KR20200001845A - A novel gene related to plant drought stress tolerance and use thereof - Google Patents

Abstract

The present invention relates to a method for increasing drought resistance which comprises a step of suppressing the expression of an AtDRUCE1, AtDRUCE2, or AtDRUCE3 gene in plants.

Description

Novel genes associated with dry stress tolerance in plants, and their use {A NOVEL GENE RELATED TO PLANT DROUGHT STRESS TOLERANCE AND USE THEREOF}

The present invention relates to novel genes associated with dry stress tolerance of plants and their use.

Higher plants face a variety of environmental stresses, such as drought, high salt, cold and heavy metals, during their lifetime due to their sedentary nature.

Environmental stresses have a significant effect on crop growth, and dry stress, particularly from water shortages, is one of the major factors in reducing crop production (Boyer, 1982; Ahuja et al., 2010).

Anomalous climatic phenomena are intensifying as the global average temperature rises due to increasing greenhouse gases and deforestation due to industrialization.

The extreme climate is reducing crop yields, which could soon lead to food problems. Even in extreme weather events caused by global warming, water shortages can be directly related to crop yields.

Recent molecular biology and genomics studies have discovered and separated various gene groups related to environmental stress. In addition, the genetic and cellular responses of plants to stress are well known.

However, knowledge of the biological functions of stress-related genes involved in resistance and sensitivity to stress in higher plants is still insufficient, and studies of stress response genes are very important to increase crop productivity.

In this regard, in order to prepare for supply and demand imbalances due to global climate change, research on the short-term and long-term water shortage of plants and research on stress-tolerant plants are being actively conducted, and protein turn-over process through the ubiquitination-26S proteasome system is being conducted. It is known to play an important role in drought stress.

Ubiquitination process consists of a series of reactions by three enzymes classified as E1 ubiquitin-activating enzyme, E2 ubiquitin conjugase, and E3 ubiquitin ligase.

Currently, the mechanism of environmental stress reaction by various E3 ubiquitin ligase has been revealed, but the study on the environmental stress reaction of the higher level E2 ubiquitin conjugase is insufficient.

The present inventors completed the present invention by discovering a new stress-related gene in Arabidopsis thaliana , a model basic plant of plant research, and first identifying gene function through the production of transformed plants.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present invention is to solve the above-mentioned problems of the prior art, an object of the present invention is to provide a method for discovering the function of the genes associated with dry stress, and to improve the resistance to dry stress and thereby to improve plants have.

According to an aspect of the present invention, there is provided a composition for enhancing dry resistance of a plant that inhibits expression of one or more selected from the group consisting of AtDRUCE1, AtDRUCE2, and AtDRUCE3.

In one embodiment, the AtDRUCE1 is composed of the amino acid sequence of SEQ ID NO: 1, the AtDRUCE2 may be composed of the amino acid sequence of SEQ ID NO: 2, the AtDRUCE3 may be composed of the amino acid sequence of SEQ ID NO: 3.

In one embodiment, the composition may comprise a nucleic acid molecule that inhibits the expression of one or more selected from the group consisting of AtDRUCE1, AtDRUCE2, and AtDRUCE3.

In one embodiment, the nucleic acid molecule may inhibit the expression of one or more nucleic acid sequences selected from the group consisting of SEQ ID NO: 4 to 6 or a nucleic acid sequence complementary thereto.

In one embodiment, the nucleic acid molecule may be T-DNA, siRNA, shRNA, miRNA, ribozyme, peptide nucleic acids (PNA) or antisense oligonucleic acid.

In one embodiment, the plant is a food crop including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and millet; Vegetable crops including Arabidopsis, Chinese cabbage, radish, peppers, strawberries, tomatoes, watermelons, cucumbers, cabbages, melons, pumpkins, green onions, onions, and carrots; Special crops, including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanuts and rapeseed; Fruit trees including apple trees, pear trees, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots and bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, and tulips; And fodder crops including lygragrass, red clover, orchardgrass, alpha alpha, tolsque cue and perennial lygragrass.

In one embodiment, the composition comprises at least one nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 3 or a nucleic acid sequence that binds to a nucleic acid sequence complementary thereto to inhibit expression; And a recombinant vector operably linked thereto.

According to another aspect of the present invention, a plant having improved dry resistance transformed with the composition is provided.

According to another aspect of the present invention, there is provided a plant having enhanced dry resistance, in which at least one expression selected from the group consisting of AtDRUCE1, AtDRUCE2, and AtDRUCE3 is suppressed.

In one embodiment, the plant may express a mutated AtDRUCE1 , AtDRUCE2 , or AtDRUCE3 gene.

According to another aspect of the present invention, there is provided a method for enhancing dry resistance of a plant, comprising: inhibiting the expression of AtDRUCE1 , AtDRUCE2 , or AtDRUCE3 genes present in genomic DNA.

In one embodiment, inhibiting the expression of the gene comprises (a) replacing or deleting one or more nucleic acid sequences constituting the gene with another sequence; (b) inserting another sequence into the nucleic acid sequence constituting the gene; Or (c) frame shifting one or more nucleic acid sequences constituting the gene.

In one embodiment, inhibiting the expression of the gene comprises (d) inhibiting constituting the gene; Or (e) inactivating mRNA transcribed from the gene.

According to one aspect of the present invention, by reducing the gene expression to increase the resistance to dry stress can improve the survival rate of the crop according to the abnormal climate, it can contribute to the increase in crop yield.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a structure and sequence analysis of the AtDRUCE protein according to an embodiment of the present invention.
Figure 2 analyzes the E2 ubiquitin conjugase activity of proteins encoded by AtDRUCE1 , AtDRUCE2 , AtDRUCE3 genes. Asterisk means DRUCE-ubiquitin adducts with anti-Myc, anti-ubiquitin antibodies.
Figure 3 analyzes the intracellular location of AtDRUCE1, AtDRUCE2, AtDRUCE 3 .
4 shows DRUCE1 , Of DRUCE2 , DRUCE3 T-DNA loss-of-function mutant alleles are depicted. FIG. 4 (A) shows an exemplary diagram and a T-DNA insertion position in the gene structure DRUCE1, DRUCE2, DRUCE3, Inverted triangles means the T-DNA insertion sites. 4 (B) shows Genotyping PCR results of single and triple loss of function mutant plants. 4 (C) shows DRUCE1 , DRUCE2 , DRUCE3 in wild-type, and mutant plants. RT-PCR results to analyze transcription levels. DRUCE10 was used as loading control.
Figure 5 shows the pore opening and closing of wild-type and transformed plants according to the dry stress.
Figure 6 analyzes the phenotype of wild-type and transformed plants according to the dry stress.

Hereinafter, with reference to the accompanying drawings will be described the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and like reference numerals designate like parts throughout the specification.

When a part is said to "include" a certain component, this means that it may further include other components, without excluding the other components, unless specifically stated otherwise.

Unless defined otherwise, molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing can be performed by conventional techniques commonly used in the field of recombinant DNA within the capabilities of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Unless defined otherwise herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art.

Various scientific dictionaries that include the terms included herein are well known and available in the art. While any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein, some methods and materials have been described. Depending on the context used by those skilled in the art, the present invention is not limited to specific methodologies, protocols, and reagents, because it can be used in various ways.

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

The present inventors have made intensive efforts to identify genes related to dry stress, and as a result, new genes, which have not been previously identified, act as negative regulators of dry stress.

The “dry stress” refers to stress due to water shortage among biological stresses and corresponds to representative stresses that reduce crop yields.

The gene has increased expression in dry stress conditions, and the translation protein of the gene is a protein having E2 ubiquitiin conjugase enzyme activity. The present inventors named the gene “ Atought -Related Ubiquitin-Conjugating Enzyme”.

The AtDRUCE1 may be composed of the amino acid sequence of SEQ ID NO: 1, the AtDRUCE2 may be composed of the amino acid sequence of SEQ ID NO: 2, and the AtDRUCE3 may be composed of the amino acid sequence of SEQ ID NO: 3.

In addition, the AtDRUCE1 gene may be encoded by the nucleic acid sequence of SEQ ID NO: 4, the AtDRUCE2 gene may be encoded by the nucleic acid sequence of SEQ ID NO: 5, and the AtDRUCE3 gene may be encoded by the nucleic acid sequence of SEQ ID NO: 6.

Plants overexpressing the gene showed reduced resistance to dry stress, whereas plants with reduced expression of the gene showed strong resistance to dry stress.

Inhibition of the expression of the gene can be carried out by inactivating the genes inherent in genomic DNA at the DNA level, mRNA level and protein level, in consideration of the characteristics of the plant can be inactivated at the DNA level and mRNA level.

A method of inactivating a gene at the DNA level, comprising: (a) replacing or deleting one or more nucleic acid sequences constituting the gene with another sequence; (b) inserting another sequence into the nucleic acid sequence constituting the gene; Or (c) frame shifting one or more nucleic acid sequences constituting the gene.

A method of inactivating a gene at the RNA level, comprising: (d) inhibiting constructing the gene; Or (e) inactivating mRNA transcribed from the gene.

Specific methods for inactivating the gene may be used without limitation mutation methods commonly used in the art, for example, homologous recombination method using a recombinant vector, single-cross recombination method, T-DNA insertion method and the like.

One aspect of the present invention provides a composition for enhancing dry resistance of a plant that inhibits expression of one or more selected from the group consisting of AtDRUCE1, AtDRUCE2, and AtDRUCE3, the composition may comprise a nucleic acid molecule that inhibits the expression of the protein have.

The composition can effectively enhance the drying resistance of the plant by inhibiting the expression of one or more selected from the group consisting of AtDRUCE1, AtDRUCE2, and AtDRUCE3.

The term “nucleic acids” is meant to encompass DNA (gDNA, cDNA and CDS) and RNA molecules inclusively, and the nucleotides that are the basic structural units in nucleic acid molecules are not only natural nucleotides, but also sugar or base sites. Modified analogues may also be included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

Specifically, the nucleic acid molecule may be, but is not limited to, T-DNA, siRNA, shRNA, miRNA, ribozyme, peptide nucleic acids (PNA) or antisense oligonucleic acid.

The “T-DNA (Tranffered DNA)” is a region of Ti plasmid that is transferred to plant cells when Agrobacterium tumefaciens , a soil bacterium with Ti plasmid, infects a plant. surrounded by a border sequence), cytokine synthetic genes, auxin synthesis genes, or opin synthesis genes, etc., which are involved in tumorigenization of plant cells by Agrobacterium .

The term "small interfering RNA" (siRNA) refers to a small nucleic acid molecule of about 20 nucleotides in size that can mediate RNA interference or gene silencing.

When the siRNA is introduced into the cell, it is recognized by a dicer protein to degrade the biomarker gene and eventually inhibit the expression of the gene. The siRNA is a method for directly chemically synthesizing a known siRNA (Sui G et al., (2002) Proc Natl Acad Sci USA 99: 5515-5520), synthesis of siRNA using transcription in an experimental environment (Brummelkamp TR et al., (2002) Science 296: 550-553), but is not limited thereto.

The “shRNA (short hairpin RNA)” refers to short hairpin RNAs in which the sense and antisense sequences of siRNA target sequences are positioned between loops of 5-9 bases.

The shRNA is to overcome shortcomings such as the high cost of biosynthesis of siRNA, short-term maintenance of RNA interference effect due to low cell transfection efficiency, and the adenovirus, lentivirus and plasmid expression vector systems from the promoter of RNA polymerase. This may be introduced into a cell and expressed, and the shRNA may be converted into an siRNA having an accurate structure by an siRNA processing enzyme (Dicer or Rnase) present in the cell to induce silencing of a target gene.

The “miRNA (microRNA)” is an RNA that plays a role in controlling gene expression in organisms, whereas the miRNA may be composed of 20 to 25 nucleotides, whereas a conventional mRNA is composed of thousands of nucleotides.

The “ribozyme” refers to RNA having an enzyme function that catalyzes a biochemical reaction such as RNA splicing, tRNA synthesis, protein synthesis, and the like.

The "peptide nucleic acids" (PNAs) refer to artificial DNA developed by organic synthesis to compensate for the biochemical instability of DNA.

The term “antisense”, also known as an antisense oligomer, is a backbone between subunits and sequences of nucleotide bases that can hybridize with target sequences in RNA by Watson-Crick base pairing to form mRNA and RNA: oligomeric heterodimers within the target sequence. It means an oligomer having.

The antisense can have exact sequence complementarity or approximate complementarity to the target sequence, block or inhibit translation of the mRNA, and alter the processing of the mRNA to produce splice variants of the mRNA.

In view of variations with biologically equivalent activity, the nucleic acid molecule may be interpreted to include sequences that exhibit substantial identity with the sequences listed in the Sequence Listing.

The substantial identity is at least 60% homology when aligning the sequence of the invention with any other sequence as closely as possible and analyzing the aligned sequence using algorithms commonly used in the art, According to one embodiment it can mean a sequence that shows 70% homology, in some embodiments 80% homology, in certain embodiments 90% homology.

Alignment methods for sequence comparison are known in the art. Various methods and algorithms for the alignment are described in 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).

NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), and is available on the Internet in blastp, blasm, It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx.

The composition may include one or more nucleic acid sequences encoding amino acid sequences selected from the group consisting of SEQ ID NOs: 1 or 3, or nucleic acid sequences that bind to and inhibit expression; And a recombinant vector operably linked thereto.

The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.

By “operably linked” is meant that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by another nucleic acid fragment. It may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation.

By "recombinant" is meant a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid or expresses a protein encoded by a peptide, a heterologous peptide or a heterologous nucleic acid.

The recombinant cell may express a gene or gene fragment which is not found in the natural form of the cell in one of the sense and antisense forms. In addition, the recombinant cell may express a gene found in a cell in a natural state, and the gene may be reintroduced into a cell by artificial means as a modified one.

By "recombinant vector" is meant bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host. Important properties of the expression vector may include origins of replication, promoters, marker genes and translation control elements.

The AtDRUCE1 , AtDRUCE2 , or The AtDRUCE3 gene has been isolated from plants (eg, Arabidopsis) and may serve to enhance the plant's resistance to certain stresses, thus having the most desirable utility for plants.

When the vector is applied to plant cells, any one commonly used in the art for gene introduction of plants can be used, for example, the SP6 promoter, the T7 promoter, the T3 promoter, the PM promoter, the ubiquitin promoter of corn, the curlies Flower Mosaic Virus (CaMV) 35S promoter, nopalin synthase (nos) promoter, Pigwater Mosaic Virus 35S promoter, sugacrein basilisform virus promoter, commelina yellow mottle virus promoter, ribulose-1,5-bis-phosphate Photoinducible promoter of carboxylase small subunit (ssRUBISCO), rice cytosol triosphosphate isomerase (TPI) promoter, adenine phosphoribosyltransferase (APRT) promoter of Arabidopsis and octopine synthase promoter It may include, but is not limited thereto.

The recombinant vector may further comprise a marker gene. The marker gene may be selected from antibiotic resistance gene, herbicide resistance gene, metabolism related gene, light emitting gene, GFP (green fluorescence protein) gene, GUS (βglucuronidase) gene and GAL (β gene).

For example, the marker gene may include a herbicide resistant Bar gene, a neomycin phosphotransferase (NPT II) gene, a hygromycin phosphotransferase gene, a phosphinothricin acetyltransferase gene, or a dihydrofolate reductase gene. It may include.

The vector system may be constructed through various methods known in the art, and specific methods thereof are described in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).

Such vectors are often used in the art, such as plasmids (eg pGA2897, pSK349, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.), phage (ex. Λλ4B, λλΔz1 and M13). Or the like (eg SV40).

In one embodiment, the recombinant vector may be an Agrobacterium binary vector.

The “binary vector” refers to a vector obtained by dividing the plasmid having a gene necessary for transferring a target nucleic acid and a plasmid having a left border (LB) and a right border (RB), which are necessary for migration, from a quantum inducible (Ti) plasmid. do. The Agrobacterium is not particularly limited as long as it is suitable for expression of the nucleic acid sequence.

According to another aspect of the present invention, a plant having improved dry resistance transformed with the composition is provided.

By “transformation” is meant any method of transferring DNA to a plant. The transformation method does not necessarily have a period of regeneration or tissue culture.

Such transformations are common for plant species, including both dicotyledonous plants as well as monocotyledonous plants, and in principle any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells.

The transformation method is a 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 ), 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 of various plant elements (DNA or RNA-coated) (Klein TM et al., 1987, Nature 327, 70), Agrobacte by plant infiltration or transformation of mature pollen or vesicles Infection with virus (incomplete) virus in infection with Leeum Tumorfaciens mediated gene (EP 0 301 316) and the like.

Plant cells into which the vector is introduced are not limited to any particular form as long as they can be regenerated into plants. For example, the cells may comprise cultured cell suspensions, protoplasts, leaf sections and callus.

The plant cells may be cultured according to a conventional method to be liquid cultures, callus, plasma cultures, and may be differentiated to be plant tissues or plants.

The transformed cells into which the recombinant vector is introduced may be re-differentiated into plants through processes such as callus induction, rooting and soil purification using standard techniques known in the art, and asexual propagation methods such as folding, grafting and seed It can be produced by the planetary breeding method using.

Cultivation of plant cells may be carried out by any method known to those skilled in the art, such as cultivation of tissue sections, callus culture of tissue sections, protoplast culture, etc., by culturing parts of plants from the mother and aseptically culturing them under appropriate conditions. The culture conditions and methods may be carried out by conditions and methods known to those skilled in the art.

Differentiating the cultured plant cells into plants is to induce differentiation of cultured plant cells in callus, protoplast form under appropriate conditions to differentiate into plant tissues or plants. Conditions and methods for differentiation are known to those skilled in the art. It can be performed by.

Specifically, the transgenic plant is AtDRUCE1 , AtDRUCE2 , or AtDRUCE3 After transformation with a recombinant vector comprising a gene can be obtained through the process of induction, rooting and soil purification of the callus according to a conventional method.

That is, the AtDRUCE1 , AtDRUCE2 , or Recombinant vectors containing the AtDRUCE3 gene can be introduced into Agrobacterium and transformed into callus using the Agrobacterium, and transgenic plants can be obtained by selecting individuals surviving herbicides.

According to another aspect of the present invention, there is provided a plant having enhanced dry resistance in which at least one expression selected from the group consisting of AtDRUCE1, AtDRUCE2, and AtDRUCE3 is suppressed, and the plant is capable of expressing a mutated AtDRUCE1, AtDRUCE2, or AtDRUCE3 gene. Can be.

The plant is mutated AtDRUCE1 , AtDRUCE2 , or By expressing the AtDRUCE3 gene, dry stress can be significantly enhanced compared to wild type.

The "plant" may include not only mature plants but also plant cells, plant tissues, callus derived from plant cells or tissues, and seeds of plants that may develop into mature plants.

The plant may include food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, and sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, peppers, strawberries, tomatoes, watermelons, cucumbers, cabbages, melons, pumpkins, green onions, onions, and carrots; Special crops, including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanuts and rapeseed; Fruit trees including apple trees, pear trees, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots and bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, and tulips; And fodder crops, including, but not limited to, lygras, red clover, orchardgrass, alphaalpha, tolsfeque and perennial lygragrass.

The method of introducing a specific nucleotide or a plant expression recombinant vector comprising the same into plant cells may be performed by various methods known in the art.

A person skilled in the art can develop a plant by culturing or culturing the transformed plant cell or seed under known and appropriate conditions.

Methods for the development or regeneration of plants from plant protoplasts or various exploits are well known in the art. Development or regeneration of plants comprising foreign genes introduced by the 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).

The plant can be confirmed whether or not transformed by a method known in the art. For example, by performing a PCR using a DNA sample obtained from the tissue of the plant, foreign genes inserted into the genome of the transformed plant can be identified.

Alternatively, Northern or Southern blotting can be performed to confirm transformation (Maniatis, et al., Molecular Cloning, A Laboratory).

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

Experimental Example 1 Acquisition of Gene Isolation

We found AtDRUCE1, AtDRUCE2 , and AB hormones induced by abiotic stress, and AtDRUCE3 gene was isolated from cDNA of Arabidopsis.

Seedlings of 10-day wild-type Arabidopsis pulverized were ground to powder in a mortar and pestle using liquid nitrogen.

Conic tube after extraction with 2ml of extraction buffer (4M guanidine-HCl 20mM, 10mM EDTA, 10mM EGTA (USB), 0.5% sarcosyl (SIGMA, pH 9) and β-mercaptoethanol (SIGMA-ALDRICH) The mixture was mixed with the same amount of PCI (phenol: chloroform: isoamyl alcohol = 25: 24: 1), shaken for 5 minutes, and centrifuged at 3,500 rpm for 25 minutes (Hanil Centrifuge, HA-1000-3).

After centrifugation, the upper organic solvent layer was removed, the same amount of PCI was mixed, and centrifugation was repeated twice. The lower aqueous solution layer was separated using two ethanol precipitation and one LiCl precipitation methods. It was.

By quantification, 2 μg of RNA was raised and single-stranded cDNA was synthesized using dT primer and MMLV reverse transcriptase (Fermentas).

20 ng of cDNA was used as a template for PCR. Two primers were used at 10 pmole, 5 μl of 10 × Taq polymerase buffer (Takara), 8 μl of dNTP mixture (1.25 mM each), and 1 unit of Taq DNA. Polymerase (Takara) was added to make a total volume of 50 μl.

PCR was performed with a Perkin Elmer DNA thermal cycler. After denaturing at 94 ° C. for 2 minutes, 30 seconds at 94 ° C., 30 seconds at 52 ° C., and 1 minute at 72 ° C. were repeated, and this procedure was performed 30 times.

After 30 repetitions, the polymerization was further performed at 72 ° C. for 5 minutes, using AtDRUCE1 , AtDRUCE2 , and electrophoresis. Amplification of the AtDRUCE3 gene was confirmed, and the DNA was sequenced again.

Experimental Example 2 Comparative Analysis of Structure and Sequence of AtDRUCE Protein

The structure of the AtDRUCE protein was plotted and multiple alignments of the analogs were performed.

Arabidopsis thaliana ) DRUCE1, DRUCE2, DRUCE3, yeast ( Saccharomyces cerevisiae ) DRUCE6, and human (homo sapiens) DRUCE6e were aligned using ClustalW.

Domain and similarity were analyzed using SMART (http://smart.embl-heidelberg.de/) and SMS (http://www.bioinformatics.org/sms2/ident_sim.html).

Referring to FIG. 1, the three proteins are E2 belonging to the DRUCE XIV group (Kraft et al. (2005)) and include a conserved DRUCE domain and a transmembrane domain.

Similarity analysis of the cross-protein sequences revealed 49.7% similarity between DRUCE1 and DRUCE2, 50.9% for DRUCE1 and DRUCE3, and 96.2% for DRUCE2 and DRUCE3 in the DRUCE domain with enzymatic activity.

The homology of the protein sequences suggests complementary functions between the three genes.

Experimental Example 3 Analysis of Protein Activity

AtDRUCE1 , AtDRUCE2 , AtDRUCE3 through in vitro E2 activity assay It was confirmed whether the protein encoding the gene functions as an E2 ubiquitin conjugase.

C- terminal membrane domain through the cut-out ^{Myc-DRUCE1ΔC 1 -273, His-} Myc-DRUCE2ΔC 1 -218, His-Myc-DRUCE3ΔC 1-212 after the tablet was expressed in E. coli, wheat UBA1 (E1) and ubiquitin (ubiquitin ) Was added and reacted.

Referring to FIG. 2, the ubiquitin-DRUCE adducts were generated in each protein as a result of the reaction, while the adducts were removed when heated with a reducing reagent DTT.

The results suggest that all three proteins have activity as actual E2 ubiquitin conjugase.

Experimental Example 4 Analysis of Intracellular Location of Proteins

FIG. 3 shows intracellular localization of DRUCE1, DRUCE2, DRUCE3 during transient expression in N. bethanmiana cells.

35S: DRUCE1 - GFP , 35S: DRUCE2 - GFP , 35S: DRUCE3 - GFP construct was co-transfected with tobacco leaves with 35: SP - mRFP -HDEL and protoplast extracted Was observed.

SP-mRFP-HDEL was used as a endoplasmic reticulum marker protein and consists of a single peptide, BIP1 (SP), monomeric RFP (mRFP), ER retention signal motif, and HDEL (Bars = 20 mm).

Analogs of the AtDRUCE protein, ScDRUCE6 (yeast) and HsDRUCE6e (human), are already known as constituent proteins of Endoplasmic Reticulum-Associated Degradation (ERAD) acting on endoplasmic reticulum (Sommer and Jentsch, 1993; Oh et al., 2006). Pole DRUCE1 has also been identified in endoplasmic reticulum (cui et al., 2012).

Tobacco ( Nicotiana) benthamiana ) and the agrobacteria system to analyze the same intracellular location of the protein by expressing the fluorescent protein-bound DRUCE.

Referring to FIG. 3, AtDRUCE1, AtDRUCE2, and AtDRUCE3 were all present in the endoplasmic reticulum and were supposed to function complementarily in the same organelles.

Experimental Example 5 Analysis of Gene Structure and Securing Mutant Plants

Prior to analyzing the phenotype of the genes according to the dry stress, the structure of the AtDRUCE1, AtDRUCE2, AtDRUCE3 genes were analyzed and T-DNA introduced mutant plants were obtained.

Seeds of alleles with T-DNA inserted into the three genes were obtained through ABRC, one for each type (FIG. 4).

DRUCE1 , via genotyping PCR (FIG. 4B) and RT-PCR experiments (FIG. 4C). The loss-of-function of the DRUCE2 and DRUCE3 mutants was confirmed.

In addition, if the three proteins have a complementary function, AtDRUCE1 , AtDRUCE2 , AtDRUCE3 In the triple loss-of-function mutant plants where all of the gene's function is lost, there is a high probability of stronger suppression of the function.

Experimental Example 6: Measurement of stomatalaperture due to dry stress

Phenotype was observed for functional analysis of the genes.

After treatment with ABA (abscisic acid), a well-known plant stress hormone, the susceptibility of wild species and mutants to ABA was analyzed by observing pore opening.

Referring to FIG. 5, when the ABA was treated after the pores were completely opened using the opening solution, the pores of the DRUCE1, DRUCE2, and DRUCE3 loss-of-function mutants were closed more than the wild species.

The DRUCE1DRUCE23DRUCE3 triple loss of function mutant further increased the pore closure effect.

The results suggest that single or triple loss of function mutants have increased sensitivity to ABA, and because ABA is produced by several abiotic stresses, the mutants are expected to increase resistance to dry stress.

Experimental Example 7: Phenotypic analysis according to dry stress

Differences in dry stress phenotype in wild species and mutants were observed.

Wild species, and DRUCE1, DRUCE2, DRUCE3, DRUCE1 DRUCE23 DRUCE3 mutants were grown for about 3 weeks before stopping the water supply for 12 days.

Two days after the water was supplied, the phenotype was observed, and the drought resistance was increased in the DRUCE1, DRUCE2, and DRUCE3 mutants (FIG. 6A).

In particular, the DRUCE1DRUCE23DRUCE3 triple loss-of-function mutant significantly increased drought resistance by more than five times compared to wild species.

Referring to FIG. 6B, the loss of mass due to water loss was analyzed in the case of single and triple loss-of-function mutants relative to wild species.

The results indicate that single-functioning mutant plants lacking the expression of AtDRUCE1 (At3g17000), AtDRUCE2 (At5g50430), or AtDRUCE3 (At1g17280) genes of Arabidopsis group XIV E2 conjugase have increased drought resistance compared to wild species. Since the loss-of-function mutants have a stronger drought resistance, it suggests that crops suitable for regions with insufficient water can be developed by introducing the strong drought resistance of the mutant plants of the gene into various food crops.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the invention is indicated by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the invention.

<110> YONSEI UNIVERSITY <120> A NOVEL GENE RELATED TO PLANT DROUGHT STRESS TOLERANCE AND USE          THEREOF <130> 18PP10489 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 309 <212> PRT <213> Arabidopsis thaliana <400> 1 Met Ala Asp Glu Arg Tyr Asn Arg Lys Asn Pro Ala Val Lys Arg Ile   1 5 10 15 Leu Gln Glu Val Lys Glu Met Gln Ala Asn Pro Ser Asp Asp Phe Met              20 25 30 Ser Leu Pro Leu Glu Glu Asn Ile Phe Glu Trp Gln Phe Ala Ile Arg          35 40 45 Gly Pro Gly Asp Thr Glu Phe Glu Gly Gly Ile Tyr His Gly Arg Ile      50 55 60 Gln Leu Pro Ala Asp Tyr Pro Phe Lys Pro Pro Ser Phe Met Leu Leu  65 70 75 80 Thr Pro Asn Gly Arg Phe Glu Thr Asn Thr Lys Ile Cys Leu Ser Ile                  85 90 95 Ser Asn Tyr His Pro Glu His Trp Gln Pro Ser Trp Ser Val Arg Thr             100 105 110 Ala Leu Val Ala Leu Ile Ala Phe Met Pro Thr Ser Pro Asn Gly Ala         115 120 125 Leu Gly Ser Val Asp Tyr Pro Lys Asp Glu Arg Arg Thr Leu Ala Ile     130 135 140 Lys Ser Arg Glu Thr Pro Pro Lys Tyr Gly Ser Pro Glu Arg Gln Lys 145 150 155 160 Ile Ile Asp Glu Ile His Gln Tyr Ile Leu Ser Lys Ala Thr Val Val                 165 170 175 Pro Lys Pro Leu Pro Leu Glu Cys Ser Gln Ala Pro Ser Ile Val Ser             180 185 190 Glu Ala His Ser Gln Val Glu Pro Gln Glu Ala Ile Thr Val Val Glu         195 200 205 Glu Arg Ser Ile Ala Thr Thr Asp Thr Ile Val Asp Asp Gln Ile Ile     210 215 220 Glu Glu Thr Ala Glu Ala Val Asn Thr Ala Ala Ser Val Val Pro Ala 225 230 235 240 Ala Ala Pro Leu Pro Ala Val Glu Val Val Val Lys Ala Ser Val Ser                 245 250 255 Gly Glu Gln Arg Met Ala Arg Arg Ala Ala Gln Lys Pro Val Asp Asp             260 265 270 Arg Leu Phe Thr Trp Ala Ala Val Gly Leu Thr Ile Ala Ile Met Val         275 280 285 Leu Leu Leu Lys Lys Phe Ile Lys Ser Asn Gly Tyr Ser Thr Gly Phe     290 295 300 Met Asp Asp Gln Ser 305 <210> 2 <211> 243 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Ala Glu Lys Ala Cys Ile Lys Arg Leu Gln Lys Glu Tyr Arg Ala   1 5 10 15 Leu Cys Lys Glu Pro Val Ser His Val Val Ala Arg Pro Ser Pro Asn              20 25 30 Asp Ile Leu Glu Trp His Tyr Val Leu Glu Gly Ser Glu Gly Thr Pro          35 40 45 Phe Ala Gly Gly Phe Tyr Tyr Gly Lys Ile Lys Phe Pro Pro Glu Tyr      50 55 60 Pro Tyr Lys Pro Pro Gly Ile Thr Met Thr Thr Pro Asn Gly Arg Phe  65 70 75 80 Val Thr Gln Lys Lys Ile Cys Leu Ser Met Ser Asp Phe His Pro Glu                  85 90 95 Ser Trp Asn Pro Met Trp Ser Val Ser Ser Ile Leu Thr Gly Leu Leu             100 105 110 Ser Phe Met Met Asp Asn Ser Pro Thr Thr Gly Ser Val Asn Thr Ser         115 120 125 Val Ala Glu Lys Gln Arg Leu Ala Lys Ser Ser Leu Ala Phe Asn Cys     130 135 140 Lys Ser Val Thr Phe Arg Lys Leu Phe Pro Glu Tyr Val Glu Lys Tyr 145 150 155 160 Ser Gln Gln Gln Val Ala Glu Glu Glu Ala Ala Thr Gln Gln Thr Thr                 165 170 175 Thr Ser Glu Asn Gln Asp Phe Pro Gln Lys Asp Asn Ala Lys Val Glu             180 185 190 Ser Glu Lys Ser Val Gly Leu Lys Lys Glu Ser Ile Gln Glu Val Gly         195 200 205 Leu Lys Glu Arg Arg Arg Asn Lys Lys Glu Ala Leu Pro Gly Trp Ile     210 215 220 Val Leu Leu Leu Val Ser Ile Val Gly Val Val Met Ala Leu Pro Leu 225 230 235 240 Leu Gln Leu             <210> 3 <211> 237 <212> PRT <213> Arabidopsis thaliana <400> 3 Met Ala Glu Lys Ala Cys Ile Lys Arg Leu Gln Lys Glu Tyr Arg Ala   1 5 10 15 Leu Cys Lys Glu Pro Val Ser His Val Val Ala Arg Pro Ser Pro Asn              20 25 30 Asp Ile Leu Glu Trp His Tyr Val Leu Glu Gly Ser Glu Gly Thr Pro          35 40 45 Phe Ala Gly Gly Phe Tyr Tyr Gly Lys Ile Lys Phe Pro Pro Glu Tyr      50 55 60 Pro Tyr Lys Pro Pro Gly Ile Thr Met Thr Thr Pro Asn Gly Arg Phe  65 70 75 80 Met Thr Gln Lys Lys Ile Cys Leu Ser Met Ser Asp Phe His Pro Glu                  85 90 95 Ser Trp Asn Pro Met Trp Ser Val Ser Ser Ile Leu Thr Gly Leu Leu             100 105 110 Ser Phe Met Met Asp Thr Ser Pro Thr Thr Gly Ser Val Asn Thr Thr         115 120 125 Val Ile Glu Lys Gln Arg Leu Ala Lys Ser Ser Leu Ala Phe Asn Cys     130 135 140 Lys Thr Pro Ala Phe Arg Lys Leu Phe Pro Glu Tyr Val Glu Lys Tyr 145 150 155 160 Asn Gln Gln Gln Leu Ala Glu Gln Ala Thr Thr Gln Leu Thr Thr Pro                 165 170 175 Glu Ser Pro Gln Lys Ser Asp Thr Lys Val Glu Ser Glu Lys Thr Ile             180 185 190 Asp Pro Thr Lys Gly Asp Ser Glu Gly Gly Leu Lys Glu Arg Lys Lys         195 200 205 Asn Asn Lys Gln Gly Leu Pro Ala Trp Ile Ile Leu Leu Leu Val Ser     210 215 220 Val Phe Gly Val Val Met Ala Leu Pro Leu Leu Gln Leu 225 230 235 <210> 4 <211> 930 <212> DNA <213> Arabidopsis thaliana <400> 4 atggcggatg agaggtataa tcggaagaat ccggcggtga agaggatttt gcaggaggtt 60 aaggagatgc aagctaatcc gtctgatgat tttatgtctc ttcctcttga ggagaatatc 120 tttgagtggc aattcgccat ccgaggtcct ggtgatactg aattcgaggg cgggatttat 180 catgggagaa ttcagttgcc tgcagattat ccattcaaac ctccttcatt catgttattg 240 acccctaacg ggcgctttga aactaacacc aagatttgct tgagcatttc aaactaccac 300 cctgagcatt ggcaaccttc atggagtgtt cggactgctt tggtggctct cattgcattt 360 atgcccacaa gtcccaatgg agcactgggc tcagtagatt atccaaagga tgagagacgt 420 acactagcca ttaaatcacg tgagacacca cctaagtatg gctctcctga acggcaaaaa 480 attattgatg agattcatca gtacatcctt agcaaagcaa ccgtggttcc aaaacctctt 540 cctctggaat gtagccaagc accttccatc gtatcagaag ctcactccca agttgagcca 600 caggaagcga taactgtagt agaagagcgg tccatcgcta caacagacac catagttgat 660 gatcaaatca tagaagagac agctgaagca gtcaatacag cagctagtgt ggttcccgct 720 gcagcacctt taccagcggt tgaagttgtg gtcaaagctt ctgtaagtgg tgagcaaagg 780 atggccagaa gagcggctca gaagccagtc gatgacagac tcttcacgtg ggcggcggtt 840 gggctcacaa tcgctatcat ggttcttctt ctgaagaagt tcataaaatc aaatggttac 900 agtactgggt ttatggatga tcagtcttga 930 <210> 5 <211> 732 <212> DNA <213> Arabidopsis thaliana <400> 5 atggcagaaa aagcttgtat aaagcgtctt caaaaagaat acagagcgct ttgcaaggaa 60 ccagtctcgc atgttgttgc tcgtccttcc ccaaatgaca ttcttgaatg gcattatgtg 120 ttggaaggca gtgagggaac gccttttgca ggtggatttt actatggaaa gatcaagttc 180 cctccagaat atccttacaa gccacctgga atcacaatga ccacaccaaa tggtcgattt 240 gtgacgcaaa agaaaatctg cttgtctatg agtgactttc atccagaaag ctggaatcca 300 atgtggtcag tgtcaagcat acttacagga cttctctcat ttatgatgga taacagcccc 360 acaacgggaa gtgtaaacac tagtgtagcc gagaaacaac ggttggcgaa gtcatctctt 420 gctttcaatt gtaaaagtgt aacattccgg aaactatttc ccgagtatgt cgagaagtac 480 agccagcaac aagtagctga agaagaagca gccactcaac agacaacaac atctgagaat 540 caggattttc ctcagaagga caatgcgaaa gtcgaatcag agaaaagcgt tggtcttaaa 600 aaggaaagta ttcaagaagt gggattgaaa gagaggagaa ggaacaagaa agaagcatta 660 ccgggttgga tagtattgtt gctagtatcg attgtcggtg ttgtaatggc gttgcctttg 720 cttcagctgt ga 732 <210> 6 <211> 714 <212> DNA <213> Arabidopsis thaliana <400> 6 atggcagaaa aggcctgtat aaaacgtctt caaaaggaat atagagcact ttgcaaggaa 60 ccagtctccc atgttgttgc tcgtccttcc ccaaatgaca ttctcgagtg gcattatgtg 120 ctggaaggca gtgagggaac accttttgca ggtggatttt actatggaaa gatcaagttt 180 cctcccgagt atccttacaa gccaccaggg attacaatga ctacaccaaa tggtcgattc 240 atgacacaga agaaaatttg tttatctatg agtgattttc atccagaaag ctggaatccg 300 atgtggtctg tatcaagcat acttacagga cttctctcat ttatgatgga taccagtccg 360 acaactggaa gtgttaacac tactgtaatt gagaaacaac ggttggctaa gtcatctctc 420 gctttcaatt gtaaaacccc agcatttcga aagctatttc cagagtatgt agagaagtac 480 aaccagcagc aactagctga gcaagctacc acacaactga caacaccaga gtctcctcaa 540 aagagcgata ccaaagtcga gtcagagaaa accatcgatc caacaaaggg agattcagaa 600 ggtggcttga aggagaggaa aaagaacaat aaacagggat tgccagcgtg gataatactg 660 ttgctagtgt cggttttcgg tgtggtaatg gcgttgcctc tgcttcaact gtga 714

Claims (13)

Promotes the drying resistance of one or more expression-resistant plants selected from the group consisting of Drought-Related Ubiquitin-Conjugating Enzyme1 (AtDRUCE1), Drought-Related Ubiquitin-Conjugating Enzyme2 (AtDRUCE2), and Drought-Related Ubiquitin-Conjugating Enzyme3 (AtDRUCE3). Composition. The method of claim 1,
The AtDRUCE1 is composed of the amino acid sequence of SEQ ID NO: 1, the AtDRUCE2 is composed of the amino acid sequence of SEQ ID NO: 2, the AtDRUCE3 is composed of the amino acid sequence of SEQ ID NO: 3. The method of claim 2,
AtDRUCE1, AtDRUCE2, AtDRUCE3 A composition comprising a nucleic acid molecule that inhibits the expression of one or more selected from the group consisting of. The method of claim 3,
The nucleic acid molecule is a composition for inhibiting the expression of one or more nucleic acid sequences selected from the group consisting of SEQ ID NO: 4 or a nucleic acid sequence complementary thereto. The method of claim 3,
Wherein said nucleic acid molecule is T-DNA, siRNA, shRNA, miRNA, ribozyme, peptide nucleic acids (PNA) or antisense oligonucleotide. The method of claim 1,
The plant may include food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, and sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, peppers, strawberries, tomatoes, watermelons, cucumbers, cabbages, melons, pumpkins, green onions, onions, and carrots; Special crops, including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanuts and rapeseed; Fruit trees including apple trees, pear trees, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots and bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, and tulips; And fodder crops comprising lygras, red clover, orchardgrass, alpha alpha, tolsque cue and perennial lygras. The method of claim 1,
One or more nucleic acid sequences encoding amino acid sequences selected from the group consisting of SEQ ID NOs. 1 to 3 or nucleic acid sequences that bind to and inhibit expression; And a recombinant vector comprising a promoter operably linked thereto. A plant having enhanced dry resistance transformed with the composition of any one of claims 1 to 7. A plant having enhanced dry resistance with at least one expression suppressed selected from the group consisting of AtDRUCE1, AtDRUCE2, and AtDRUCE3. The method of claim 9,
Mutated AtDRUCE1 , AtDRUCE2 , or Plant expressing the AtDRUCE3 gene. Inhibiting the expression of AtDRUCE1 , AtDRUCE2 , or AtDRUCE3 gene present in genomic DNA; Method of enhancing the dry resistance of a plant comprising a. The method of claim 11,
Inhibiting the expression of the gene comprises (a) replacing or deleting one or more nucleic acid sequences constituting the gene with another sequence; (b) inserting another sequence into the nucleic acid sequence constituting the gene; Or (c) frame shifting one or more nucleic acid sequences constituting the gene. The method of claim 11,
Inhibiting the expression of the gene comprises (d) inhibiting constituting the gene; Or (e) inactivating mRNA transcribed from said gene.

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