KR20140052625A - COLD -INDUCIBLE OsDhn1 GENE AND PROTEIN ENHANCING COLD TOLERANCE - Google Patents

Abstract

The present invention relates to an OsDhn1 gene and protein which enhance low temperature tolerance. The present invention provides: a composition which includes a gene delivery system with a nucleotide sequence encoding an amino acid sequence of a second sequence on a sequence list as an active ingredient, and which enhances the resistance of plants against abiotic stress: a transformed plant cell or a transformed plant in which the composition is introduced; and a production method of the transformed plant cell or the transformed plant.

Description

Cold-Inducible OsDn1 Gene and Protein Enhancing Cold Tolerance < RTI ID = 0.0 >

The present invention relates to low temperature-inducible OsDn1 genes and proteins that promote low temperature tolerance.

Abiotic stresses, such as cold, dryness and high salinity, cause morphological, physiological, biochemical and molecular changes in plants and ultimately affect plant growth and productivity (Wang et al. 2001). In addition, these stresses induce the expression of various genes that play important roles in stress tolerance, regulation of other gene expression, and stress signaling (Shimano et al., 1999; Shinozaki and Yamaguchi-Shinozaki, 2000; al., 2002; Shinozaki et al., 2003). Among these genes, the dehydrin gene encoding two families of LEA (Late Embryogenesis Abundant) proteins is expressed in dehydrated tissues such as nourishment tissues under seeding or abiotic stress (reviewedd by Close, 1996; Allagulova, 2003). The dihydrin protein is present in various cellular tissues such as the cytoplasm, nucleus, mitochondria, vacuole, and plasma membrane periphery (reviewd by Rorat, 2006). Although the function of dehydrin in plants has not been elucidated yet, it has been thought of as a molecular chaperon to protect proteins, enzyme activities, nucleic acids and membrane structures under stress (Bray, 1993; Close, 1996 ; Koag et al., 2003; Peng et al., 2008; Hara et al., 2009).

So far, a number of multi-organisms have been developed in a variety of organisms such as angiosperms, gymnosperms, pteridophytes, bryophytes, fungi, algae and cyanobacteria. (Campbell and Close, 1997; Close 1997; Li et al. 1998; Mtwisha et al. 1998; Shih et al. 2008). Also, dihydrins include a number of Responsive to Abscisic acid (RAB) and cold-regulated proteins such as RAB16, COR47 and WCOR410 (Kusano et al., 1992; Danyluk et al., 1994; Welin et al., 1995; Nylander et al., 2001). In many studies, expression of dihydrins has been reported to be associated with cold, dry, and salt stress (reviewed by Rorat, 2006). Therefore, a method of producing a stress tolerant plant by transplanting a dehydrin gene has been attempted. For example, tobacco over-expressing the citrus dihydrine gene CuCOR19 and maize ZmDHN2b showed tolerance to cold stress (Hara et al., 2003; Xing et al., 2011). Arabidopsis overexpressing the dihydrin genes such as ERD10 , LTI30 , RcDhn5, and DHN- 5 showed resistance to various abiotic stresses such as freezing and salting (Puhakainen et al., 2004; Brini et al. 2007; Peng et al., 2008).

Transplantation in rice using the dihydrin gene has not been reported so far. On the other hand, it has been reported that transgenic rice expressing other LEA proteins such as HVA1 , PMA1959 , PMA80 and OsLEA3-1 are increased resistance to dry stress (Xu et al., 1996; Rohila et al., 2002; Cheng et al., 2002; Xiao et al., 2007). Rice is the most important and major edible crop for mankind. Therefore, the stress-response mechanism in rice is very important in promoting tolerance to stress. To this end, the study of various abiotic stress-responsive genes in rice is essential. Among the abiotic stress-responsive genes of rice, OsDhn1 has been reported to encode dry-induced SK3 type dehydrins such as Arabidopsis COR47 / RD17 and ERD10, wheat WCOR410 (Lee et al., 2005).

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

As a result of intensive efforts to discover genes capable of promoting tolerance to cold stress, the present inventors have found that rice ( Oryza sativa Dehydrin 1) gene, which is induced by stress, from sativa , and confirmed that the plant transformed with this gene exhibits enhanced tolerance against low temperature, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a cold-inducible OsDn1 protein which promotes resistance to low temperatures.

It is another object of the present invention to provide a nucleic acid molecule comprising a nucleotide sequence encoding a cold-inducible OsDn1 protein.

It is yet another object of the present invention to provide a vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a cold-inducible OsDn1 protein.

It is another object of the present invention to provide a transformant transformed by the vector of the present invention.

It is another object of the present invention to provide a method for producing transgenic plants with enhanced cold stress tolerance.

Another object of the present invention is to provide a transgenic plant having low temperature stress tolerance.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing a low temperature-inducible OsDn1 protein, which comprises the amino acid sequence of the second sequence of the sequence listing, and which, when overexpressed in the plant, Or more of the amino acid sequence homology.

As a result of efforts to discover genes capable of promoting tolerance to cold stress, the present inventors have found that OsDhn1 ( Oryza sativa Dehydrin 1) gene induced by stress from rice ( Oryza sativa ) Showed enhanced resistance to low temperatures.

As used herein, the term " cold stress " means stress due to low temperature, preferably 15 DEG C or less, more preferably 12 DEG C or less, and most preferably 10 DEG C or less.

The low-temperature-inducible OsDhnl protein of the present invention has substantial identity to the above-mentioned amino acid sequence and at the same time is interpreted to include an amino acid sequence within a range capable of enhancing tolerance to cold stress. The above-mentioned substantial identity is determined by aligning the amino acid sequence of the present invention with any other sequence as much as possible, and analyzing the aligned sequence using a program commonly used in the art (for example, ClustalX and PROSIS) Means an amino acid sequence that exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology.

According to another aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding said cold-inducible OsDn1 protein.

As used herein, the term " nucleic acid molecule " has the meaning inclusive of DNA (gDNA and cDNA) and RNA molecules. In the nucleic acid molecule, the nucleotide which is a basic constituent unit is not only a natural nucleotide, Also included are analogues (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

Most preferably, the nucleic acid molecule of the present invention comprises the nucleotide sequence of SEQ ID NO: 99-971 in the nucleotide sequence of SEQ ID NO: 1. The nucleic acid molecules of the present invention that encode the cold-inducible OsDhnl protein are also interpreted to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences described above. The above-mentioned substantial identity can be determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible, and analyzing the aligned sequence using a program commonly used in the art (for example, ClustalX and DNASIS) Means a nucleotide sequence which, in one case, exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology.

According to another aspect of the present invention, the present invention provides a vector comprising a nucleotide sequence encoding the above cold-inducible OsDn1 protein. According to a preferred embodiment of the present invention, the vector of the present invention comprises (a) a nucleotide sequence encoding a cold-inducible OsDn1 protein and (b) a vector comprising a promoter operably linked to said nucleotide to provide.

As used herein, the term " operably linked " refers to a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The sequence will control the transcription and / or translation of the different nucleic acid sequences.

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

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, p _L ^λ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc. ), A ribosome binding site for initiation of translation and a transcription / translation termination sequence. When E. coli is used as a host cell, E. coli The promoter and operator site of the tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol . , 158: 1018-1024 (1984)) and the left promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, . Ann Rev Genet, 14:. . 399-445 (1980)) may be used as a control region.

The vectors that can be used in the present invention include plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19 which are frequently used in the art, phages such as λgt4λB, λ -Charon,?? Z1, M13, etc.) or a virus (e.g., SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell (for example, a metallothionein promoter) or a mammalian virus Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk Promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences to facilitate the purification of the cold-inducible OsDn1 protein expressed therefrom. Fusion sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA). Because of the additional sequence for such purification, proteins expressed in the host are rapidly and easily purified through affinity chromatography.

The vector of the present invention may be a selection marker and may include an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The gene of the present invention has been isolated from plants and has the most favorable utility for plants since it can act to enhance plant tolerance to cold stress. Thus, the vector of the present invention comprises (i) a nucleotide sequence encoding a cold-inducible OsDn1 protein; (Ii) a promoter operatively linked to the nucleotide sequence of (i) above and acting on plant cells to form RNA molecules; And (iii) a plant expression vector comprising a 3'-non-detoxified site that acts in plant cells to cause polyadenylation of the 3'-end of the RNA molecule.

According to a preferred embodiment of the present invention, a promoter suitable for the present invention may be any of those conventionally used in the art for transgenic introduction of a plant, for example, a ubiquitin promoter of corn, a cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase (nos) promoter, Piguet mosaic virus 35S promoter, water crab basil lipid viral promoter, combellinella yellow mothball virus promoter, ribulose-1, 5-bis- (TPI) promoter, adenine phosphoribosyl transferase (APRT) promoter of Arabidopsis, and the tryptophan synthase promoter of ssRUBISCO, a cysteine triphosphate isomerase (TPI) promoter of Arabidopsis thaliana, do.

According to a preferred embodiment of the present invention, the 3'-non-detoxified site causing polyadenylation in accordance with the present invention is derived from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3 'end) Bevan et al., Nucleic Acids Researcher , 11 (2): 369-385 (1983)), derived from the Octopine synthase gene of Agrobacterium tumefaciens, the 3 'end of the protease inhibitor I or II gene of tomato or potato, and CaMV 35S Terminator is included.

Optionally, the vector additionally carries a gene encoding a reporter molecule (e.g., luciferase and beta -glucuronidase). In addition, the vector of the present invention can be used as a selection marker for a gene resistant to antibiotics (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) (e.g., neomycin phosphotransferase ( npt II ) Gomycin phosphotransferase ( hpt ), etc.).

According to another aspect of the present invention, there is provided a transformant transformed by the above-mentioned vector of the present invention.

A host cell capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any host cell known in the art, and examples include E. coli JM109, E. coli Bacillus subtilis, Bacillus subtilis, and Bacillus strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, and Salmonella typhimurium, ≪ RTI ID = 0.0 > T. < / RTI > Marcesons and various Pseudomonas spp.

When the vector of the present invention is transformed into eukaryotic cells, Saccharomyce cerevisiae , insect cells, human cells (e.g., Chinese hamster ovary, W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells.

The method of delivering the vector of the present invention into a host cell may be carried out by the CaCl ₂ method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9: 2110-2114 (1973) , Hanahan, D., J. MoI . Biol . , 166: 557-580 (1997)), one method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9: 2110-2114 (1983)) and electroporation method (Dower, WJ et al., Nucleic. Acids Res . , 16: 6127-6145 (1988)). In addition, when the host cell is a eukaryotic cell, microinjection (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology , 52: 456 electroporation (Neumann, E. et al, EMBO J., 1:. 841 (1982)), liposome-mediated transfection method (Wong, TK et al, Gene , 10:87 (1980).), DEAE- Dextran treatment (Gopal, Mol . Cell Biol . , 5: 1188-1190 (1985)), and the gene can be introduced into the host cell by Yang et al., Proc . Natl . Acad . Sci . , 87: 9568-9572 have.

The vector injected into the host cell is expressed in host cells, in which case a large amount of cold-inducible OsDn1 protein is obtained.

According to another aspect of the present invention, the present invention provides a method for producing a plant cell, comprising: (a) transforming a plant cell or a plant tissue with the vector of the present invention; (b) screening the transformed plant cell or plant tissue; And (c) regenerating the plant from the transformed plant cell or plant tissue to obtain a transformed plant.

According to another aspect of the present invention, the present invention provides a transgenic plant having low temperature stress tolerance.

The explant used in the present invention is a plant cell or a plant tissue, and when plant tissue is used, a callus is preferably used.

In the method of the present invention, transformation of plant cells can be carried out according to conventional methods known in the art, including electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982) ), Particle bombardment (Yang et al., Proc . Natl . Acad . Sci . , 87: 9568-9572 (1990)) and Agrobacterium-mediated transformation (U.S. Patent Nos. 5,004,863, 5,349,124 and 5,416,011) . Of these, Agrobacterium-mediated transformation is most preferred.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent such as a metabolic inhibitor, an antibiotic and a herbicide. Plant cells that stably contain a marker gene that is transformed and conferring selectative resistance are grown and divided in the above cultures. Exemplary labels include, but are not limited to, hygromycin phosphotransferase gene, glycophosphate tolerance gene and neomycin phosphotransferase (nptII) system.

Methods for the development or regeneration of plants from plant protoplasts or from various expansions are well known in the art. The development or regeneration of plants containing foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (U.S. Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

The method of the present invention is applied to various plants, but preferably applies to crop plants such as rice, barley, wheat and corn, more preferably to rice, and most preferably to Japonica cultivars.

In the present invention, a preferred transformation method is carried out using an Agrobacterium system, more preferably Agrobacterium tumefaciens ) -binary vector system.

In a method of using an Agrobacterium system, a specific embodiment includes the following steps: (a ') introducing Agrobacterium tumefaciens, which can be inserted into the genomic DNA of a plant cell and have the following sequence, Faces ( Agrobacterium tumefaciens ): (i) a nucleotide sequence encoding a cold-inducible OsDn1 protein; (Ii) a promoter operatively linked to the nucleotide sequence of (i) above and acting on plant cells to form RNA molecules; (Iii) a 3'-non-detoxified site that acts in plant cells to cause polyadenylation of the 3'-end of the RNA molecule; (b ') Regenerating the infected explant in a regeneration medium to obtain a transformed plant.

Transformation of plant cells is carried out with Agrobacterium tumefaciens containing Ti plasmids (Depicker, A. et al., Plant cell transformation by Agrobacterium plasmids. In Genetic Engineering of Plants, Plenum Press, New York (1983) ).

More preferably, binary vector systems such as pBin19, pRD400, pRD320, pGA1611 and pGA1991 are used (An, G. et al., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, (Ii) a structural gene operably linked to said promoter, and (iii) a promoter operably linked to said promoter, wherein said promoter is selected from the group consisting of: The vector further carries a gene encoding a reporter molecule (e.g., luciferase and glucuronidase). Examples of promoters used in the binary vector include CaMV 35S But are not limited to, promoters, 1 promoter, 2 promoter and nopaline synthase (nos) promoter.

Infection of the explant by Agrobacterium tumefaciens involves methods known in the art. Most preferably, the infection process comprises co-culturing the culture of Agrobacterium tumefaciens with an explant. Whereby Agrobacterium tumefaciens is infected into plants.

The explant transformed by Agrobacterium tumefaciens is regenerated in the regeneration medium, which ultimately forms a transgenic plant.

The transformed plants according to the present invention are confirmed to be transformed by methods known in the art. For example, PCR using DNA samples from tissues of transformed plants can identify foreign genes inserted into the genome of the transgenic plant. Alternatively, Northern or Southern blotting can be performed to confirm the transformation (Maniatis et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for improving abiotic stress resistance of a plant comprising as an active ingredient a gene carrier comprising a nucleotide sequence encoding the amino acid sequence of the second sequence of the sequence listing, A transgenic plant cell or plant, and a method for producing the transgenic plant cell or plant.

(b) The nucleotide sequence used in the present invention is involved in increasing the resistance of a plant to drought stress, and when the plant is transformed using the nucleotide sequence, the resistance of the plant to stress is increased, And when applied to rice, it is possible to produce rice which is resistant to drying stress, thereby maximizing the productivity.

Figure 1 shows the construction of OsDn1 over-expressed binary vector. OsDhnl < / RTI &gt; full length was subcloned into the binary vector pGA1611 under the maize ubiquitin promoter. P _ubi , maize ubiquitin promoter; P _35S , CaMV 35S promoter; Tnos, the terminator sequence of the nopaline synthase gene; T7, Terminator sequence in Transcript 7; hph , hygromycin phosphotransferase gene; RB (right border sequence) and LB (left border sequence), Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens- derived Ti plasmid, respectively.
Figure 2 shows Northern blot analysis of OsDn1 in transgenic plants. Total RNA of T1 plants was isolated from agarose gel, blotted onto nylon membrane, and hybridized with radioactive isotope labeled OsDn1 probe. Numbers and WT represent the transgenic strain and the wild type, respectively. EtBr (Ethidium Bromide) - The stained rRNA band indicates that RNA is loaded in equal amounts.
Fig. 3 shows the Southern blot analysis result of OsDn1OX . 5 ㎍ of genomic DNA of selected 2, 4, 11, and 22 overexpressed hosts were treated with Hind III and hybridized with a probing maize ubiquitin promoter. Indicates the size and location of Hind III treated lambda DNA fractions.
FIG. 4 shows RT-PCR (Reverse Transcription-Polymerase Chain Reaction) results of OsDhnl OX. RT-PCR analysis was carried out using primers for OsDhn1 and lip9 with cDNA of seedlings of 3 OX strain, NT (non-transgenic segregant) and WT (wild type) as template. RAc1 (Rice Actin Gene) and OseEF- 1a (elongation factor gene) were used as PCR control.
FIG. 5 shows the results of analysis of stress tolerance of the passage OsDn1 OX with chlorophyll fluorescence. (A) 7 hours of dry treatment of transformants (No. 4, No. 11 and No. 22), NT and WT; (b) cold treatment at 4 占 폚 for 24 hours; (c) 200 mM NaCl treatment for 3 days; And (d) Fluorescence changes were monitored for 100 μM MV treatment for 3 hours. The results were expressed as 'mean ± standard deviation' obtained from more than 4 experiments. * Indicates significant differences between OX and NT, and # indicates significant differences between OX and WT (Student t -test, P <0.05).
Figure 6 shows the representative flower of OsDn1OX after drying treatment. The transgenic plants (nos. 4 and 11), NT and WT were treated with 7 days of water-free drying stress and grown for one month.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and methods

Transgenic rice production

OsDhn1 overexpressed transgenic rice plants , Osdhn1 full-length cDNA (Genbank accession no. AY786415, Sequence Listing 1) was transformed into KpnI and SacI (Kim et al., 2003) of binary vector pGA1611 (Kim et al., 2003) controlled by maize ubiquitin promoter Site to construct a rice transformation vector pSK169 (Fig. 1).

The Japonica rice variety, Dongjin, was transformed by Agrobacterium-mediated co-culture and regenerated in media containing 40 mg / L hygromycin B (Hiei et al., 1994; Lee et al. , 1999). The regenerated plants were transferred to the paddy fields of the greenhouse and the seeds were obtained for each line. To select homozygous transformation lines, more than 20 seeds of T2 generation were germinated on a filter paper containing hygromycin-B using the method of Xiong and Yang (2003). After incubation at 28 ° C for 10 days, the number of surviving nannies was counted and determined as homozygous lines.

Southern blot and Northern blot analysis

Chen and Roland (1999) were used to isolate genomic DNA from leaves. The DNA (5 μg) was digested with restriction enzyme (12 hours at 37 ° C), electrophoresed on 0.8% agarose gel and separated using a high-bond-N membrane (Amersham, UK). For Northern blot analysis, total RNA was isolated using Tri reagent (Molecular Research Center, USA). Total RNA (30 μg) was electrophoresed on 1.3% agarose gel and transferred to the membrane. Hybridization of DNA and RNA was carried out using a radioisotope labeled probe prepared by the random priming method (Feinberg and Vogelstein, 1983). After hybridization, the membrane was washed with 2 x SSC, 0.1% SDS at room temperature for 15 minutes, 1 x SSC, 0.1% SDS at room temperature for 15 minutes, and 0.1 x SSC, 0.1% SDS at room temperature for 15 minutes. Hybridization signals were confirmed with an image analyzer (BAS-1500, Fuji, Japan) and exposed to Hyperfilm ^TM MP (Amersham, UK).

RT-PCR analysis

Total RNA was isolated using RNeasy Mini Plant kit (QIAGEN, Germany) and cDNA was synthesized according to manufacturer's instructions using Sprint RT Complete kit (CLONTECH, USA). The gene was amplified by performing PCR under the following conditions: 5 minutes at 94 ° C, then 1 minute at 94 ° C, 1 minute at 56 ° C, and 1 minute at 72 ° C for 22-40 cycles, 7 minutes at 72 ° C. A forward primer (5'-AGCTCAAACAAGTCAAGAGC-3 ') and a reverse primer (5'-AAGCACCAAACTAACACACG-3') were used for OsDhn1 amplification and a forward primer 5'-ATGGCCACTCCTGCTCCC-3 'and a reverse primer 5'-CCAGCCCAAAACCAATACAA- 3 'was used. RAc1 forward primer (5'-CATGCTATCCCTCGTCTCGACCT-3 ') and RAc1 (Jain et al. 2006) were used for the amplification of rice actin RAc1 (McElroy et al. 1990) and the kidney factor gene OseEF1- Reverse primer (5'-CGCACTTCATGATGGAGTTGTAT-3 '), OseEF1-a positive primer (5'-CCACACCTCCCACATTGCCGTC-3') and reverse primer (5'-ACGACACAGTGCACACAGCGAG-3 '

Stress-immunity test

To investigate abiotic stress-tolerance, we analyzed chlorophyll fluorescence and wilting ratios of transgenic plants. For the fluorescence assay, the youngest leaves of 7-day old seedlings were cut into 5 cm lengths and then stressed as follows: (1) air drying for 7 days at a relative humidity of 70% or less (drought stress); (2) floating at 4 ° C in distilled water for 24 hours (cold stress); Or (3) suspended in a solution of 100 μM methyl viologen (MV) for 12 hours under dark conditions and floated for 3 hours under light conditions of 70 μmol m ^-2 sec ^-1 or less Oxidative stress). To induce salinity stress, 8 days old seedlings were placed in 200 mM NaCl solution and chlorophyll fluorescence was measured 3 days later. After a dark adaptation time of 30 minutes, fluorescence signals were measured using Plant Efficiency Analyzer (Hansatech, UK). The Fv / Fm ratio was measured using a photosystem (Genty et al., 1989) and the degree of seedling damage was evaluated. To analyze forgery, 7 day old seedlings were treated under the following conditions: (1) restored for the next 7 days without water for 3 days; (2) allowed to stand at 4 DEG C for 4 days and then restored for 6 days; Or (3) 100 mM NaCl solution for 7 days and then recovered for 7 days. After the recovery period, dead plants and healthy plants were counted and compared with the control group.

Experiment result

Production of overexpressed transgenic rice plants of OsDhn1

To construct OsDhn1 overexpressed rice (OsDhn1 OX), the constructed binary vector (pSK169) contains the OsDhN1 gene controlled by the ubiquitin promoter (Fig. 1). Nineteen lines of transgenic plants were produced through Agrobacterium-mediated transgenic transformation, of which 18 lines were matured and seeded. They did not show any morphological changes under normal growth conditions. Northern blot analysis of leaf samples of T1 plants grown under normal conditions showed that 15 lines of 18 lines of OsDhn1 expression were higher than wild-type plants (Fig. 2). Additional experiments were performed by selecting plant lines 2, 4, 11, and 22 that exhibited a high expression rate for OsDhnl. Southern blots were performed on selected plant lines 2, 4, 11, and 22 to confirm that 8, 2, 3, and 1 copies of T-DNA were inserted, respectively (FIG. 3). Germination tests were performed in a medium containing hygromycin to select homozygous offspring, and eventually, line 4, 11 and 22 were selected.

OsDhn1  For overexpressing plants RT - PCR  analysis

The overexpression of OsDHn1 in selected over-expressing plant lines was analyzed by RT-PCR. The results, 4, 11 and 22 times, but the O sDhn1 strongly expressed in a plant line, NT (non-transgenic segregant) and wild type (WT, wild type) in the control group OsDhn1 Expression was not confirmed (Fig. 4).

The expression of lip9 was also analyzed. Lip9 known as selective splicing form of OsDhn1 are encoding the two segments instead of three K- K- segment present in OsDhn1 protein (Lee et al., 2005) . Expression of lip9 showed the same level without big difference in OsDhn1 overexpression plants and control plants (Fig. 4). These results indicate that the overexpressed plant line of the present invention overexpresses the OsDhn1 full length and accumulates intact SK ₃ -type Dhn (dehydrin) protein in the cells. OsDn1 protein accumulation was analyzed by immunoblotting with anti-K segment antibodies.

OsDhn1  Stress-Tolerance Assessment in Overexpressed Plants

OsDhn1 In order to evaluate the stress-tolerance of over-expressing plant lines, chlorophyll fluorescence of plant leaves was measured and the activity of the photochemical system was checked under stress conditions. Under normal growth conditions, the ratio of Fv / Fm was OsDn1 And about 0.8 in overexpressed plants and control seedlings. Under drought stress caused by air drying, OsDhn1 The Fv / Fm values of the overexpressed plant lines 4 and 22 were significantly higher than those of the NT and WT control groups. After 5 hours of drought stress treatment, the Fv / Fm values of WT and NT were decreased to 0.55 ± 0.08 and 0.57 ± 0.06, respectively, whereas OsDhn1 The Fv / Fm values of the two lines of overexpressing plants were 0.69 ± 0.05 and 0.66 ± 0.04, which showed a Fv / Fm value of 20% higher than the control plant (FIG. 5a). In addition, after 6 hours and 7 hours of drought stress treatment, OsDhn1 The Fv / Fm values of the overexpressed plants were 35% and 26% higher than those of the control plants, respectively. On the other hand, during cold stress treatment, OsDhn1 The Fv / Fm values of the overexpressed plants were not significantly different from those of the control group. After 24 hours of cold stress treatment, the Fv / Fm values of WT and NT decreased to 0.36 ± 0.02 and 0.40 ± 0.02, respectively, and OsDhn1 The Fv / Fm values of the two lines of overexpressing plants were 0.37 ± 0.02 and 0.37 ± 0.05, similar to those of the control plants (FIG. 5b). Similar to cold stress treatment, the ratio of Fv / Fm due to salt stress and MV-induced oxidative stress was OsDhn1 Over-expressing plants and control plants (Fig. 5c-5d).

A wilting analysis was performed to assess stress tolerance in whole seedlings. When water was not given for 3 days as a drought stress and then recovered under normal growth conditions, OsDhn1 The wilting ratio of the overexpressed plants was lower than that of the control plants. Three OsDhn1 The rate of streaking on the fourth leaf of the over-expressing plant line was 7.1%, 62.5%, and 51.3%, respectively, and this ratio was lower than that of the control plant by 30% (Table 1). OsDhn1 Only the overexpressed plant line 4 showed a significantly lower stomach rate than the NT plant. OsDhn1 Over-expressing plants lines 11 and 22 showed lower stoichiometry than NT plants but no significant differences (P <0.05, χ ² test). The cells were exposed to cold stress conditions for 4 days and recovered for 6 days under normal growth conditions. Then, three OsDhn1 The stunning rates for the third leaf of the overexpressed plant line were 40.0%, 46.2% and 66.7%, respectively, and these results are significantly lower than those of the WT plants showing completely seeded phenotype. These OsDhn1 The overcorrection rate of overexpressed plants was almost similar to that of NT plants.

process No.4 No.11 No.22 NT WT dry 2/28
(7.1%) 20/32
(62.5%) 60/117
(51.3%) 58/89
(65.2%) 148/148
(100.0%) cold 10/25
(40.0%) 12/26
(46.2%) 20/30
(66.7%) 49/96
(51.0%) 117/117
(100.0%) salt 66/68
(97.1%) 74/75
(98.7%) 88/89
(98.9%) 116/120
(96.7%) 184/184
(100.0%)

After 100 mM NaCl solution was treated to induce saline stress, OsDn1 As a result of measuring the stunning ratio of overexpressed plants, OsDhn1 The overage rate of overexpressed plants was not significantly different from that of NT and WT plants. OsDhn1 Except for line 4 of the overexpressed plants, the rate of overcorrection was 3% lower than that of WT plants.

30-day-old OsDhn1 Drought stress induces overexpression of mature plants (after the water stopped 7 days 7 days recovering in normal growth conditions), and analysis of the above, coordination, OsDhn1 Overexpressed plant line 4 did not decline and showed a wholly healthy phenotype as compared to control plants. OsDhn1 Overexpressed plant line 11 showed a stunning rate of about 31%, while NT and WT plants showed 42% and 54% stunning rates, respectively. Representative forms of the transgenic plants that appeared after stopping watering for 7 days are shown in Fig. Taken together, these results indicate that overexpression of OsDhN1 significantly improves tolerance to drought stress in plants.

Argument

The Dhn (Dehydrin) gene encodes a lysine-rich K-segment and is strongly expressed in response to abiotic stress. Because of this feature, Dhn is believed to play a role in protecting cells under abiotic stress. A number of in vitro assays and transfection experiments have confirmed the cytoprotective effect of Dhn . However, the specific role of Dhn is unknown (reviewed by Rorat, 2006). According to the rice genome and microarray database, the rice genome contains at least 8 Dhn genes, which are strongly expressed in response to abiotic stress. These rice Dhn gene of OsDhn1 is drought-induced SK ₃ - is known to code the type Dhn, known to have a downstream target genes high similarity with the Arabidopsis COR47 / RD17 and ERD10 known as of the CBF / DREB1 transcription factors (Lee et al ., 2005). The expression of OsDhn1 , also known as Dip1 , is actually increased in CBF / DREB1 transgenic rice plants , and these transgenic rice plants show a strong resistance to abiotic stress (Oh et al., 2005; Ito et al., 2006; Xu et al., 2011). In addition, OsDhn1 It has been confirmed that the promoter contains a cis-acting element which is presumed to have abiotic stress-reactivity and has a strong drought-inducing property (Lee et al., 2005; Yi et al., 2010).

The present inventors have taken into account the interaction of stress-responsive and abiotic stress-responsive transcription factors and predicted that OsDn1 would play a role in stress responses and resistance. To illustrate this prediction, the present inventors constructed and overexpress OsDn1 overexpressed plants and performed various analyzes. The constitutive expression of OsDhn1 in OsDn1 overexpressed plants was confirmed by RT-PCR analysis. Expression of lip9, an alternative splicing form of OsDhn1 , was not significantly different between OsDn1 overexpressing plants and control plants. The K-segment is considered to be an important site for dihydrin function, OsDhN1 contains three K-segments, while lip9 contains two K-segments. Lip9 was first identified from rice callus (Aguan et al., 1991) and OsDhn1 was identified from rice seedlings (Lee et al., 2005). This fact implies that the intracellular function of OsDhn1 differs from that of lip9 . Thus, the selective phenotype of OsDn1 overexpressing plants of the present invention It is entirely due to the overexpression of ectopic expression of OsDhN1 .

As a result of chlorophyll fluorescence and streak rate measurement, OsDn1 overexpressing plants showed strong resistance to drought stress (FIGS. 5 and 6, Table 1). Especially, when evaluated by seedling stage and ripening stage, drought tolerance is higher in mature stage than in seedling stage. On the other hand, the stunning rate of the OsDhn1 overexpressed plants was somewhat lower than that of the WT plants, but the strong tolerance to cold stress and salt stress was not confirmed in the analysis of the present invention (Table 1). These results suggest that OsDhn1 protects against drought stress rather than cold or salt stress. In addition, an endogenous OsDhn1 gene with increased expression under drought stress conditions will contribute to induce drought tolerance in cooperation with ectopic overexpression of the OsDhn1 gene. Therefore, the endogenous OsDhn1 gene, which is induced by drought, is expected to be associated with drought tolerance.

A number of studies have been reported so far on dihydrogen overexpressed transgenic plants with increased stress tolerance. However, the specific function of the dihydrin gene for stress tolerance is not yet known. In vitro studies have shown that dihydrin binds to proteins, lipids, and metals, and has various activities such as radical-scavenging and cold protection. Of these activities, SK _n- type dihydrins are known to be essential for plant cold adaptation (reviewed by Rorat, 2006). In particular, the expression of SK ₃ type dihydrins such as wheat WCOR410 and citrus CuCOR19 is associated with cold tolerance and transgenic plants containing these genes exhibit increased cold stress tolerance (Danyluk et., 1998; Houde et Yin et al., 2006; Xing et al., 2011). On the other hand, the OsDn1 overexpressed plants of the present invention did not show strong resistance to cold stress, despite SK ₃ type dihydrin coding. Under cold stress, OsDhn1 expression was temporarily increased at the seedling stage (Lee et al., 2005), and this result may be one reason for explaining low cold stress tolerance of OsDHn1 overexpressing plants. On the other hand, the experimental conditions of the cold tolerance test of the present invention may not be suitable for OsDn1 overexpressed plants. In response to cold stress at 12 ℃, the expression of OsDhn1 was increased in leaves and flowers of mature plants (Lee et al., 2005). In addition, the present inventors have not conducted an experiment for cold stress tolerance of OsDn1 over-expressing mature plants. Therefore, it is necessary to confirm the function of OsDhn1 for various abiotic stress tolerance through further experiments using transgenic plants at various stages of development.

Abiotic stress often causes oxidative stress that functionally and structurally denatures proteins (Smirnoff 1998). Thus, the present inventors have evaluated the oxidative stress resistance by analyzing chlorophyll fluorescence in the seedlings treated with the Gen ROS- generation or chemical methyl Biology to investigate the role of OsDhn1 for oxidative stress. As a result, there was no significant difference in Fv / Fm values between OsDn1 overexpressed plants and control plants (FIG. 5d). These results indicate that OsDn1 is not involved in the radical-scavenging activity.

In conclusion, the present inventors have found that OsDhn1 overexpressing transgenic rice has a strong resistance to drought stress and OsDhn1 gene plays an important role in rice drought tolerance. Although the function of OsDhNl at the molecular level has not been described in the present invention, it is expected that stress tolerance is induced by the molecular chaperone function of OsDhNl . The molecular function of OsDhn1 to stress tolerance should be clarified through further studies.

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While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry-University Cooperation Foundation Sogang University <120> Cold-Inducible OsDhn1 Gene and Protein Enhancing Cold Tolerance <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1117 <212> DNA <213> Orysa sative Dehydrin1 <400> 1 gtcacagctc aaacaagtca agagcgaata gttcttgctg atctgttgtt tgattacttt 60 agttctcgag aggctttagc tgaatccatc gatcgatcat ggaggatgag aggaacacgg 120 agagccacca gggtggcgag gctgcagagc aggtggaggt gaaggacagg ggcctcttcg 180 acaacctcct tggcaggaag aaggacgatc agccggagga gaagaagcat gaggaggagc 240 ttgtcaccgg catggagaag gtctccgtgg aagagccaaa gaaggaggag caccacgccg 300 agggcgagaa gaaggagagc ctcctctcca agctgcaccg atccagctcc agctccagct 360 cgtcgagtga tgaggaagag gaggtgatcg atgacaacgg cgaggtggtc aagaggaaga 420 agaagaaggg gctcaaggag aagatcaagg agaagctgcc cggccacaag gaccatgccg 480 gtgagcatgc tcctccgccc gcggcgacgg gcttccccgc gccggctccg cctgcatccg 540 tggtgacggc cgcgcccacg ccagctcctg ctcccgtggt gactcacggc gatcaccacc 600 acgacaccgc cgtccccgtg gagaagatcg agggtgatca cgccaagacg gaggcgaccc 660 tgccacgtgc acccgaggag gagaagaagg gcttcctcga caagatcaag gagaagctgc 720 ccggcggcca caagaagccg gaagacgcaa ctgctgtgcc gccgccggcc gcctcaccgg 780 ctgctcctgc cactactccg gcgccagcac acccaccgcc ggctacagag gaagtgagca 840 gcccggatgg gaaggagaag aagggtatac tgggcaagat catggagaaa ctgcccggtt 900 accacaaggg ctccggcgag gaagacaaga ccgccgccgc cgccaccggc gagcacaaga 960 gcagcgctta attggggcgt gtgtgagacc aggccatggt tggaatttgg aagtgtttgg 1020 cgtgtgttag tttggtgctt tttctgcact gcagctttgt taagttcgtg tcaagattgg 1080 tcaaggcctg gtcagcgaaa aaaaaaaaaa aaaaaaa 1117 <210> 2 <211> 290 <212> PRT <213> Oryza sativa Dehydrin1 <400> 2 Met Glu Asp Glu Arg Asn Thr Glu Ser His Gln Gly Gly Glu Ala Ala   1 5 10 15 Glu Gln Val Glu Val Lys Asp Arg Gly Leu Phe Asp Asn Leu Leu Gly              20 25 30 Arg Lys Lys Asp Asp Gln Pro Glu Glu Lys Lys His Glu Glu Glu Leu          35 40 45 Val Thr Gly Met Glu Lys Val Ser Val Glu Glu Pro Lys Lys Glu Glu      50 55 60 His His Ala Glu Gly Glu Lys Lys Glu Ser Leu Leu Ser Lys Leu His  65 70 75 80 Arg Ser Ser Ser Ser Ser Ser Ser Ser Asp Glu Glu Glu Glu Val                  85 90 95 Ile Asp Asp Asn Gly Glu Val Val Lys Arg Lys Lys Lys Lys Gly Leu             100 105 110 Lys Glu Lys Ile Lys Glu Lys Leu Pro Gly His Lys Asp His Ala Gly         115 120 125 Glu His Ala Pro Pro Ala Ala Thr Gly Phe Pro Ala Pro Ala Pro     130 135 140 Pro Ala Ser Val Val Thr Ala Ala Pro Thr Pro Ala Pro Ala Pro Val 145 150 155 160 Val Thr His Gly Asp His His His Asp Thr Ala Val Pro Val Glu Lys                 165 170 175 Ile Glu Gly Asp His Ala Lys Thr Glu Ala Thr Leu Pro Arg Ala Pro             180 185 190 Glu Glu Glu Lys Lys Gly Phe Leu Asp Lys Ile Lys Glu Lys Leu Pro         195 200 205 Gly Gly His Lys Lys Pro Glu Asp Ala Thr Ala Val Pro Pro Pro Ala     210 215 220 Ala Ser Pro Ala Ala Pro Ala Thr Pro Ala Pro Ala His Pro Pro 225 230 235 240 Pro Ala Thr Glu Glu Val Ser Ser Pro Asp Gly Lys Glu Lys Lys Gly                 245 250 255 Ile Leu Gly Lys Ile Met Glu Lys Leu Pro Gly Tyr His Lys Gly Ser             260 265 270 Gly Glu Glu Asp Lys Thr Ala Ala Ala Ala Thr Gly Glu His Lys Ser         275 280 285 Ser Ala     290

Claims (8)

A low temperature-inducible OsDn1 protein which promotes tolerance to cold stress in the plant when overexpressed in a plant, or a protein that shows an amino acid sequence homology of 80% or more with the protein, when the plant overexpresses the amino acid sequence of the second sequence.
9. A nucleic acid molecule comprising a nucleotide sequence encoding the cold-inducible OsDn1 protein of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence is the nucleotide sequence of SEQ ID NO: 99-971 in the nucleotide sequence of SEQ ID NO: 1.
3. A vector comprising the nucleic acid molecule of claim 2 or 3.
A transformant transformed by the vector of claim 4.
A method for producing a transgenic plant having enhanced cold stress tolerance comprising the steps of:
(a) transforming a plant cell or plant tissue with the vector of claim 4;
(b) screening the transformed plant cell or plant tissue; And
(c) regenerating the plant from the transformed plant cell or plant tissue to obtain a transformed plant.
The method according to claim 6, wherein the plant is rice, barley, wheat, or corn.
A transgenic plant having improved tolerance to cold stress produced by the method of claim 6 or 7.

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