription factor that regulates the expression of other genes, and is effectively transferred to a plant transformant expression vector including the genes thereof without affecting general growth characteristics. Plants with high resistance to various stresses can be obtained. Therefore, it is expected to contribute greatly to the increase in yield of economic crops and the like. In addition, the present invention will be useful in the search for new environmental stress resistance genes by revealing a plant resistance mechanism in which expression is regulated by this transcription factor.

1 is a result of a northern blot experiment showing that the OsbHLH148 gene is induced by rice with the environmental stress resistance hormone ABA.
Figure 2 shows the structure of the DNA (P _35S : cauliflower mosaic virus (CaMV) 35S promoter) recombined rice OsbHLH148 gene into plant transformation expression vector pBI111L (GB removed from pBl121).
3 is a result of confirming the constant expression of OsbHLH148 by Northern blot experiment by selecting nine lines showing kanamycin resistance after transferring the recombinant pCaOsbHLH148 DNA to the Arabidopsis.
Figure 4 is a result of performing a genomic Southern blot using the OsbHLH148 gene site as a probe to confirm whether the OsbHLH148 gene is correctly inserted into the transformed Arabidopsis.
Lane WT: Wild Type Baby Pole (Col-0)
Lanes # 2 ~ # 7, # 9: Transgenic Arabidopsis Line
FIG. 5 shows the results of culturing in a medium containing 3 μM ABA to measure ABA sensitivity of transformed Arabidopsis overexpressing OsbHLH148 gene.
WT: Wild-type Baby Pole
T-3, T-5: transgenic Arabidopsis ( 35S :: OsbHLH148 )
Figure 6 is a result of observing the leaves of the transformed and transformed Arabidopsis while the natural drying, the above (A) is a measure showing the weight change according to the outflow of water, showing that the moisturizing properties of the transgenic plant improved. , (B) is a photograph showing the plant state after 120 minutes in this state.
WT: Wild-type Baby Pole
T-3, T-5: transgenic Arabidopsis ( 35S :: OsbHLH148 )
FIG. 7 is a photograph showing the results of investigating the resistance of plants to low moisture stress after 7 days of stopping hydration of the transgenic and non-transgenic Arabidopsis grown 4 weeks.
WT: Wild-type Baby Pole
T-3, T-5: transgenic Arabidopsis ( 35S :: OsbHLH148 )
8 shows genomic Southern blot analysis of the rice transformed with OsbHLH148 gene (top) and recombinant vector (bottom) for rice transformation.
9 shows OsbHLH148 Northern blot analysis of rice transformed with the gene.
Lane WT: Wild Rice
Lanes 1 to 2 and 6 to 11: transgenic rice line
10 shows expression results of OsbHLH148 gene according to environmental stress treatment time.
Figure 11 shows the results of a drought resistance test for rice transformed with OsbHLH148 gene.
WT: Wild Rice
T-2, T-9, T-11: Transgenic Rice Line
12 is a result of measuring Fv / Fm value for rice transformed with OsbHLH148 gene.
WT: Wild Rice
T-2, T-9, T-11: Transgenic Rice Line

In order to achieve the object of the present invention, the present invention is a transcription factor OsbHLH148 ( Oryza that regulates the expression of genes involved in rice-derived stress resistance sativa basic helix-loop-helix 148) protein.

In the present invention, "transcription factor" is used as a generic name to mean a protein that acts on the promoter region of a gene so that the gene is transcribed into mRNA. In addition, "OsbHLH148" represents a protein present in rice as one of the "transcription factors", and the gene encoding the enzyme protein is expressed as " OsbHLH148 gene."

The range of OsbHLH148 protein according to the present invention is rice ( Oryza sativa ) includes a protein having an amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent 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.

The present invention also provides a gene encoding the OsbHLH148 protein. The gene of the present invention is characterized by regulating the expression of genes involved in stress resistance, and includes both genomic DNA and cDNA encoding the OsbHLH148 protein. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1 . The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a recombinant vector comprising the OsbHLH148 gene according to the present invention. The recombinant vector of the present invention may preferably be the pCaOsbHLH148 vector described in FIG. 2 or the recombinant vector described in FIG. 8, but is not limited thereto.

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” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of recombinant vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer a portion of themselves, the so-called T-region, to plant cells. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

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

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

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Octopine gene terminator, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a host cell transformed with the recombinant vector of the present invention. Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

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

The method of carrying the vector of the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

The present invention also provides a method of increasing resistance to environmental stress in a plant, comprising the step of transforming plant cells with the recombinant vector of the present invention to overexpress the OsbHLH148 gene.

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 the hybrid DNA according to the present invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

The present invention also provides a transgenic plant which is transformed with the recombinant vector of the present invention to increase resistance to environmental stress. The environmental stress may be drought, salty or cold, but is not limited thereto, and is preferably drought stress. The transforming plant is a food crop selected from the group consisting of rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum; 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; Apple trees, pears, jujubes, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops selected from the group consisting of lysis, redclover, orchardgrass, alphaalpha, tolsquescue and perennial lygragrass.

The present invention also provides a seed obtained from a transgenic plant with increased resistance to environmental stress.

The invention also comprises the steps of transforming a plant cell with a recombinant vector according to the invention; And

Provided is a method for producing a transformed plant having increased resistance to environmental stress, comprising the step of regenerating the transformed plant from the transformed plant cells.

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 . In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

The present invention also provides a composition for increasing the resistance of plants to environmental stress, comprising the OsbHLH148 gene of the present invention. In the composition of the present invention, the OsbHLH148 gene may be composed of the nucleotide sequence of SEQ ID NO: 1. The composition of the present invention includes the rice-derived OsbHLH148 gene as an active ingredient, by transforming the gene into a plant, it is possible to increase the plant's resistance to environmental stress. The plant is as described above.

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

Example  1: rice Osbhlh148  Molecular Biology of Genes

The history of the study of OsbHLH148 , a rice gene used in the present invention, and its molecular biological function will be described in detail.

Osbhlh148 cDNA was obtained by amplification by polymerase chain reaction (PCR) in wild type rice (breed: Nakdong). Rice was treated with 200 μM ABA or 100 μM methyljasmonate (MeJA) for 1 hour and then total RNA was phenol / SDS / LiCl method (Carpenter and Simon, 1998, Methods Mol. Biol. 82, 85-89). CDNA was obtained by reacting this RNA sample with reverse transcriptase Superscript III purchased from Invitrogen, using an oligo d (T) of 18 base pairs. For the purpose of amplifying the amount of cDNA, 5'-ATGCAAATGGAGTCG-3 '(SEQ ID NO: 3) as a forward primer and 5'-TCAAAACACATTTTG-3' (SEQ ID NO: 4) as a reverse primer were used. PCR amplification reaction was performed using polymerase.

The amplified cDNA was extracted from the gel and blunt end ligated to Enzynomics's pTOP blunt V2 vector for subcloning. The nucleotide sequence of the obtained full-length OsbHLH148 cDNA was analyzed and confirmed to be consistent with the amino acid sequence of the sequence number (AK071734) and the protein (LOC_Os03g53020) of the gene (Os03g0741100) of the rice gene database.

The OsbHLH148 gene (cDNA) is composed of 900 bases (including end codons) as shown (SEQ ID NO: 1), and the OsbHLH148 transcription factor encoded by this gene has a 299 amino acid sequence (SEQ ID NO: 2). OsbHLH148 transcription factor is a DNA binding protein with a basic helix-loop-helix (bHLH) structure.

Analysis of cis -acting elements on the OsbHLH148 promoter sequence through a database search showed that a number of elements responded to ABA, drought, jasmonic acid, ethylene, salicylic acid and wounds. It appeared to exist. Accordingly, the activation pattern of OsbHLH148 gene expression for various plant hormones was observed through Northern blot analysis. ABA was treated with Hoagland medium to a final concentration of 200 μM to soak up the roots of rice grown for 2 weeks. OsbHLH148 gene reached maximum expression when ABA was treated for 1 hour and maintained for 3 hours (FIG. 1). Therefore, the expression of this gene was judged to be very sensitive to various external stresses.

The gene was not induced by treatment with the disease-inducing hormone salicylic acid. OsJAmyb and Os are the control genes for stress on ABA. Expression of OsAOC gene was also observed. The rRNA was observed for each lane in order to prove that the same amount of RNA was compared in this electrophoretic experiment.

Example  2: Osbhlh148  Arabidopsis transformation experiments overexpressing genes

In order to prepare a transgenic Arabidopsis overexpressing the OsbHLH148 gene, the OsbHLH148 gene was fused to the CaMV35S promoter, which induces constant expression of the gene (FIG. 2). The vector contains the gene NPTII , which is resistant to the antibiotic kanamycin, between LB and RB, the gene sequences that represent the boundaries of the T-DNA moiety inserted into the plant genome. It is. This DNA construct was named pCaOsbHLH148. The structure of pCaOsbHLH148 is shown in FIG.

These DNA constructs were referred to as Agrobacterium tumerfaciens Agrobacterium containing transgenic DNA was selected in a medium containing C58C1 ( Agrobacterium tumefaciens C58C1) and containing kanamycin, rifampicin and gentamicin. According to the floral dip transformation, submerged the stalk of the Arabidopsis sage for 3 minutes in this strain culture medium, then placed in an enclosed container at almost 100% relative humidity, raised in growth for 24 hours, and then opened and opened. It was raised upright. Seeds obtained from these plants were germinated in a medium containing kanamycin to select kanamycin resistant plants, and after receiving the seeds from them, germinating and selecting them in kanamycin medium, transplanting the resistant transformants into the soil, the second generation of seeds Got. This was again screened and tested on third generation seed without kanamycin susceptible individuals. First, Northern blot analysis using the OsbHLH148 gene DNA as a probe resulted in 9 individuals identified as overexpressing the gene at all times (FIG. 3).

Genomic Southern blot analysis was performed to confirm the number and location diversity of the genes of interest inserted into these transgenic Arabidopsis (FIG. 4). This gene was isolated from rice and was not detected in non-transformants because it was not present in Arabidopsis. No. 3, 5, lines were inserted into plant chromosomal DNA with each T-DNA inserted into each line. Are different from each other.

Example  3: Osbhlh148  Phenotype Observation of Transgenic Arabidopsis Depending on Gene Expression Change

In view of the expression of OsbHLH148 in rice by ABA treatment, OsbHLH148 was found to be a transcription factor that activates ABA-dependent action. Germination rates were observed in MS medium containing 3 μM ABA to determine how the overexpression of OsbHLH148 gene responds to ABA, a plant hormone associated with environmental stress. There was no difference in germination and growth of wild-type Arabidopsis (WT, Col-0) and OsbHLH148 gene overexpression in medium without ABA, but overexpression of OsbHLH148 gene was sensitive to ABA in medium containing 3 μM ABA. It was confirmed to be hypersensitve (FIG. 5). This means that overexpression of the OsbHLH148 gene may be resistant to environmental stress.

Example  4: Osbhlh148  Environmental Stress Resistance Test of Transgenic Arabidopsis Depending on Gene Expression Amount

First, the degree of water loss of the overexpression of OsbHLH148 gene was compared with wild type Arabidopsis. The weight of the plants grown for 4 weeks was naturally dried at 23 ° C. except for the roots. Overexpression of the OsbHLH148 gene showed less water loss than wild-type Arabidopsis (Figure 6). To reaffirm this result, plants grown for 4 weeks were dried on plant growth at 23 ° C. and 30% humidity. After 10 days of soaking in the same volume of water, the wild type Arabidopsis withered, but the overexpression of the OsbHLH148 gene showed resistance (FIG. 7). To clarify this, after 10 days of drying, the same volume of water was observed and 3 days later, the wild-type Arabidopsis died but the overexpression of OsbHLH148 gene was alive.

Example  5: Osbhlh148  Rice Transformation Experiment Overexpressing Genes

In order to analyze the function of genes OsbHLH148 OsbHLH148 A transgenic rice with overexpressed genes was developed. First, the OsbHLH148 gene was fused to an OsCc1 promoter that induces constant expression of the gene (FIG. 8). The vector contains the BAR gene, which confers resistance to the herbicide, basta, for the selection of transgenic rice between LB and RB, the gene sequences that represent the boundaries of the T-DNA region inserted into the plant genome. It can be used to select plants for which genes have been successfully transferred. The DNA constructs were transformed into Agrobacterium tumefaciens LBA4404 ( Agrobacterium tumefaciens). LBA4404 ), and Agrobacterium containing the introduced DNA was selected in a medium containing tetracycline and spectinomycin. The strain culture was wild type rice ( Oryza sativa L. cv. Nakdong) callus was differentiated in a medium containing cytotaxy and phosphinothricin. During the differentiation, the transformed rice, in which the roots and leaves were developed, was grown in a greenhouse to obtain first-generation transformed seeds. The first-generation seeds were grown in paddy soil, and then transgenic rice was selected by basa treatment, and the second-generation seeds were obtained after ripening. The same procedure was repeated to test the third generation seed without bastard susceptibility.

Example  6: Osbhlh148  Selection of Transgenic Rice Overexpressing Genes

Genomic DNA of the third generation of transgenic rice grown in greenhouse paddy soil for 2 weeks, treated with Eco RI restriction enzyme and Southern blot using MAR gene as a probe 2, 8, 11 It was confirmed that 1 copy, 1 copy, 1, 9, 10 copy of OsbHLH148 gene was inserted into transgenic rice 1 (Fig. 8). The expression level of the OsbHLH148 gene in these transgenic rice was confirmed by Northern blot, and the highest expression was found in 2 and 9 transgenic rice, and relatively moderate in 11 transgenic rice. , 1, 8, 10 times the lowest expression in transgenic rice (Fig. 9). Based on the above results, future experiments were carried out using transgenic rice 2, 9 and 11.

Example  7: Osbhlh148 Expression patterns of environmental stress in genes

The wild-type rice grown for 2 weeks in the greenhouse was treated with various environmental stresses to investigate the expression of OsbHLH148 gene (FIG. 10). In the case of drought stress, the experimental group was naturally dried at 23 ° C. and treated for each hour, and showed the highest expression after 1 hour. In the case of salt stress, the experimental group treated with rice roots in salted water was obtained the highest expression after 1 hour. In case of cold stress, the experimental group treated with light for 24 hours at 4 ℃ plant growth showed the highest expression after 12 hours. In the case of wounding stress, the experimental group treated with the end of the leaf corresponding to one-fourth of the total leaf area was treated hourly, and showed the highest expression after one hour. OsbHLH148 gene induces rapid expression of environmental stress in rice and drought stress resistance of Arabidopsis transformants, suggesting that overexpressing OsbHLH148 gene may be resistant to drought stress.

Example  8: Osbhlh148  Drought Resistance Test of Rice Transformed with Gene

Osbhlh148 In order to determine whether the overexpressed rice is resistant to environmental stress, a drought resistance experiment was performed. Drought conditions were given by four days of transgenic rice and wild type rice grown in a greenhouse for 4 days, and OsbHLH148 transgenic rice was more resistant than wild type rice and returned to water after 10 days. As a result, wild type rice was mostly dried and OsbHLH148 gene-transformed rice was recovered again (FIG. 11).

The photochemical efficiency of PSII was measured by examining the chlorophyll content (Fv / Fm; Fv, variable chlorophyll fluorescence, Fm, maximum chlorophyll fluorescence) to support the drought resistant phenotype of OsbHLH148 gene transgenic rice. As a result, rice transformed with OsbHLH148 gene had 32-48% higher Fv / Fm value than wild type rice (FIG. 12).

<110> SNU R & DB FOUNDATION <120> OsbHLH148 gene enhancing drought stress tolerance of plant and          uses according <130> PN11041 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 900 <212> DNA <213> Oryza sativa <400> 1 atgcaaatgg agtcgtacta cggcgcgttc cacgccgacg aggccgcgtt cttcttcccc 60 caccacgtgc cggcgtcgcc ggagctgccg ttcggcctca ttgcgtcgcc ggagccggag 120 ccggagccgg agcaggcggc ggcggaggcg aggcagagcg cgttccagga gtacggcggc 180 gcggtgcacg ccggagcgcc ggcggcggcg ggggctgtca ccaccggtgg gacgaacatc 240 caccggaggg tgatggacgt gctgggcagg atgggcggcg gcggcggcgg gggggagaag 300 ggggagggtg aggagatgga ggaggaggag gaggtgccgc agcggcggcg gcgcgggcag 360 ggcgccgacg tggagagcag ccgcggtttc cgccacatga tgcgcgagcg ccagcgccgc 420 gagaagctca gccagagcta cgccgacctc tacgccatgg tctcctctcg ctccgagggg 480 gacaagaact cgatcgtgca gtcggcggcg atctacatcc acgagctgaa ggtcgcgagg 540 gaccagctgc agaggaggaa cgaggagctg aaggcccaga tcatgggcca cgacgagcag 600 cagccgtgcg tcacggtgca gttcgaggtc gacgagccgt cgtcgtcgat cgactccatg 660 atcgccgcgc tccggcggct caagagcatg agcgtcaagg cgcgcgggat ccggtcgagc 720 atgtccggga acaggctgtg gacggagatg aacgtcgaga ccacgattgc ggcttgtgaa 780 gtggaaaagg cagtggaaga ggctctcaag gaagtagaga ggaaccagcc tgacagcgat 840 gccccatttc ctggaagcaa aggctggaca cagacatctc atgtgcaaaa tgtgttttga 900                                                                          900 <210> 2 <211> 299 <212> PRT <213> Oryza sativa <400> 2 Met Gln Met Glu Ser Tyr Tyr Gly Ala Phe His Ala Asp Glu Ala Ala   1 5 10 15 Phe Phe Phe Pro His His Val Pro Ala Ser Pro Glu Leu Pro Phe Gly              20 25 30 Leu Ile Ala Ser Pro Glu Pro Glu Pro Glu Pro Glu Gln Ala Ala Ala          35 40 45 Glu Ala Arg Gln Ser Ala Phe Gln Glu Tyr Gly Gly Ala Val His Ala      50 55 60 Gly Ala Pro Ala Ala Ala Gly Ala Val Thr Thr Gly Gly Thr Asn Ile  65 70 75 80 His Arg Arg Val Met Asp Val Leu Gly Arg Met Gly Gly Gly Gly Gly                  85 90 95 Gly Gly Glu Lys Gly Glu Gly Glu Glu Met Glu Glu Glu Glu Glu Val             100 105 110 Pro Gln Arg Arg Arg Arg Gly Gln Gly Ala Asp Val Glu Ser Ser Arg         115 120 125 Gly Phe Arg His Met Met Arg Glu Arg Gln Arg Arg Glu Lys Leu Ser     130 135 140 Gln Ser Tyr Ala Asp Leu Tyr Ala Met Val Ser Ser Arg Ser Glu Gly 145 150 155 160 Asp Lys Asn Ser Ile Val Gln Ser Ala Ala Ile Tyr Ile His Glu Leu                 165 170 175 Lys Val Ala Arg Asp Gln Leu Gln Arg Arg Asn Glu Glu Leu Lys Ala             180 185 190 Gln Ile Met Gly His Asp Glu Gln Gln Pro Cys Val Thr Val Gln Phe         195 200 205 Glu Val Asp Glu Pro Ser Ser Ser Ile Asp Ser Met Ile Ala Ala Leu     210 215 220 Arg Arg Leu Lys Ser Met Ser Val Lys Ala Arg Gly Ile Arg Ser Ser 225 230 235 240 Met Ser Gly Asn Arg Leu Trp Thr Glu Met Asn Val Glu Thr Thr Ile                 245 250 255 Ala Ala Cys Glu Val Glu Lys Ala Val Glu Glu Ala Leu Lys Glu Val             260 265 270 Glu Arg Asn Gln Pro Asp Ser Asp Ala Pro Phe Pro Gly Ser Lys Gly         275 280 285 Trp Thr Gln Thr Ser His Val Gln Asn Val Phe     290 295 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 atgcaaatgg agtcg 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 tcaaaacaca ttttg 15

Claims (11)

The transcription factor OsbHLH148 ( Oryza, which regulates the expression of genes involved in rice-derived stress resistance, consisting of the amino acid sequence of SEQ ID NO: 2) sativa basic helix-loop-helix 148) protein. Gene encoding the OsbHLH148 protein of claim 1. 3. The gene according to claim 2, wherein the gene consists of the nucleotide sequence of SEQ ID NO: 1. The gene of claim 2, wherein the gene has 95% or more sequence homology with 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 5. A method of increasing resistance to environmental stress in a plant, comprising the step of transforming the plant cell with the recombinant vector of claim 5, overexpressing the OsbHLH148 gene. A transgenic plant transformed with the recombinant vector of claim 5 to increase resistance to environmental stress. 10. The transgenic plant of claim 8, wherein said environmental stress is drought, salt or cold. Seed of the transgenic plant according to claim 8. A composition for increasing the resistance of plants to environmental stress, comprising the gene of claim 2.

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