KR101197465B1 - OsABF1 gene from Oryza sativa and uses thereof - Google Patents

Abstract

The present invention relates to stress resistance related to OsABF1 ( Oryza) derived from rice sativa abscisic acid responsive element-binding factor 1) protein, gene encoding the protein, recombinant vector containing the gene, host cell transformed with the recombinant vector, plant cell by transforming the recombinant vector to stress resistance of the plant To enhance the stress resistance of the transformed plant and its seed produced by the method, a method for producing a stress-resistant transformed plant using the recombinant vector and to improve the stress resistance of the plant comprising the gene It relates to a composition.

Description

OsABF1 gene from Oryza sativa and uses approximately}

The present invention relates to a rice-derived OsABF1 protein and genes encoding the same, and more specifically, to rice-derived stress-related OsABF1 ( Oryza sativa abscisic acid responsive element-binding factor 1) protein, gene encoding the protein, recombinant vector containing the gene, host cell transformed with the recombinant vector, plant cell by transforming the recombinant vector to stress resistance of the plant To enhance the stress resistance of the transformed plant and its seed produced by the method, a method for producing a stress-resistant transformed plant using the recombinant vector and to improve the stress resistance of the plant comprising the gene It relates to a composition.

Plant growth and productivity are greatly affected by environmental stresses such as drying, high salt and low temperatures. When exposed to abiotic stress conditions, plants undergo various changes from physiological adaptation to gene expression. Among many stress-inducible genes are genes such as osmoprotectants, chaperones and detoxification enzymes that directly protect against environmental stress. In addition to these genes, transcription factors and protein kinases that regulate gene expression and signaling during the stress response (Seki et al, 2003, Curr Opin Biotech 14, 194-199). Thus, timely expression of stress-responsive genes is critical to the plant's ability to survive under different environmental stress conditions (Yamaguchi-Shinozaki and Shinozaki, 2006, Annu Rev Plant Biol 57, 781-803; Chinnusamy et al. , 2007, Trends Plant Sci 12, 444-451; Shinozaki and, 2007, J Exp Bot 58,221-227).

Many experiments involving transcriptome analysis have identified a number of transcription factors that respond to abiotic stress, including bZIPs, zinc finger proteins, AP2 / EREBPs, bHLHs and NACs (Chen et al. , 2002, Plant Cell 14, 559-574; Seki et al, 2002, Funct Integr Genomics 2, 282-291; Rabanni et al, 2003, Plant Physiol 133, 1755-1767; Oh et al, 2005, Plant Physiol 138, 341-351; Nijhawan et al, 2008, Plant Physiol 146, 333-350; Oh et al, 2009, Plant Physiol 150, 1368-1379). It acts as a major mediator of plant resistance to the combined-acting factor (cis -acting elements), and thereby non-biotic stress (Meshi and Iwabuchi, 1995, - a variety of transcription factors specific system in the promoter of stress responsive gene Plant Cell Physiol 36, 1405-1420; Yanagisawa, 1998, J Plant Res 111, 363-371; Liu et al, 1999, Eur J Biochem 262, 247-257; Riechmann and Ratcliffe, 2000, Curr Opin Plant Biol 3, 423 -434; Fode et al, 2008, Plant Cell 20, 3122-3135). Thus, the stress-induced transcription factors form complex signaling networks in response to abiotic stress.

bZIP proteins include a number of transcription factor families that have a bZIP domain consisting of a basic region and leucine zippers (Hurst, 1994, Protein Profiles 1, 123-168; Jakoby, et al, 2002, Trends Plant Sci 7, 106-111). Conserved basic regions are involved in sequence-specific DNA binding, while less conserved leucine zipper regions contain amphipathic sequences in coiled-coil form and have dimerization specificity. To give. The leucine zipper sequence thus mediates homo- and / or heterodimerization of bZIP transcription factors. In plants, bZIP transcription factor genes can be expressed in a variety of ways, including organ-specific, stimulus-responsive, development-dependent, and cell cycle-specific. (Schindler et al, 1992, EMBO J 11, 1261-1273; Minami et al, 1993, Plant Mol Biol 23, 429-434; de Vetten and Ferl, 1995, Plant J 7, 589-601; Chern et al, 1996, Plant J 10, 135-148; Uno et al, 2000, Proc Natl Acad Sci USA 97, 11632-11637; Rodriguez-Uribe and O'Connell, 2006, J Exp Bot 57, 1391-1398).

Abscisic acid, a plant hormone, plays an essential role in the plant's adaptive response to abiotic stress, including dryness, low temperature, and high salts. Apsis acid is also involved in various aspects of plant growth and development such as seed maturation, dormancy, inhibition of cell division and germination (Leung and Giraudat, 1998, Annu Rev Plant Physiol Plant Mol Biol 49, 199-222 Yamaguchi-Shinozaki and Shinozaki, 2005, Trends Plant Sci 10, 88-94; Christmann et al, 2006, Plant Biol 8, 314-325; Kim, 2007, J Plant Biol 50, 117-121). Many bZIP transcription factors are known to be involved in abscitic acid and / or stress signaling (Lopez-Molina et al, 2002, Plant J 32, 317-328; Fujita et al, 2005, Plant Cell 17, 3470-3488). . bZIP transcription factors interact with specific ABA-responsive elements (ABREs) with the conserved motif PyACGTGGC in the promoter region of many abscitic acid-induced genes, thereby causing Transactivation of gene expression (Niu et al, 1999, Plant Mol Biol 41, 1-13; Kim et al, 1997, Plant J 11, 1237-1251). Thus, bZIP transcription factors may be represented as abscid acid reactive factor-binding factors (ABFs) or ABRE-binding proteins (AREBs). In Arabidopsis, 13 of 75 bZIP transcription factors belong to the A group which contains the ABF gene of 10 subfamily.

Expression of the Arabidopsis bZIP transcription factor family genes ABF2 / AREB1, ABF4 / AREB2 and ABF3 is upregulated in vegetative tissues by abscitic acid, dehydration and salt stress. In transient experiments with protoplasts, transcription of the reporter gene induced by ABRE is also activated by the bZIP transcription factor (Nakashima et al, 2006, Plant Mol Biol 60, 51-68). Constitutive overexpression of ABF3 in Arabidopsis and rice showed enhanced drying resistance (Kang et al, 2002, Plant Cell 14, 343-357). In addition, overexpression of the rice regulators OsbZIP23 and OsbZIP72, positive regulators of abscitic acid signaling, enhances abiotic stress tolerance (Lu et al, 2008, Planta 229, 605-615; Xiang et al, 2008, Plant Physiol 148, 1938). -1952). ABF protein also plays a role in the growth and development of plants. For example, Arabidopsis abi5 mutants show a decrease in sensitivity to abscitic acid by interrupting abscitic acid signaling during germination of the seed, indicating that AtABI5 links gene expression and abscitic signaling in the seed ( Finkelstein and Lynch, 2000, Plant Cell 12, 599-609). In monocotyledonous plants, the rice and barley homologs of AtABI5, TRAB1 and HvABI5, physically interact with the corresponding AtABI3 homologues OsVP1 and HvVP1, and regulate abscidic acid-induced gene expression (Hobo et al, 1999, Proc Natl Acad Sci USA 96, 15348-15353; Nakamura et al, 2001, Plant J 26, 627-635; Casaretto and Ho, 2003, Plant Cell 15, 271-284). In addition, the bZIP transcription factor OsABI5 is involved in rice fertility and stress tolerance (Zou et al, 2008, Plant Mol Biol 66, 675-683). The data suggest that ABF proteins act on conserved ABA signaling pathways in dicotyledonous and monocotyledonous plant species.

Rice is one of the most important major crops and a model of monocotyledonous plant species. However, while many bZIP transcription factors involved in stress signaling are currently identified in Arabidopsis, few of the 89 known bZIP transcription factors in rice have been functionally identified.

In the present invention, the inventors describe abiotic stress-induced bZIP transcription factors in rice. OsABF1 It was confirmed. Expression patterns of OsABF1 were investigated in rice shoots and roots exposed to various stress conditions. ABRE binding activity and transactivation ability of OsABF1 were evaluated using a yeast one-hybrid system. To investigate the in vivo function of OsABF1 , T-DNA insertion mutants of OsABF1 were analyzed under salt and dry treatment. In addition, expression of known acetic acid reactive genes was investigated in these mutants. The role of OsABF1 in rice absic acid-dependent abiotic stress signaling was investigated.

Korea Patent Registration No. 10-0599211 discloses abiotic stress resistance gene is disclosed that the Oslti32, is a stress tolerance gene OsMSRPK1 disclosed in Korea Patent Registration No. 10-0781075 call.

The present invention is derived from the above demands, and the present inventors have found that OsABF1 derived from rice. The present invention was completed by confirming that genes are upregulated under abiotic stress treatment.

In order to solve the above problems, the present invention is a rice ( Oryza stress-related OsABF1 ( Oryza ) derived from sativa ) sativa abscisic acid responsive element-binding factor

The present invention also provides a gene encoding the OsABF1 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 enhancing stress resistance of plants by transforming the recombinant vector into plant cells.

In addition, the present invention provides a transgenic plant with enhanced stress resistance produced by the above method and a seed thereof.

The present invention also provides a method for producing a stress-resistant transformed plant using the recombinant vector.

In addition, the present invention provides a composition for enhancing stress resistance of a plant comprising the gene.

The rice-derived OsABF1 gene of the present invention has increased expression under the treatment of absic acid and various abiotic stresses. Therefore, the OsABF1 gene may be useful for developing transgenic plants that are resistant to environmental stress.

Figure 1 shows the phylogenetic analysis of Arabidopsis and rice-derived Group A bZIP protein and OsABF1 protein. The number at the fork is the bootstrap value as a percentage.
2 shows the expression pattern of OsABF1 gene in shoots and roots of rice seedlings. (A) Expression of OsABF1 gene in rice seedlings at 0, 2, 4, 8, 12 and 24 hours under normal conditions (distilled water) without stress treatment. (B) Expression of OsABF1 gene in shoots and roots of rice seedlings after 0, 2, 4, 8, 12 and 24 hours after anoxic treatment and 4 hours (R4) and 12 hours (R12) recovery from the stress. (CG) Start stress in shoots and roots of rice seedlings during dry (10% polyethylene glycol), salt (250 mM sodium chloride), oxidative stress (10 mM hydrogen peroxide), low temperature (4 ° C.) conditions, and absticic acid (100 μM) treatment Expression pattern of OsABF1 gene after 0, 2, 4, 8, 12 and 24 hours. (H) Shoots of rice seedlings after 12 hours under anoxic, dry (10% polyethylene glycol), salt (250 mM sodium chloride), absic acid (100 μM), oxidation (10 mM hydrogen peroxide) or low temperature (4 ° C.) stress conditions Expression pattern of OsDEG10 in. The rice actin1 gene was used as an internal control.
Figure 3 shows the subcellular location of GFP-OsABF1 and OsABF1-GFP fusion proteins in transfected maize lobe protoplasts. (A) GFP-OsABF1; (B) OsABF1-GFP. Chlorophyll autofluorescence and NLS-RFP were used as chloroplast and nuclear markers, respectively. To distinguish from GFP (green) and RFP (red) fluorescence, false color (blue) was used for chlorophyll autofluorescence.
4 shows a transactivation assay of OsABF1 protein. (A) Vectors used in yeast one-hybrid assay. (B) transactivation activity in yeast. Beta-galactosidase activity is expressed in Miller units. pYESTrp2 / AtABF3 and pYESTrp2 empty vectors were used as positive and negative controls, respectively. Bars indicate SD values.
5 shows the survival rate of OsABF1 mutant plants under abiotic stress. OsABF1 from 2 - (A) mutant alleles Osabf1 -1 and Osabf1 Schematic diagram of genomic structure and T-DNA insertion site. Boxes and solid lines represent exons and introns, respectively. The location of T-DNA is indicated by a triangle. (B) Analysis of the expression of OsABF1 by RT-PCR 12 h after treatment with absic acid (100 μM) in wild type rice plants Hwayoung (HY) and Dongjin (DJ), and corresponding T-DNA mutants. (C) Survival rate of Osabf1 mutant when treated with high salt (250 mM sodium chloride). (D) Typical phenotypes of wild-type and T-DNA mutants treated with high salt. The left and right plants are live and dead wild type plants, respectively. (E) Survival of dehydrated Osabf1 mutants. (F) Typical phenotype of dehydrated wild type and T-DNA mutants. The left and right plants are typical live and dead plants from each line, respectively.
6 is Osabf1 RT-PCR analysis of Apsis acid / stress regulatory genes in mutant and wild type rice plants is shown. (A) Hwayoung and Osabf1 −1 ; (B), and Dongjin Osabf1 -2. Expression of the marker gene was analyzed in mutants and control plants after 0, 2, 4, 8, 12 and 24 hours of treatment with absic acid (100 μM). The rice actin1 gene was used as an internal control.

In order to achieve the object of the present invention, the present invention consists of an amino acid sequence represented by SEQ ID NO: 2, rice (( Oryza stress-related OsABF1 ( Oryza ) derived from sativa ) sativa abscisic acid responsive element-binding factor

The range of OsABF1 protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from rice 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. "Substantially homogeneous physiological activity" means activity that enhances the stress resistance of plants.

The invention also includes fragments, derivatives and analogues of OsABF1 protein. As used herein, the terms “fragment”, “derivative” and “analogue” refer to a polypeptide that possesses substantially the same biological function or activity as the OsABF1 polypeptide of the invention. Fragments, derivatives, and analogs of the present invention comprise (i) polypeptides substituted with one or more conservative or nonconservative amino acid residues (preferably conservative amino acid residues), wherein the substituted amino acid residues are encoded by a genetic code. Or (ii) a polypeptide having substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of a polypeptide, such as polyethylene glycol) A polypeptide derived from a bound mature polypeptide, or (iv) an additional amino acid sequence (eg, a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) and It may be a polypeptide derived from said polypeptide bound. Such fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

The present invention also provides a gene encoding the OsABF1 protein. The gene of the present invention may be DNA or RNA encoding the OsABF1 protein. DNA includes cDNA, genomic DNA or artificial synthetic DNA. DNA can be single stranded or double stranded. The DNA may be a coding strand or a non-coding strand.

 Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

Polynucleotides encoding a mature polypeptide represented by SEQ ID NO: 2 include a coding sequence encoding only a mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences encoding noncoding sequences.

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.

The invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequences as described herein, or fragments, analogs, and derivatives thereof. Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitutional variants, deletional variants, and insertional variants. As is known in the art, an allelic variant is an alternative to a polynucleotide, which may comprise one or more substituted, deleted or inserted nucleotides and which does not result in a substantial functional change in the polypeptide encoded by the variant Do not.

In addition, the present invention provides a poly-hybridized hybridization with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with a nucleotide sequence of SEQ ID NO. It relates to nucleotides. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" are (1) hybridization and washing under lower ionic strength and higher temperature such as 0.2 x SSC, 0.1% SDS, 60 ° C; Or (2) in the presence of a denaturant such as 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll and 42 ° C; Or (3) at least 80%, preferably at least 90%, more preferably at least 95%. In addition, the biological function and activity of the polypeptide encoded by the hybridizable polynucleotide is the same as the biological function and activity of the mature polypeptide represented by SEQ ID NO: 2.

In accordance with an embodiment of the invention, it should be understood that the OsABF1 gene is preferably rice derived. However, embodiments of the present invention have a high homology (e.g., 60% or more, ie 70%, 80%, 85%, 90%, 95%, even 98% sequence identity) with the rice OsABF1 gene and other It also includes other genes derived from plants. Methods for sequencing, and means for determining sequence identity or homology (e.g., BLAST) are well known in the art.

The present invention also provides a recombinant vector comprising the OsABF1 gene according to the present invention.

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.

In the present invention, the polynucleotide sequence encoding OsABF1 protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors comprising OsABF1 protein-encoding DNA sequences and appropriate transcriptional / translational control signals can be constructed by methods well known to those of skill in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to a plant cell when present in a suitable host, such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly. Preferably, the recombinant vector may be, but is not limited to, a pKBS1-1 vector, a pBI101 vector, and a pCAMBIA vector.

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. The host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains.

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 .

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 method for enhancing stress resistance of a plant comprising the step of transforming the recombinant vector into a plant cell and overexpressing an OsABF1 gene. Preferably, the stress may be, but is not limited to, anoxic, dry, salt, oxidative or cold stress.

In addition, the present invention provides a transgenic plant with enhanced stress resistance produced by the above method and a seed thereof. Preferably, the plant may be a monocotyledonous plant or a dicotyledonous plant, but is not limited thereto.

The monocotyledonous plants include Alismataceae, Hydrocharitaceae, Juncaginaceae, Schuchzeriaceae, Pomomotontonaceae, Najadaceae, Zosteraceae, Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae, Iridaceae, Burmanniaceae, Juncaceae, Commelinaceae , Eriocaulaceae, Panaxaceae (Gramineae, Gramineae, Poaceae), Araceae, Lemnaceae, Sparganiaceae, Typhaceae, Cycloaceae, Cyperaceae, Musaceae ), Ginger (Zingiberaceae), Cannaceae (Cannaceae) or Orchidaceae (Orchidaceae), but is not limited thereto.

The dicotyledonous plants are Asteraceae (Dolaceae, Diapensiaceae), Asteraceae (Clethraceae), Pyrolaceae, Ericaceae, Myrsinaceae, Primaceae (Primulaceae), Plumbaginaceae, Persimmonaceae (Ebenaceae) , Styracaceae, Stink bug, Symplocaceae, Ash (Oleaceae), Loganiaceae, Gentianaceae, Menyanthaceae, Oleaceae, Apocynaceae , Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Labiatae, Solanaceae, Scrophulariaceae , Bignoniaceae, Acanthaceae, Sesame (Pedaliaceae), Fructose (Orobanchaceae). Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, (Perox Adoxaceae), Valerianaceae, Dipsacaceae, Campanaceae ( Campanulaceae, Compositae, Myricaceae, Sapaceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Elmaceae, Moraceae , Urticaceae, Santalaceae, Mistletoe, Lothanthaceae, Polygonaceae, Landaceae, Phytolaccaceae, Nyctaginaceae, Pomegranate, Azizaceae (Portulacaceae), Caryophyllaceae, Chinopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cassia family, Cecidiphyllaceae, Ranunculus eae), Berberidaceae, Lardizabalaceae, Bird breeze (Mentaceae, Menispermaceae), Nymphaeaceae, Ceratophyllaceae, Cabombaceae, Saururaceae , Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Camellia, Theaceae, Guttaiferae, Droseraceae, Papaveraceae ), Capparidaceae, Cruciferaceae (Mustaceae, Cruciferae), Planeaceae (Plataceae, Platanaceae), Verruaceae, Hamamelidaceae, Pheasant (Snaphaceae, Crassulaceae), Panaxaceae (Saxifragaceae) Eucommiaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae Euphorbiaceae), star moss (Callitrichaceae), Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Mapleaceae, Aceraceae, Sapindaceae, Mapleaceae Hippocastanaceae, Sabiaceae, Balsam, Balsaminaceae, Aquifoliaceae, Nova, Celastraceae, Staphyleaceae, Buxaceae, Empetraceae, Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Adenaceae, Thymelaeaceae, Eraeagnaceae (Flacourtiaceae), Violaceae, Passifloraceae, Tamaricanaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Buddha (Lythraceae), Pomegranate Punica ceae), Onnagraceae, Haloragaceae, Bataceae (Alangiaceae), Dogwood (Hornaceae, Cornaceae), Arboraceae (Agaraceae) or Umbelliferae (Apiaceae) It may be, but is not limited thereto.

In addition, the present invention comprises the steps of transforming plant cells with the recombinant vector; And

It provides a method for producing a stress-resistant transformed plant comprising the step of regenerating a plant from the transformed plant cells.

The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tyumeo Pacific Enschede may be mediated by (Agrobacterium tumefiaciens). 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.

Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989, Momillan, N. Y.).

The present invention also provides a composition for enhancing stress resistance of a plant, comprising the OsABF1 gene of the present invention. In the composition of the present invention, the OsABF1 gene may be composed of the nucleotide sequence of SEQ ID NO: 1. The composition of the present invention contains an OsABF1 gene derived from rice as an active ingredient, and the plant is capable of enhancing stress resistance of the plant by transforming the gene OsABF1 into a plant. 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.

Materials and methods

Plant materials, growth conditions and stress treatment

Rice ( Oryza sativa L. cultivars Dongjin and Hwayoung) Seeds were sterilized with 70% ethanol and soaked in distilled water for one day, followed by plant growth chambers (28 ± 1 ℃, 80% relative humidity and 14 hours-light / 10 hours-cancer photoperiod). Incubated for 14 days. The 14 day old seedlings were placed under anaerobic conditions for 0, 2, 4, 8, 12 and 24 hours, followed by 4 and 12 hours recovery period from the stress. Oxygen removal was performed using pure nitrogen gas in AtmosBag ^™ -inflatable polyethylene isolation chambers (Sigma-Aldrich, Milwaukee, WI) (Kato-Noguchi and Morokuma 2007). For other stress treatments, the surface of the seed was sterilized and immersed in distilled water and transferred to a growth chamber (28 ± 1 ° C., 80% relative humidity and 14 hours-light / 10 hours-light photoperiod) for 14 days. Incubated at. The seedlings are then dried (0% 2, 4, 8, 12 and 24 hours) (10% polyethylene glycol), salt (250 mM sodium chloride), oxidative stress (10 mM hydrogen peroxide), low temperature (4 ° C.) and abscitic acid ( And 100 μM).

The T-DNA insertion mutant lines of OsABF1 , Osabf1 -1 and Osabf1 -2 can be found in the rice T-DNA insertion sequence database (Jeong et al, 2006, Plant J 45, 123-132; http://www.postech.ac .kr / life / pfg / risd / index.html). Osabf1 -1 and homozygous (homozygous) of the line is OsABF1 Osabf1 -2 gene were isolated by PCR screening using a specific and T-DNA-specific primers.

cDNA Cloning

OsABF1 full-length cDNA clones from rice ( O. sativa L. cv. Dongjin) drying of the seedling-treated total RNA extracted from the shoot and the primer pair, 5 '- AAGCTTATGATGGCGTCGAGGGTG-3' ( forward; SEQ ID NO: 3) and 5'-GGTACCCTACCACTCCATCGAGTT - 3 '(reverse; RT using the SEQ ID NO: 4) Separated by PCR. The PCR product was inserted into the pLUG-TA vector (iNTRON Biotechnology, Seoul, Korea) and the cDNA sequence was registered in GenBank (Registration No .: GQ904238).

RT - PCR

Total RNA was extracted from the stress-treated seedlings using Trizol reagent (BRL, Grand island, NY). PrimeScritpt ^TM First strand cDNA was synthesized from 2 μg of purified total RNA using Reverse Transcriptase (Takara Bio Inc., Shiga, Japan). Oligo (dT) was used as a primer and the RT reaction was carried out in a total volume of 25 μl at 42 ° C. for 1 hour. Gene specific primers were used to investigate the expression pattern of OsABF1 , including other transcripts of rice seedlings under different stress treatments. OsDEG10 was used as a positive PCR marker to confirm other abiotic stress treatment effects (Park et al, 2009, Biochem Biophys Res Commun 380, 597-602).

For gene expression analysis, total RNA was also isolated from the T-DNA mutant line and the absic acid-treated seedling shoots of their respective wild-type plants. The rice actin gene was amplified as an internal control to quantify the relative amount of cDNA (McElroy et al, 1990, Plant Cell 2, 163-171). Primer sequences used for RT-PCR analysis of OsABF1 gene, regulatory genes and rice actin are shown in Table 1.

Primers used for RT-PCR analysis gene Primer sequence (5 '→ 3') OsABF1
Forward: TCG CAC ACG GCA TCG GAT CT (SEQ ID NO: 5) Reverse: AGT TGC GTG ACC AGC GAC TC (SEQ ID NO: 6) OsDEG10
Forward: AGC GGA TCG ACA AGT TAT TG (SEQ ID NO: 7) Reverse: AAC ACA ACC AGC ACC TCA TG (SEQ ID NO: 8) SKC1
Forward: AGC TCT GCC GAT GAA GAC CAG (SEQ ID NO: 9) Reverse: AGT TGG CGA ACG TCG AGA CGA (SEQ ID NO: 10) Asr1
Forward: ACC ACC TGT TCC ACC ACA AGA (SEQ ID NO: 11) Reverse: TCT TCT CGT GGT GCT CGT GGA (SEQ ID NO: 12) SalT
Forward: AGG ACA TCA GTG TGC CAC CCA (SEQ ID NO: 13) Reverse: TGC GTC GAT AAG CGT TCC AGA (SEQ ID NO: 14) OsNAC
Forward: TGT ACG GAG AGA AGG AGT GGT (SEQ ID NO: 15) Reverse: TCA TCC AAC CTG AGG CTG TTC (SEQ ID NO: 16) OsLEA3
Forward: ATA CCA AGG AGG CGA CGA AGG (SEQ ID NO: 17) Reverse: TCA TCC CCA GCG TGC TCA TCA (SEQ ID NO: 18) OsABA45
Forward: TCT TCA ACA CCT GGA GCC GCA (SEQ ID NO: 19) Reverse: ACG GCC TTG TCA TAG CTA ACG (SEQ ID NO: 20) Actin1
Forward: GAT ATG GAG AAG ATC TGG CA (SEQ ID NO: 21) Reverse: TAG CTC TTC TCC ACG GAG GA (SEQ ID NO: 22)

System analysis ( Phylogenetic analysis )

Clustal W, Pole BioInformatique Lyonnasis (PBIL) program (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html) was used to perform multiple sequence alignments. The phylogenetic tree was constructed using MEGA software version 4.0 by the neighbor-joining method (Tamura et al, 2007, Mol Biol Evol 24, 1596-1599). Bootstrap analysis was performed with 1,000 replicates, and the bootstrap values were expressed as percentages.

GFP  Of fusion proteins Subcellular  location( subcellular localization )

The full-length cDNA of the OsABF1 gene was selected from the primer pair, 5'-CACCATGATGGCGTCGAGGGTGATGGCG-3 '(forward; SEQ ID NO: 23) and 5'-CTACCACTCCATCGAGTTTGTTCT-3' (reverse, N-terminal GFP fusion; SEQ ID NO: 24) or 5'-CCACTCCATCGAGTTTGTTCTTCT Amplified by PCR using 3 ′ (reverse, C-terminal GFP fusion; SEQ ID NO: 25). PCR products were inserted into the pENTR / D-TOPO vector (Invitrogen, Carlsbad, CA). The validated cDNA insert was then subcloned into p2FGW7 for N-terminal GFP fusion or p2GWF7 for C-terminal GFP fusion using LR clonase (Invitrogen) (Karimi et al, 2002, Trends Plant Sci 7 , 193-195). The resulting GFP-OsABF1 and OsABF1-GFP fusion constructs induced by the CaMV35S promoter were transported into corneous mesophyll protoplasts using polyethylene glycol (PEG) -calcium mediated methods (Hwang and Sheen, 2001, Nature 413, 383-389). Subsequently, an incubation of 12 to 24 hours was performed to allow for transient expression. Chlorophyll autofluorescence and OsHXK5NLS-RFP were used as markers of chloroplasts and nuclei, respectively (Cho et al, 2009, Plant Physiol 149, 745-759). Expression of the fusion construct was monitored using confocal microscopy (LSM 510 META, Carl Zeiss, Jena, Germany).

Yeast One- hybrid  Experiment

The cDNA sequence of the entire OsABF1 open reading frame (ORF) excluding the stop codon, the C-terminal bZIP region (OsABF1ΔN) excluding the stop codon, and the N-terminal region (OsABF1ΔC) excluding the bZIP domain was determined by PCR using an appropriate primer pair. Each amplified. The PCR product was cloned and sequenced using the pLUG-TA cloning vector system and then in frame with a pYESTrp2 vector comprising a GAL4 DNA binding domain to construct pYESTrp2 / OsABF1, pYESTrp2 / OsABF1ΔN, and pYESTrp2 / OsABF1ΔC. Fused to fit. The yeast strain carrying the pYC7-Int plasmid was lacZ It was used as a reporter system. The pYC7-Int / ABRE construct was prepared by inserting the Em1a trimer (GGACACGTGGCG) into the Sma I site of pYC7-Int. pYESTrp2 / OsABF1, pYESTrp2 / OsABF1ΔN and pYESTrp2 / OsABF1ΔC were transformed into yeast cells carrying pYC7-Int or pYC7-Int / ABRE. In addition, pYESTrp2 empty vector and pYESTrp2 / AtABF3 (Choi et al, 2000, J Biol Chem 275, 1723-1730) were transformed into yeast cells as negative and positive controls, respectively. The transformants were incubated at 30 ° C. conditions in SD-Ura-Trp plates until positive clones were observed. Positive clones identified were streaked in fresh SD-Ura-Trp plates to purify colonies.

In yeast  Transactivation Assay ( transactivation assay )

Four to five yeast colonies were incubated overnight at 30 ° C. in SD liquid medium. The culture was then diluted several times in fresh yeast extract-peptone-dextrose (YPD) medium and incubated at 30 ° C. for 3-5 hours. A ₆₀₀ was then measured at the end of the growing season, 1.5 mL of each culture was harvested by simple centrifugation and 1.5 mL of Z buffer (60 mM Na ₂ HPO ₄ -7H ₂ O, 40 mM NaH ₂ PO ₄ -H ₂ O, it was suspended in _{10 mM KCl, 1 mM MgSO 4} ? 7H 2 O, pH 7.0). The culture was re- harvested by centrifugation and resuspended in 0.3 mL of Z buffer and divided into 0.1 mL aliquots, three freeze-thawed using liquid nitrogen to lyse cells. An aliquot of Z buffer (100 mL Z buffer and 0.27 mL beta-mercaptoethanol) supplemented with beta-mercaptoethanol was then added to the preparation. The reaction was initiated at 30 ° C. by the addition of 0.16 mL of O-nitrophenyl β- D-galactopyranoside (ONPG) stock solution (4 mg / mL). After the color turned yellow, the reaction was terminated by adding 0.4 mL of 1 M Na ₂ CO ₃ . Beta-galactosidase activity at A ₄₂₀ is expressed in Miller units.

Stress Tolerance in Rice Mutants

For the saline treatment, rice plants were hydroponicly grown for 14 days in a growth chamber (28 ± 1 ° C., 80% relative humidity and 14 hours-light / 10 hours-light photoperiod) and then in 250 mM sodium chloride solution. Moved for 3 days. The plant was then transferred for 12 hours to normal growth conditions. For dehydration treatment, rice plants incubated for 14 days were transferred to dishes for 12 hours, then rehydrated and incubated for 14 days. Then the number of plants growing was counted.

Example  One: OsABF1 Identification of transcription factors

We performed microarray experiments to monitor rice genes whose expression changed under abiotic stress conditions (data not shown). From this analysis, we selected and identified OsABF1 , a bZIP transcription factor found to be induced by various types of abiotic stress. Using the specific genes that are based on; (LOC_Os01g64730 http://blast.jcvi.org/euk-blast/index.cgi?project=osa1) primers, 801 bp cDNA clone of OsABF1 is TIGR Rice Genome Annotation database Separated from rice seedlings. Sequence analysis shows that OsABF1 is identical to the predicted gene OsZIP12 from the OsZIP family list (Nijhawan et al, 2008, Plant Physiol 146, 333-350). Comparison of other cloned bZIP proteins with OsABF1 showed the highest homology with OsbZIP40, GBF4 and AtbZIP13, previously classified transcription groups as Group A bZIPs (Lu et al, 2008, Planta 229). , 605-615). The basic region of the OsABF1 bZIP domain showed high sequence similarity with the basic domains of the three bZIP proteins. The leucine zipper region of OsABF1 contains five heptad repeats. Thus, OsABF1 can be classified as one of the Group A bZIPs that make up the ABF family.

To assess the divergence of OsABF1 from other bZIPs, a phylogenetic tree for group A bZIPs of Arabidopsis and rice was constructed. The analysis shows that all group A bZIPs can be classified into three subgroups, including rice and Arabidopsis bZIPs, respectively (FIG. 1). It is therefore proposed that the divergence of the subgroups estimates the divergence of the monocotyledonous and dicotyledonous plants.

Example  2: Abiotic  Under stress treatment OsABF1 Expression

Under various treatments such as anaerobic, dry, salt, cold, osmotic and oxidative stress, and absic acid, the expression pattern of OsABF1 gene was analyzed by RT-PCR in shoots and roots of rice seedlings (FIG. 2). OsABF1 in untreated control samples No transcript was detected (FIG. 2A). In contrast, OsABF1 was upregulated more strongly in shoots than root after 4 hours in response to anoxic treatment (FIG. 2B). In dry and salt-treated seedlings, OsABF1 Expression was induced high after 2 hours (FIGS. 2C and 2D). OsABF1 under oxidative stress (hydrogen peroxide) and low temperature (4 ° C) treatment Genes were similarly induced within 2 hours, reached maximum levels after 8-12 hours, and then gradually decreased (Figures 2D and 2F). The results demonstrate that OsABF1 is rapidly upregulated under all abiotic stress conditions tested . In addition, the inventors examined whether OsABF1 responded to the treatment of phytohormonal abscisic acid and found that it was highly induced within 2 hours in abscidic acid-treated seedlings (FIG. 2G). OsDEG10 , a well-known abiotic stress-inducing gene, was used as a positive control in the experiments, and the transcript levels of the gene were increased under conditions of anaerobic, salt, dry, sodium chloride, methyl viologen and low temperature. It is already known (Park et al, 2009, Biochem Biophys Res Commun 380, 597-602). Analysis of the present invention confirmed that OsDEG10 is expressed within 12 hours in all stress-treated rice seedlings (FIG. 2H).

Example  3: OsABF1 of Subcellular  location

To determine the subcellular location of OsABF1 protein, we prepared GFP-OsABF1 and OsABF1-GFP fusion constructs under the control of the CaMV35S promoter. The construct was then expressed in maize lobe protoplasts. The results of the experiment showed that OsABF1-GFP and GFP-OsABF1 fusion proteins are expressed in the nucleus of corn protoplasts, as confirmed by colocalization of the fusion protein with the nuclear marker OsHXK5NLS-RFP ( 3A and 3B) (Cho et al, 2009, Plant Physiol 149, 745-759). The data thus indicate that OsABF1 is a nuclear protein that can act as a transcription factor that regulates the expression of downstream genes.

Example  4: OsABF1 Transactivation ability

Yeast one-hybrid experiments were performed to test the transactivation ability of OsABF1. The yeast GAL1 OsABF1, OsABF1 ΔC, and OsABF1 ΔN sequences into the yeast expression vector containing the B42 pYESTrp2 domain under the control of the promoter was cloned (Fig. 4A). Thus, expression of the cDNA insert fused to the B42 activation domain can be induced by glucose. This construct was used to transform the yeast strain pYC7-Int with a trimer of the EM1a factor, which is a conserved ABRE domain. In yeast strains transformed with pYESTrp2 / OsABF1 cultured in SD-Ura-Trp, beta-galactosidase activity was found to be three times higher than the control constructs (FIG. 4B). The transactivation capacity remained for OsABF1ΔC, but showed no requirement for the N-terminal region in yeast strains containing OsABF1ΔN. Thus, the results of the present invention suggest that OsABF1 binds to the cis-acting factor comprising the ABRE core sequence and thereby induces transcription of downstream genes. In this respect it is noteworthy that no enzyme activity is detected in the assay of the present invention for the pYC7-Int strain without the ABRE promoter (data not shown).

Example  5: OsABF1 Analysis of Mutants

To investigate the function of OsABF1 during abiotic stress conditions, the present inventors have hwayoung (HY), and Dongjin (DJ) of -1 and Osabf1 Osabf1 each made of, independent T-DNA mutant alleles of the two OsABF1 from wild-type plants - 2 was separated. Osabf1 -1 includes T-DNA insertions in the second exon, while Osabf1 -2 has this insertion in the first intron (FIG. 5A). Absence of OsABF1 transcript in homozygous mutants was confirmed by RT-PCR, and then stress tolerance tests were performed on the plants (FIG. 5B). In this experiment, the mutants and corresponding wild-type plants were hydroponicly grown for 14 days and transferred to 250 mM sodium chloride solution and further incubated for 3 days. After saline treatment, the plants were recovered for 12 days in hydroponic solution. No mutant plants survived the treatment, while 22% to 29% of wild type plants survived at the end of the experimental period (FIGS. 5C and 5D). The result is Osabf1 It proves that mutants are more susceptible to high salts than wild type plants.

We performed a parallel stress resistance assay for dehydration by exposing the 14-day-old seedlings to dry conditions for 12 hours and then recovering in hydroponic solutions for 14 days. The results showed that 23% to 42% of mutant plants and 54% to 65% of wild type plants survived (Figures 5E and 5F). Thus, wild type plants are more resistant to dehydration than Osabf1 mutants. The results also suggest that OsABF1 plays an important role in enhancing the resistance of rice plants to abiotic stress conditions including high salt and dehydration.

Example  6: Osabf1  In mutants Absis Expression of stress-regulated genes

Apsis acid-dependent and independent regulatory systems are known to be involved in the regulation of stress-responsive gene expression (Yamaguchi-Shinozaki and Shinozaki, 2005, Trends Plant Sci 10, 88-94; 2006, Annu Rev Plant Biol 57, 781- 803). To assess the possible regulatory role of OsABF1, we compared the expression patterns of abscisity / stress-regulated genes in Osabf1 mutants and wild-type rice seedlings treated with Apsis acid. Six abiotic stress-induced genes were selected to investigate the regulatory functions of OsABF1: OsABA45 (LOC_Os12g29400), Asr1 (LOC_Os02g33820), SalT (LOC_Os01g24710) induced by low temperature, drying, high salt and abscitic acid in rice seedlings ) And OsNAC (LOC_Os01g66120) (Rabanni et al, 2003, Plant Physiol 133, 1755-1767); OsLEA3 (LOC_Os05g46480), which encodes a late embryogenesis abundant protein induced by absic acid; And SKC1 (LOC_Os01g20160) (Zou et al, 2008, Plant Mol Biol 66, 675-683), which encodes a cation transporter involved in salt reactions in rice seedlings. Interestingly, OsNAC , OsLEA3 , and OsABA45 in wild-type plants exposed to Apsis acid Upregulated expression of the gene, Osabf1 In mutants it was markedly inhibited (FIG. 6). No significant difference in expression profile was found for other genes. The data indicate that OsABF1 plays a regulatory role during the expression of specific abscisic acid / stress-induced genes.

Attach an electronic file to a sequence list

Claims (11)

delete delete delete delete delete SEQ ID NO: consisting of 2 amino acid sequence of rice (Oryza sativa) was transformed with the origin of the stress tolerance related OsABF1 (Oryza sativa abscisic acid responsive element -binding factor 1) a recombinant vector comprising a gene encoding a protein in a plant cell OsABF1 A method of regulating stress tolerance of a plant, comprising regulating the expression of a gene. The method of claim 6, wherein the stress is anaerobic, dry, salt, oxidative or cold stress. A transgenic plant with controlled stress tolerance prepared by the method of claim 6. Seeds of plants according to claim 8. Transforming a plant cell with a recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 2, Oryza sativa abscisic acid responsive element-binding factor 1 (OSABF1) protein related to stress resistance derived from rice ( Oryza sativa ) ; And
Method for producing a stress-controlled transformed plant comprising the step of regenerating the plant from the transformed plant cells. delete

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