KR20180045630A - Novel Gene Related to Plant Drought Stress Tolerance and Use Thereof - Google Patents

Abstract

The present invention provides an OsDPU gene which increases resistance to drought stress in plants and a transgenic rice plant in which an expression of the gene is degraded. OsDPU is a rice-specific E3 ligase protein and the transgenic plant with reduced transcription level of the corresponding genes shows drought resistance. The transgenic rice plant with the degraded expression of OsDPU is expected to contribute to increased crop yields by increasing a survival rate of crops through enhanced resistance to drought stress in plants and will have a very high utility value as crops in response to climate change.

Description

Novel Gene Related to Plant Drought Stress Tolerance and Use Thereof "

The present invention relates to a composition for promoting drought stress tolerance of a plant containing a gene involved in drought stress tolerance.

Due to the increase in greenhouse gases caused by industrialization and the destruction of forests, the average temperature of the Earth rises, and the abnormal weather phenomenon is increasing. As a result, the production of crops is declining, which is likely to lead to food problems (Wang et al., 2001). Among the abnormal climate phenomena caused by global warming, water shortage has a characteristic to be directly related to crop production. Therefore, interest in research and production of transgenic rice plants resistant to drought is increasing (Bae et al., 2011, Cho et al., 2008). E3 ligase is a protein that can induce protein turnover mainly through proteasome by attaching a small protein called ubiquitin to a specific substrate (Kraft et al., 2005; Stone et al., 2005). These E3 ligases are expected to turnover stress proteins in drought stress situations and to be resistant to drought. Therefore, E3 ligase with strong transcription level is selected in drought stress situation in rice, and transcription of selected E3 ligase Transgenic plants with reduced levels were produced.

The present inventors first extracted the stress-related U-box type E3 ligase, OsDPU (Drought induced Plant U-box type E3 Ligase), from rice and investigated gene function through research and transgenic plant production.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

1. Bae, H., Kim, S. K., Cho, S. K., Kang, B.G., and Kim, W.T. (2011) Overexpression of OsRDCP1, a rice RING domain-containing E3 ubiquitin ligase, increased tolerance to drought stress in rice (Oryza sativa L.). Plant Sci. 180, 775-782. 2. Arabidopsis PUB22 and PUB23 are homologous U-box E3 ubiquitin ligases that play combinatory roles in response to drought stress. Plant Cell. 20, 1899-1914. 3. Kraft, E., Stone, SL, Ma, L., Su, N., Gao, Y., Lau, OS, Deng, XW, and Callis J. (2005) Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol. 139,1597-1611. 4. Rice-type ubiquitin ligase family of Arabidopsis, Plant Physiology, J. of the Department of Plant Physiology, Hankuk University of Agriculture, 137, 13-30 5. Wang, W. X., Vinocus, B., Shoseyov, O., and Altman, A. (2001) Biotechnology of plant osmotic stress tolerance: physiological and molecular considerations. Acta Hort., 560, 285-292.

Therefore, the inventors of the present invention have sought to improve the productivity of plants by identifying the genes capable of improving tolerance to drought stress in plants. As a result, it has been found that when the expression of OsDPU (Drought induced Plant U-box type E3 Ligase) gene of rice is lowered, the above-described enhanced tolerance to drought stress can be obtained.

Accordingly, an object of the present invention is to provide a composition for enhancing drought stress tolerance of a plant comprising a nucleic acid molecule that inhibits the expression of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 .

(A) a nucleotide sequence which binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or the sequence of SEQ ID NO: 1 and inhibits the expression of the amino acid sequence of SEQ ID NO: 2; (b) a promoter that is operatively linked to the nucleotide sequence and that forms RNA molecules in plant cells; And (c) a recombinant vector for plant expression comprising a poly A signal sequence that acts on plant cells to cause polyadenylation of the 3'-terminal of the RNA molecule. The present invention also provides a composition for enhancing drought stress tolerance of a plant .

It is another object of the present invention to provide a plant cell having improved drought stress tolerance transformed with the composition of the present invention.

It is another object of the present invention to provide a plant having enhanced drought stress tolerance transformed with the composition of the present invention.

It is another object of the present invention to provide a method for enhancing drought stress tolerance of a plant, which comprises introducing the composition of the present invention into plant cells.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Accordingly, the present invention provides a composition for enhancing drought stress tolerance of a plant comprising a nucleic acid molecule that inhibits the expression of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

According to a preferred embodiment of the present invention, the nucleotide sequence may be a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the sequence of SEQ ID NO: 1.

According to another preferred embodiment of the present invention, the nucleic acid molecule may be T-DNA, siRNA, shRNA, miRNA, ribozyme, PNA (peptide nucleic acids) or antisense oligonucleotide.

According to another preferred embodiment of the present invention, the nucleic acid molecule may be T-DNA.

According to another preferred embodiment of the present invention, the plant may be a terminal plant.

According to another preferred embodiment of the present invention, the plant may be rice flour.

(A) a nucleotide sequence which binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or the sequence of SEQ ID NO: 1 and inhibits the expression of the amino acid sequence of SEQ ID NO: 2; (b) a promoter that is operatively linked to the nucleotide sequence and that forms RNA molecules in plant cells; And (c) a recombinant vector for plant expression comprising a poly A signal sequence which acts on plant cells to cause polyadenylation of the 3'-terminal of the RNA molecule. The present invention also provides a composition for enhancing drought stress tolerance of a plant.

The present invention also provides a plant cell having enhanced drought stress resistance transformed with the composition of the present invention.

The present invention also provides a plant having enhanced resistance to drought stress induced by the composition of the present invention.

Also, the present invention provides a method for enhancing drought stress tolerance of a plant, comprising introducing the composition of the present invention into plant cells.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide a transgenic rice plant having reduced expression of OsDPU gene and a heterologous gene which increases tolerance to drought stress.

 The plants of the present invention are expected to increase the survival rate of crops through improved tolerance to drought stress by lowering the expression of OsDPU gene which increases tolerance to drought stress and are expected to contribute to the increase of crop yield, . ≪ / RTI >

In addition, the present invention provides a plant improved in drought stress tolerance through knock-down of the gene, and a CRISPR / CAS9 system, which is a technique for solving the side effects of a recombinant plant, And thus it is expected that the same non-GMO crop resistant to drought stress as the above gene can be provided.

1A shows a CDS sequence diagram of OsDPU.
FIG. 1B shows the results of an experiment in which the level of mRNA expression of OsDPU in an abiotic stress condition was confirmed.
FIG. 2A shows a schematic diagram of a border portion of a binary vector pGA2897 prepared to produce OsDPU-expressing plant. HPT: Hygromycin resistant gene
FIG. 2b shows the result of genomic Southern blot analysis to select lines of the transgenic plants.
FIG. 3A shows the results of RT-PCR to determine whether the OsDPU mRNA levels of the two independent line OsDPU-expressing transgenic plants were actually reduced under normal conditions and when the drought stress was treated .
Figure 3b is a photograph of wild type (WT) and pUbi: RNAi-OsDPU plant lines # 1 and # 2.
Figures 3c and 3d show the results of planting two types of wild type (WT) and pUbi: RNAi-OsDPU plant lines # 1 and # 2, respectively, in one flower pot, growing for about 6 weeks and giving drought stress for 5 days to 7 days .

Hereinafter, the present invention will be described in more detail.

As described above, in the case of a new variety genetically modified organism (GMO) generally developed by genetic recombination technology, side effects such as stability and ecosystem disturbance appeared, and a new alternative plant was needed for this.

Therefore, the present inventors sought to solve the above-mentioned problems by providing a gene capable of improving resistance to drought stress in plants. As a result, it was confirmed that the knock-down plant of the present invention was resistant to drought stress, and the same effect was obtained when the knock-out plant was made using the CRISPR / CAS9 system, which is a non-GMO production technique I can expect to see you. Therefore, there is an effect of providing a plant which can increase crop yield by increasing tolerance to drought stress, without any side effects such as stability and ecosystem disturbance, which are side effects of conventional recombinant plants.

Unlike animals, plants do not have the ability to move away from these stresses, so they have a sophisticated internal control system to actively cope with stress. Should be established. The stress experienced by a plant is divided into biological stress derived from insect pests and abiotic stress derived from the environment. In particular, the term "drought stress" of the present invention refers to drought during abiotic stress, And are representative stresses that reduce crop yields.

The present invention provides a composition for enhancing drought stress tolerance of a plant comprising a nucleic acid molecule that inhibits the expression of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

The nucleotide sequence may be a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the sequence of SEQ ID NO:

According to the present invention, the expression of the nucleotide sequence of SEQ ID NO: 1 of the present invention is increased when a drought, a high salt or a water-deficient stress is applied after various abiotic stresses are applied to a plant, There was no increase in expression when ABA was added. Despite these expression patterns, it was confirmed that the suppression of the expression of the gene significantly increased the survival rate even in a drought stress environment as demonstrated in the following examples. The present inventors named this gene OsDPU (Drought induced Plant U-box type E3 Ligase). Specifically, as shown in FIG. 3D, it was confirmed that the survival rate of the plant with low expression of the gene was greatly increased even in the environment where drought stress was applied.

The nucleotide sequence used in the present invention is preferably, but not limited to, the nucleotide sequence described in the attached Sequence Listing.

Variations in nucleotides do not cause changes in the protein. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (e.g., by codon degeneration, six codons for arginine or serine), or codons that encode biologically equivalent amino acids ≪ / RTI >

The nucleic acid molecule may be a molecule such as T-DNA, siRNA, shRNA, miRNA, ribozyme, PNA (peptide nucleic acids) or antisense oligonucleotide.

The term "nucleic acids" of the present invention has the meaning inclusive of DNA (gDNA, cDNA and CDS) and RNA molecules, and nucleotides which are basic constituent units in nucleic acid molecules are not only natural nucleotides, (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

The term "T-DNA (Tranffered DNA) " of the present invention refers to a region of a Ti plasmid that migrates to plant cells when the soil bacteria Agrobacterium tumefaciens with a Ti plasmid is infected with plants, and has an incomplete linear repeat arrangement of 25 base pairs Border sequence, and border sequence). On the T-DNA, cytokine synthesis gene, auxin synthesis gene or opine synthesis gene related to the in vitro transplantation of plant cells by Agrobacterium exist.

The term "siRNA (small interfering RNA) " of the present invention means a small nucleic acid molecule of about 20 nucleotides in size that can mediate RNA interference or gene silencing. When siRNA is introduced into a cell, dicer protein and degrades the biomarker gene, thereby inhibiting gene expression. Such siRNA may be prepared by a direct chemical synthesis of known siRNAs (Sui G et al., (2002) Proc Natl Acad Sci USA 99: 5515-5520), a synthetic method of siRNA using transcription in laboratory conditions (Brummelkamp TR et al., 2002 ) Science 296: 550-553), but the present invention is not particularly limited thereto.

The term "shRNA (short hairpin RNA)" of the present invention means a short hairpin RNA having a sense and antisense sequence of siRNA target sequence located between loops composed of 5-9 bases . The shRNA is used to overcome the disadvantages of high cost biosynthesis cost of siRNA, short time maintenance of RNA interference effect due to low cell transfection efficiency, and the like by using adenovirus, lentivirus and plasmid expression vector system from the RNA polymerase promoter This shRNA can be used to induce silencing of the target gene by converting it into an siRNA having the correct structure by the siRNA processing enzyme (Dicer or Rnase) present in the cell.

The term "miRNA (microRNA)" of the present invention refers to an RNA that plays a role in regulating gene expression in an organism, while a normal mRNA is composed of several thousand nucleotides, while a miRNA is composed of 20 to 25 nucleotides have.

The term "ribozyme" of the present invention means RNA having an enzyme function that catalyzes a biochemical reaction such as RNA splicing, tRNA synthesis, and protein synthesis.

The term "PNA (peptide nucleic acids) " of the present invention means artificial DNA developed by organic synthesis to complement biochemical instability of DNA.

The term "antisense" of the present invention is also referred to as an antisense oligomer, and includes a sequence of a nucleotide base capable of hybridizing with a target sequence in RNA by Watson-Crick base pair formation to form an RNA: oligomer heterodimer in the target sequence, Means an oligomer having a backbone between subunits. The antisense can have an exact sequence complement or approximate complement to the target sequence, block or inhibit the translation of the mRNA, and alter the processing of the mRNA producing splice variants of the mRNA. Thus, the antisense of the present invention may preferably be an antisense oligomer complementary to the polynucleotide of the biomarker gene of the present invention. The antisense can be used in a manner that prevents or suppresses the expression of a carcinogen gene by administration to a subject in a conventional manner. For example, a method of mixing an antisense oligodeoxynucleotide with a poly-L-lysine derivative by electrostatic attraction and administering the mixture to a vein of a subject (JS Kim et al., J controlled Release 53, 175-182 , 1998) may be used but are not particularly limited thereto.

Given the mutation having the above-mentioned biological equivalent activity, the nucleic acid molecule used in the present invention coding for the abiotic stress-inducible OsDPU protein (SEQ ID NO: 1) exhibits substantial identity with the sequence described in the sequence listing Sequence is also included. The above-mentioned substantial identity is determined by aligning the sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. 60% homology, 70% homology according to one embodiment, 80% homology according to some embodiments, and 90% homology according to a particular embodiment.

Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

(A) a nucleotide sequence which binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or the sequence of SEQ ID NO: 2 and inhibits the expression of an amino acid of the second sequence of the sequence listing; (b) a promoter that is operatively linked to the nucleotide sequence and that forms RNA molecules in plant cells; And (c) a recombinant vector for plant expression comprising a poly A signal sequence which acts on plant cells to cause polyadenylation of the 3'-terminal of the RNA molecule. The present invention also provides a composition for enhancing drought stress tolerance of a plant.

The term "operably linked" of the present invention means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, Will control the transcription and / or translation of the other nucleic acid sequences.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this can be found in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

The vectors that can be used in the present invention include plasmids such as pGA2897, pSK349, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19 which are frequently used in the art, phages ? 4B,? -Charon,? Z1 and M13, etc.) or viruses (e.g. SV40 and the like).

The gene of the present invention has been isolated from a plant (for example, rice) and has the most favorable utility for plants since it can act to enhance plant tolerance to drought stress. Therefore, when the vector of the present invention is applied to a plant cell, any promoter suitable for the present invention may be any of those conventionally used in the art for gene transfer of a plant. For example, SP6 promoter, T7 promoter , The T3 promoter, the PM promoter, the ubiquitin promoter of corn, the Cauliflower Mosaic Virus (CaMV) 35S promoter, the nopaline synthase (nos) promoter, the Piguet mosaic virus 35S promoter, the water crane basiliform virus promoter, the combine melanogaster virus Promoter, ribofluose-1, 5-bis-phosphate carboxylase small subtilt (ssRUBISCO) light-inducible promoter, rice cytosolic triosulfate isomerase (TPI) promoter, adenine phosphoribosyl transferase (APRT) promoter and an octopine synthase promoter The. According to a specific embodiment of the present invention, the promoter used in the present invention is a cauliflower mosaic virus (CaMV) 35S promoter.

According to one embodiment of the present invention, the poly A signal sequence causing the 3'-terminal polyadenylation according to the present invention is derived from the nopaline synthase gene of Agrobacterium tumefaciens (NOS 3 'end (Bevan et al. Nucleic Acids Research, 11 (2): 369-385 (1983)), derived from the tryptophan synthase gene of Agrobacterium tumefaciens, protease inhibitor I of tomato or potato 3 'terminus, the CaMV 35S terminator and the OCS terminator (octopine synthase terminator) sequence. According to a particular embodiment of the invention, the 3'-terminal polyA signal sequence which results in polyadenylation in accordance with the invention is an OCS terminator.

Alternatively, the vector may further carry a gene encoding a reporter molecule (e.g., luciferase and beta -glucuronidase). In addition, the vector of the present invention may be used as a selection marker, such as an antibiotic (e.g. neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) resistance gene (for example, neomycin phosphotransferase (npt) Mycophosphotransferase (hpt), and the like).

According to one embodiment of the present invention, the recombinant vector for plant expression of the present invention is an Agrobacterium binary vector.

The term "binary vector" of the present invention includes a plasmid having a left border (RB) and a right border (RB) necessary for movement in a Ti (tumor inducible) plasmid and a plasmid having a gene necessary for transferring a target nucleotide Vector illustration. The transforming Agrobacterium of the present invention may be any strain suitable for expression of the nucleotide sequence of the present invention. According to a specific embodiment of the present invention, Agrobacterium strain for plant transformation in the present invention is Agrobacterium Agrobacterium tumefaciens GV3101.

Methods for introducing the recombinant vectors of the present invention into Agrobacterium can be carried out through various methods known to those skilled in the art and include, for example, particle bombardment, electroporation, transfection A lithium acetate method, a heat shock method, and a freeze-thaw method. However, the present invention is not limited thereto. According to certain embodiments of the present invention, electroporation is used.

The present invention also provides a plant cell having enhanced drought stress resistance transformed with the composition of the present invention.

The present invention also provides a plant having enhanced drought stress tolerance transformed with the composition of the present invention.

The term "plant (or plant) " of the present invention is understood not only to include mature plants but also plant cells, plant tissues, plant cells or tissues, seeds of calli and plants derived from tissues and the like which develop into mature plants.

The plants to be transformed in the present invention include food crops including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats and millet; Vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep, grapes, citrus, persimmon, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops including ragras, red clover, orchardgrass, alpha-alpha, tall fescue and perennialla grasses. According to one embodiment of the present invention, the plant to be transformed in the present invention is a monocotyledon. According to a specific embodiment of the present invention, the plant to be transformed in the present invention is rice (Orysa sartiva L.).

Methods for introducing a specific nucleotide or a recombinant vector for plant expression including the same into a plant cell can be carried out by various methods known in the art.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent such as a metabolic inhibitor, an antibiotic and a herbicide. Plant cells that stably contain a marker gene that is transformed and conferring selectative resistance are grown and divided in the above cultures. Exemplary labels include, but are not limited to, the hygromycin phosphotransferase gene, the glycophosphate tolerance gene, and the neomycin phosphotransferase (nptII) system. Plasmids can be transformed by inserting exogenous polynucleotides into vectors such as plasmids and viruses, and Agrobacterium bacteria can be used as a mediator (Chilton et al. Cell 11: 263: 271 (1977), USA (1985), and the direct exogenous polynucleotide can be introduced into plant cells to transform plants (Lorz et al., Mol. Genet. 199: 178-182; ). For example, electroporation (Neumann, E., et al., EMBO J., 1: 841 (1982)), microparticle bombardment , Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and polyethylene glycol-mediated uptake. Generally, a method widely used for transforming plants is a method of infecting plant cells or seeds with Agrobacterium tumefaciens transformed with an exogenous polynucleotide (see U.S. Patent No. 5,004,863, 5,349,124 and 5,416,011).

One of ordinary skill in the art can cultivate or plant transformed plant cells or seeds under suitable known conditions to develop into plants. Methods for the development or regeneration of plants from plant protoplasts or from various expansions are well known in the art. Development or regeneration of plants containing foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (see US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

According to one embodiment of the present invention, the transformation of the present invention is carried out using an Agrobacterium-mediated Agrobacterium-mediated system. According to another embodiment, Agrobacterium tumefaciens-binary vector (Depicker, A., et al., Plant cell transformation by Agrobacterium plasmids. In Genetic Engineering of Plants, Plenum Press, New York (1983)).

Infection of the explant by Agrobacterium tumefaciens includes methods known in the art. According to an embodiment of the present invention, the infection process includes a step of culturing Agrobacterium tumefaciens culture with rice callus. Through this, Agrobacterium tumefaciens is infected into plants.

Explants transformed by Agrobacterium tumefaciens are regenerated in regeneration medium, which ultimately forms transgenic plants.

The transformed plants according to the present invention are confirmed to be transformed by methods known in the art. For example, PCR using a DNA sample obtained from the tissue of a transformed plant can identify a foreign gene inserted into the genome of the transgenic plant. Alternatively, Northern or Southern blotting can be performed to confirm the transformation (Maniatis, et al., Molecular Cloning, A Laboratory

(Methods of Enzymology, Vol. 153, (1987)) for producing transformed plant cells and transgenic plants of the present invention.

Since the transgenic plant cells and plants of the present invention are produced using the vectors of the present invention described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification

According to another aspect of the present invention, there is provided a method for enhancing drought stress tolerance of a plant, comprising introducing the composition of the present invention into plant cells.

Since the method of the present invention shares the aforementioned composition with related amino acid sequences, nucleotide sequences, and recombinant vectors for plant expression comprising them, common in the context of the above compositions, It is omitted.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

[Example]

Example 1. Ubi: Production of Transgenic Rice Plants Decreased in Expression of RNAi-OsDPU

The plasmid was transformed into Agrobacterium tumefaciens LBA4404 in order to express the transfection vector pGA2897 produced in the rice plant to lower the expression of the OsDPU gene.

The seeds of Dongjinbyeo, a wild species, were sterilized in 40% lactose for 30 minutes, washed with sterilized water 5 times or more, and then washed with 3% sucrose, CHU medium (4 g / L, Duchedfa Biochem), casamino acid Callus was induced in N6D medium consisting of L-proline (2.9 g / L), 2,4D (2 mg / L) and 0.002% gelite. The callus of the induced rice was transformed with the previously transformed Agrobacterium tumefaciens LBA4404 and the transformed callus was selected using hygromycin B antibiotic. Transgenic plants were obtained from selected callus.

Example 2 Expression of OsDPU Gene Expression in Transgenic Rice Plants Undergone OsDPU RNAi Expression

Freeze with liquid nitrogen and mix well with wild-type and OsDPU RNAi-expressing transgenic rice plant leaves with 2 ml / g of RNA extraction buffer. Add 200 μl of chloroform to the mixture, mix again, centrifuge at 4 to 13000 rpm, and mix the supernatant with equal volume of precipitation buffer. The total RNA was extracted by centrifuging the mixed solution at 4 to 13000 rpm.

Single-stranded cDNA was synthesized by oligo dT primer and TOPscript reverse transcriptase (enzynomics) according to the manufacturer's instructions to synthesize cDNA from 2 μg of total RNA per sample. RT-PCR was performed using the synthesized cDNA as a template. The primers used for the analysis are shown in Table 1.

* Primer used for RT-PCR to confirm transcription level and expression of OsDPU mRNA, primer used for vector construction for transgenic plant, primer sequence used for genomic Southern blot to select independent line to be.

As shown in Fig. 1B, OsUBQ10 was used as a loading control and OsRab16b was used as a positive control. The increased level of OsRab16b mRNA indicates that the rice was well treated with abiotic stress. OsDPU showed no change in mRNA expression level at low temperature stress and ABA hormone, but the amount of mRNA expression was strongly increased in PEG, NaCl, and Drought stress.

Example 3: Ubi: RNAi-OsDPU Expression Decrease Independent Line Selection of Transgenic Rice Plants

3-1. Genomic DNA isolation and genomic Southern blot

Wild type and OsDPU RNAi expression reduction Genomic DNA extracted from the leaves of transgenic rice plants using the CTAB method is cut with BamHI restriction enzyme for 12 hours. It is electrophoresed on 0.8% agarose gel and transferred to nylon membrane. Hybridization was performed using HPT probes labeled with 32P on this membrane, and independent lines were selected by sensitizing them to IP plates.

As a result, it was confirmed that OsDPU mRNA level was decreased in the plants with decreased OsDPU expression under both normal and drought stress conditions. As shown in Fig. 3a, OsPU mRNA levels were more decreased in line # 1 (wild type) than in line # 2 (pUbi: RNAi-OsDPU).

As shown in FIGS. 3C and 3D, pUbi: RNAi-OsDPU # 1 was 40% (FIG. 3C) when the wild type survived to about 15% , and pUbi: RNAi-OsDPU # 2 survived 30% (FIG. 3D).

<110> Industry-Academic Cooperation Foundation. Yonsei University <120> Novel Gene Related to Plant Drought Stress Tolerance and Use          Thereof <130> 1061331 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> CDS of Drought induced Plant U-box type E3 Ligase <400> 1 atggcgctgc tggcgcggag ggcgcggaag gcggtgatgg cgaaggcgcc ggcgccgctg 60 ctgcagaaga ggggtggcgg cgcggcggcg gagctggcga tcccggcgca cttccggtgc 120 ccgatctcgc tggacctgat gcgcgacccg gtgacggcgc cgacggggat cacgtacgac 180 agggagggga tcgaggcgtg gctggacacc gggcgcgcgg tgtgccccgt cacccacgcg 240 ccgctccggc acgaggacct cgtccccaac cacgccatcc gccgcgtcat ccaggactgg 300 tgcgtcgcca accggtcccg cggcgtggag cgcatcccca cgcccaagat ccccgtcacg 360 cccgtgcagg cctccgagct gctgttcgac gtcgcggagt cggcggcgcg gcgcggcgcg 420 gcggggcgcg ccgccggggc ggtcgccagg gtccgggcgc tcgcgaggga cagcgagcgg 480 aaccggcggt gcttcgtgtc cgtcggcacg gggcgcgtgc tggccgcggc gttcgagtct 540 ctcgccgccg ccggggaggc gggcgttctc gaggacgtcc tggcggcatt ggtctgcatg 600 atgccgttgg acgaggaggc ggccagggtc ttggcctcgt ctagctcgat gggttcactc 660 gtcgccattg ccaagcacgg gagcttggcc ggaaggctga acgctgtgct ggcgatcaag 720 gaggccgtgt cgcgggacgg agcgttcgtg gacctggcgg atgacaaggt cgacaaggtc 780 gtcgacgcgc tggtcgtgat catcaaggct ccgatctgcc cgcaggccac caaggctgcc 840 atggtcgcca cctaccacct ggcgagctcc gacgagcgcg tcgcggcgag ggtagcatcc 900 acggggctcg tcccaacgct catcgaggcc ctcgtagacg ccgacaagag cgtgtccgag 960 aaggccctgg cggtgctcga cgcaatgctc gcctcggagg aaggccgcgc gagcgcgcgg 1020 ggccacgccc tcgcaatgcc ggccctcgtg aagaagatgt tccgcgtgtc cgacgtggcc 1080 accgagctcg ccgtgtcggc gatgtggcgg ctcggctgca aggcctcctc cggcgacgag 1140 gaggccgccg cgacggggtg cctggtcgag gcgctgcgtg tgggcgcgtt ccagaagctt 1200 ctcctcctcc tgcaggtggg ctgcagggac gcgaccaagg agaaggccac tgagctgctc 1260 aagatgctca acaagcacaa agggttgggg gagtgtgtcg acgctgtgga tttcagaggg 1320 ctcaataggc tgtcatga 1338 <210> 2 <211> 445 <212> PRT <213> Artificial Sequence <220> <223> Drought induced plant U-box type E3 ligase <400> 2 Met Ala Leu Leu Ala Arg Arg Ala Arg Lys Ala Val Met Ala Lys Ala   1 5 10 15 Pro Ala Pro Leu Leu Gln Lys Arg Gly Gly Gly Ala Ala Ala Glu Leu              20 25 30 Ala Ile Pro Ala His Phe Arg Cys Pro Ile Ser Leu Asp Leu Met Arg          35 40 45 Asp Pro Val Thr Ala Pro Thr Gly Ile Thr Tyr Asp Arg Glu Gly Ile      50 55 60 Glu Ala Trp Leu Asp Thr Gly Arg Ala Val Cys Pro Val Thr His Ala  65 70 75 80 Pro Leu Arg His Glu Asp Leu Val Pro Asn His Ala Ile Arg Arg Val                  85 90 95 Ile Gln Asp Trp Cys Val Ala Asn Arg Ser Ser Gly Val Glu Arg Ile             100 105 110 Pro Thr Pro Lys Ile Pro Val Thr Pro Val Gln Ala Ser Glu Leu Leu         115 120 125 Phe Asp Val Ala Glu Ser Ala Ala Arg Arg Gly Ala Ala Gly Arg Ala     130 135 140 Ala Gly Ala Val Ala Arg Val Ala Ala Leu Ala Arg Asp Ser Glu Arg 145 150 155 160 Asn Arg Arg Cys Phe Val Ser Val Gly Thr Gly Arg Val Leu Ala Ala                 165 170 175 Ala Phe Glu Ser Leu Ala Ala Ala Gly Glu Ala Gly Val Leu Glu Asp             180 185 190 Val Leu Ala Ala Leu Val Cys Met Met Pro Leu Asp Glu Glu Ala Ala         195 200 205 Arg Val Leu Ala Ser Ser Ser Ser G Met Ser Ser Val Val Ala Ile Ala     210 215 220 Lys His Gly Ser Leu Ala Gly Arg Leu Asn Ala Val Leu Ala Ile Lys 225 230 235 240 Glu Ala Val Ser Arg Asp Gly Ala Phe Val Asp Leu Ala Asp Asp Lys                 245 250 255 Val Asp Lys Val Val Asp Ala Leu Val Val Ile Ile Lys Ala Pro Ile             260 265 270 Cys Pro Gln Ala Thr Lys Ala Ala Met Val Ala Thr Tyr His Leu Ala         275 280 285 Ser Ser Asp Glu Arg Val Ala Ala Arg Val Ala Ser Thr Gly Leu Val     290 295 300 Pro Thr Leu Ile Glu Ala Leu Val Asp Ala Asp Lys Ser Val Ser Glu 305 310 315 320 Lys Ala Leu Ala Val Leu Asp Ala Met Leu Ala Ser Glu Glu Gly Arg                 325 330 335 Ala Ser Ala Arg Gly His Ala Leu Ala Met Pro Ala Leu Val Lys Lys             340 345 350 Met Phe Arg Val Ser Asp Val Ala Thr Glu Leu Ala Val Ser Ala Met         355 360 365 Trp Arg Leu Gly Cys Lys Ala Ser Ser Gly Asp Glu Glu Ala Ala Ala     370 375 380 Thr Gly Cys Leu Val Glu Ala Leu Arg Val Gly Ala Phe Gln Lys Leu 385 390 395 400 Leu Leu Leu Gln Val Gly Cys Arg Asp Ala Thr Lys Glu Lys Ala                 405 410 415 Thr Glu Leu Leu Lys Met Leu Asn Lys His Lys Gly Leu Gly Glu Cys             420 425 430 Val Asp Ala Val Asp Phe Arg Gly Leu Asn Arg Leu Ser         435 440 445

Claims (10)

A composition for promoting tolerance to drought stress in a plant comprising a nucleic acid molecule that inhibits the expression of a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 2.
2. The composition of claim 1, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary to the sequence of SEQ ID NO:
2. The composition of claim 1, wherein the nucleic acid molecule is T-DNA, siRNA, shRNA, miRNA, ribozyme, PNA (peptide nucleic acids) or antisense oligonucleotides.
4. The composition of claim 3, wherein the nucleic acid molecule is T-DNA.
The composition according to claim 1, wherein the plant is a monocotyledon.
6. The composition of claim 5, wherein the plant is rice.
(a) a nucleotide sequence which binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or the sequence of SEQ ID NO: 1 and inhibits expression of the amino acid sequence of SEQ ID NO: 2; (b) a promoter that is operatively linked to the nucleotide sequence and that forms RNA molecules in plant cells; And (c) a recombinant vector for plant expression comprising a poly A signal sequence that acts in plant cells to cause polyadenylation of the 3'-terminal of the RNA molecule.
A plant cell having enhanced drought stress tolerance transformed with the composition of any one of claims 1 to 7.
A plant having enhanced drought stress tolerance transformed with the composition of any one of claims 1 to 7.
A method for enhancing drought stress tolerance of a plant, comprising introducing the composition of any one of claims 1 to 7 into a plant cell.

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