KR101350225B1 - RsERF1 gene from Raphanus sativus and uses thereof - Google Patents

Abstract

The invention of the mud ( Raphanus) sativus ) -related RsERF1 related to environmental stress resistance ( Raphanus sativus ethylene responsive transcription factor 1 ) A plant comprising a protein, a gene encoding the protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, and a plant cell transformed with the recombinant vector to overexpress the RsERF1 gene. environmental stress method for enhancing the resistance, the recombinant vector a method for producing environmental stress resistance is enhanced transgenic plant using, transformed with the environmental stress resistance prepared by the method it promotes conversion plant and of its seed and gaetmu derived RsERF1 It relates to a composition for enhancing environmental stress resistance of a plant comprising a gene.

Description

RsERF1 gene from Raphanus sativus and uses approximately

The present invention relates to RsERF1 gene and its use derived from mud , and more particularly to mud ( Raphanus) sativus ) -related RsERF1 related to environmental stress resistance ( Raphanus sativus ethylene responsive transcription factor 1 ) A plant comprising a protein, a gene encoding the protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, and a plant cell transformed with the recombinant vector to overexpress the RsERF1 gene. environmental stress method for enhancing the resistance, the recombinant vector a method for producing environmental stress resistance is enhanced transgenic plant using, transformed with the environmental stress resistance prepared by the method it promotes conversion plant and of its seed and gaetmu derived RsERF1 It relates to a composition for enhancing environmental stress resistance of a plant comprising a gene.

Various environmental stresses, such as plant pathogens, drought, high concentrations of salt, low temperatures and high temperatures, inhibit plant growth and act as one of the factors limiting the yield of crops in many important agricultural sectors. When a plant is exposed to environmental stress such as high concentration of salt, high temperature, low temperature, and drought, plant disease, intracellular osmotic pressure or ionic imbalance occur, thereby inhibiting plant growth and photosynthesis. Plants have evolved their defense against environmental stress. As a defense against plant pathogens, the synthesis of plant hormones such as salicylic acid, jasmonic acid and ethylene, the synthesis of antibacterial compounds such as phytoallexin, cell wall enforcement and various There is expression of disease resistant genes. In addition, defense mechanisms against high-salt salt stress include the maintenance of ion homeostasis by several transporters located in the cell membranes of plants and the activation of salt overly sensitive (SOS) signaling systems that are resistant to high salt salt stress. (Zhu et al., 2003 Curr. Opin. Plant Biol 6: 441-445).

Environmental stresses that inhibit plant growth The most severe stress is osmotic stress caused by various external conditions such as dehydration, high salt and cold. Since only a slight increase in resistance to osmotic stress can have a significant impact on agricultural productivity and yield, many studies have been conducted on the regulation mechanism of osmotic stress and its associated regulatory factors. Recent studies have shown that plants have precise mechanisms as part of their adaptive response to osmotic stress, and one of the important mechanisms of these stress adaptive responses is the transcriptional activation of genes encoding proteins required for adaptive response (Liu et et al. al., 2000 Proc. Natl. Acad. Sci. USA 97: 3730-3734). In addition, in order to understand the mechanism for adapting to osmotic stress, a number of genes induced by expression of specific stresses have been isolated, and their characteristics have been extensively studied.

On the other hand, there are various types of terrain from the coastal region of Korea to the alpine area of over 2,700m, and the natural environment is so varied that there are various kinds of plants that adapt and inhabit there, and there are about 4,000 unique native plants. These native plants can be used to restore the environment such as reclaimed land and deserted land, and wild plant resources are of great value for future medicine development, discovery of useful genes and breeding of ornamental flowers. Unique ecosystems, such as salt coastal salt marshes, are a rare confined area of the world, and are very likely to find useful genetic resources or new materials that cannot be found in terrestrial ecosystems. It is very important to develop a useful genetic source of salt plants because planting salt-resistant plants that are highly adaptable to tidal flats or reclaimed land is used to restore salt marshes and these salt-tolerant genes must be obtained from salt plants.

On the other hand, mud mud is one of the salt plants that grow on the sandy beaches by the sea, and it is remarkable as a useful genetic source with excellent environmental stress resistance. The mud has its origin in the crop, Raphanus sativus . It is said that the seeds of radish were blown away by wind and grew into a different shape than the original radish. It can be seen in the southern provinces and on Jeju Island, and grows in groups on the roadside, on the beach, or in the sand. It is similar to radish, but its roots are thinner and harder than radish, and smaller than radish leaves but strong. Specifically, the leaf is a pinnate leaf that comes out of the root and has small leaves on both sides of the petiole divided into bird feathers. Beautiful flowers are planted for ornamental purposes, soak kimchi with leaves and roots or eat them in miso soup. In civilian use, it is used to treat coughs, indigestion, and bronchitis.

Korea Patent Registration No. 10-0796164 discloses a ssieyi emal and blood 1 (CaMRP1) encoding a membrane receptor-like proteins gene of chili and environmental stress-resistant transgenic plant using the same are disclosed, Korea Patent Publication No. 10-2000-0072720 No. Discloses a novel transcriptional regulator of plants that enhances resistance to osmotic stress, but the RsERF1 gene derived from the mud of the present invention and its use have not been identified.

The present invention been made by the request as described above, the present inventors have gaetmu (Raphanus sativus) derived RsERF1 ( Raphanus sativus ethylene responsive transcription factor 1 ) The present invention was completed by confirming that environmental stress resistance is enhanced in Arabidopsis plants transformed with a recombinant vector containing a gene.

In order to solve the above problems, the present invention provides an environmental stress resistance-related RsERF1 derived from seaweed ( Raphanus sativus ) ( Raphanus sativus ethylene responsive transcription factor 1 ) protein.

The present invention also provides a gene encoding the protein.

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

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

In addition, the present invention provides a method for enhancing the environmental stress resistance of a plant comprising the step of transforming the plant cell with the recombinant vector overexpressing the RsERF1 gene.

In addition, the present invention provides a method for producing a transgenic plant having enhanced environmental stress resistance, comprising transforming plant cells with the recombinant vector and regenerating the plant from the transformed plant cells.

In addition, the present invention provides transgenic plants and seeds thereof improved in environmental stress resistance produced by the above method.

The present invention also provides a composition for enhancing environmental stress resistance of plants comprising the RsERF1 gene derived from mud , consisting of the nucleotide sequence of SEQ ID NO: 1.

RsERF1 derived from the mud radish of the present invention Genes play a role in regulating environmental stress resistance, and by controlling the expression of these genes in plants, it is possible to develop transgenic plants with increased environmental stress resistance, and economic crops to which they are applied are reclaimed or deserted barrens. It can be useful for the development of agriculture and plant seed industry because it can overcome the cultivation environment and greatly increase the yield even in the harsh natural environment changed due to extreme weather.

Figure 1 shows the cDNA sequence of the RsERF1 gene isolated from the mud .
Figure 2 shows the amino acid sequence deduced from the cDNA base sequence of RsERF1 .
3 is a diagram comparing amino acid sequences of other plants having homology with amino acid sequences deduced from the cDNA base sequence of RsERF1 . The portion indicated by lines represents the AP2 domain, and the portion marked with stars represents the well-conserved alanine (133) and aspartic acid (138) of the ERF. Nuclear targeting signal The NLS (nucleus localization signal) consists of a basic amino acid called 'KRKRK'. AEE75492, Arabidopsis thaliana ); AAS20427, Capsicum annuum ); AAK95687, Tomato ( Solanum lycopersicum ).
Figure 4 shows the results of the evolutionary distance by showing the amino acid sequence of another plant having homology with the amino acid sequence deduced from the cDNA base sequence of RsERF1 as a phylogenetic tree.
5 is RsERF1 In order to determine the expression of genes, total RNA was isolated from the leaves treated with high salt stress (0.2 M NaCl) and RT-PCR (A) and Northern blot analysis (B) were performed.
6 is RsERF1 It is a result of analyzing the position in the cell by expressing the GFP recombinant protein containing the protein encoding the gene in the tobacco protoplasts.
7 is Escherichia coli prepared by cloning RsERF1 behind GST. coli ) expression vector pDEST15.
FIG. 8 shows the results of analyzing the amount of expression of the recombinant protein including the entire RsERF1 gene in Escherichia coli over time, followed by protein electrophoresis (SDS-PAGE) (M, molecular size marker; V, vector; T , Total protein; Sol, soluble protein; In, insoluble protein).
9 shows the results of Western analysis using GST antibody after protein electrophoresis (SDS-PAGE) to confirm the expression and expression level of RsERF1 using recombinant proteins expressed in E. coli (V, vector; T , Total protein; Sol, soluble protein).
10 is a result of analysis by protein electrophoresis (SDS-PAGE) to confirm whether the purified purified by sequentially using the recombinant protein expressed in Escherichia coli 1 G by using a GST-tag purification column by 1ml to be.
11 is an experiment for artificially preparing a short oligonucleotide containing a recombinant protein GST- RsERF1 and a DNA binding recognition site expressed in E. coli, and confirms the binding to the DNA binding recognition site, protein electrophoresis ( Electrophoretic mobility shift assays (EMSAA) using DNA labeled with radioisotope (γ ³² P-ATP) followed by SDS-PAGE (GCC, GCC-box; mGCC, mutated GCC; DRE) , DRE motif; mDRE, mutated DRE motif).
12 shows the results of analyzing the transcriptional activity of RsERF1 in yeast. (A) Schematic of the position of the AP2 domain in RsERF1 . (B) The cloned expression of the RsERF1 gene into an expression vector containing a GAL4 DNA binding domain is shown. Self RsERF1 the gene in order to determine whether the activity after cloning a gene RsERF1 the GAL4 DNA binding domain expression vector, were transformed into MaV203 yeast strain, and screening for the transformed gene RsERF1 SD (Leu-) in SD solid medium (Leu After growth in-, Ura-, His) solid medium, growth was confirmed, beta-galactosidase (β-galactosidase) in SD (Leu-, Ura-, His-, + β-X-gal) medium This is the result of checking the activity. WT: positive control, m1 and m2: negative control.
13 is RsERF1 Results for overexpressing transgenic plants. (A) It is a schematic diagram of the overexpression vector of RsERF1 for plant transformation. RsERF1 The cDNA was cloned into the plant transformation vector pB2GW7 and then transformed into Arabidopsis. (B) DNA was extracted from the Arabidopsis transformant leaves, and the result of the transformation was confirmed by PCR. +: 35S- RsERF1 plasmid (positive control),-: negative control.
14 is RsERF1 RT-PCR was performed to compare RsERF1 and stress-related gene expression of overexpressed transgenic Arabidopsis with control plants. After extracting RNA from control plants and RsERF1 overexpressed Arabidopsis, synthesizing cDNA, PCR products amplified with each primer were electrophoresed on agarose gel.
15 is a comparative analysis of the root growth and live weight of the control plants and RsERF1 transgenic plants osmotic stress (0.2 M mannitol), salinity stress (0.15M NaCl) and non-stressed plants (A) and comparing them to the exact figures One graph is (B).

In order to achieve the object of the present invention, the present invention is composed of the amino acid sequence of SEQ ID NO: 2, Raphanus sativus ) to enhance the environmental stress resistance of RsERF1 ( Raphanus) sativus ethylene responsive transcription factor 1 ) provides the protein.

The range of RsERF1 protein according to the present invention includes proteins having an amino acid sequence represented by SEQ ID NO: 2 isolated from muds and functional equivalents of the proteins. 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 environmental stress resistance.

The present invention is also RsERF1 Fragments, derivatives and analogues of proteins. As used herein, the terms “fragment”, “derivative” and “analogue” refer to a polypeptide that possesses substantially the same biological function or activity as the RsERF1 polypeptide of the invention. The fragments, derivatives, and analogs of the present invention may be used in conjunction with (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted (the substituted amino acid residues are encoded 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 the polypeptide, such as polyethylene glycol) (Iv) an additional amino acid sequence (e. G., A leader sequence, a secretory sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) and a polypeptide derived from the mature polypeptide Or a polypeptide derived from said polypeptide. Such fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

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 coding for 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. The nucleotide mutant includes a substitution mutant, a deletion mutant, and an insertion mutant. 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 is the RsERF1 Thereby providing a gene encoding a protein. Gene of the present invention is RsERF1 DNA or RNA encoding the protein. DNA includes cDNA, genomic DNA, or artificial synthetic DNA. The DNA may 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 comprise the nucleotide sequence of SEQ ID NO: 1. Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The present invention also relates to a polynucleotide which hybridizes with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with the nucleotide sequence of SEQ ID NO: 1 described above. 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.

According to an embodiment of the invention, RsERF1 It should be understood that the gene is preferably derived from mud. However, an embodiment of the present invention is a silkworm RsERF1 But also other genes from other plants with high homology to the gene (e. G., 60% or more, i.e., 70%, 80%, 85%, 90%, 95%, or even 98% . Methods for sequencing, and means for determining sequence identity or homology (e.g., BLAST) are well known in the art.

The present invention is also the RsERF1 A recombinant vector containing the gene is provided.

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 re-introduced into the cell by an artificial means as modified.

The recombinant plant expression vector of the present invention can be used as a transient expression vector capable of transient expression in a plant into which the foreign gene is introduced and a plant expression vector capable of permanently expressing the foreign gene in the introduced plant.

Binary vectors that can be used in the present invention may be any binary vector containing RB (right border) and LB (left border) of T-DNA capable of transforming plants when present with Ti plasmid of A. tumefaciens . Preferably, pBI101 (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pCAMBIA vectors, and the like, which are frequently used in the art, may be used.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is 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. Particularly preferred forms of the Ti-plasmid vector are described in EP 0 120 516 B1 and US Patent 4,940,838, It is a vector. Other suitable vectors that can be used to introduce a gene according to the invention into a plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinotricin, antibiotic resistant genes such as kanamycin, Ampicillin , G418, Bleomycin, hygromycin, chloramphenicol, Genes, but are not limited thereto.

In a plant expression vector according to an embodiment 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, constitutive promoters do not limit selectivity.

In a plant expression vector according to an embodiment of the present invention, the terminator can be a conventional terminator, such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agro And the terminator of the Octopine gene of Agrobacterium tumefaciens , but is not limited thereto.

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

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

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

The method of carrying the vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973), when the host cell is a prokaryotic cell). ), Hanahan method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like. In addition, when the host cell is a eukaryotic cell, microinjection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (1973)), Electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), and gene balm (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) The vector can be injected into the host cell.

The present invention is by transforming the plant cell with a recombinant vector including the gene RsERF1 provides a method for enhancing the environmental stress resistance of a plant, comprising overexpressing a gene RsERF1.

In the present invention, "environmental stress" refers to an external factor that lowers the growth or productivity of a plant, and is roughly divided into biotic stress and abiotic stress. Biological stresses typically include pathogens, and abiotic stresses include high concentrations of salt, dry, low temperature, high temperature and oxidative stress. The term " environmental stress resistance "refers to a trait that inhibits or delays plant growth or productivity deterioration due to such environmental stresses.

In the method according to an embodiment of the present invention, the environmental stress may be salt or osmotic stress, but is not limited thereto.

Also, the present invention provides a method for producing a plant cell, comprising the steps of: transforming a plant cell with the recombinant vector; And regenerating the plant from the transformed plant cell. The present invention also provides a method for producing a transgenic plant having enhanced environmental stress resistance.

In the method according to an embodiment of the present invention, the environmental stress may be salt or osmotic stress, but is not limited thereto.

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.).

In addition, the present invention provides transgenic plants and seeds thereof improved in environmental stress resistance produced by the above method.

Plants to which the method according to the invention may be applied include monocotyledons or dicotyledons. Examples of the monocotyledonous plants include, but are not limited to, rice, wheat, barley, bamboo shoots, corn, taro, asparagus, onion, garlic, leek, leek, Examples of dicotyledonous plants include cedar, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mud, watermelon, melon, cucumber pumpkin , Gourds, strawberries, soybeans, green beans, kidney beans and peas, and preferably, but not limited to Arabidopsis.

The present invention also provides a composition for enhancing environmental stress resistance of a plant comprising the RsERF1 gene, consisting of the nucleotide sequence of SEQ ID NO: 1. The composition is RsERF1 consisting of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient Gene, and by transforming the gene into a plant, the environmental stress resistance of the plant can be increased.

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

Example  One : RsERF1 ( Raphanus sativus ethylene responsive transcription  factor 1 Gene identification

Seaweed ( Raphanus sativus ) seeds were collected from Daejeon Korea Biotechnology Research Institute. After germination of the muds, the leaves were harvested for about 6 weeks in the greenhouse, soaked in NaCl (200 mM) and treated for 0, 3, 6 and 24 hours, after which total RNA was extracted by liquid nitrogen. MRNA was isolated from the total RNA, synthesized by cDNA, and synthesized by dsDNA for adapter connection. Then, cloned into pBluescript-SK and transformed into SOLR cells, cDNA library (Macrogen) was prepared. A total of 340 were analyzed in the mud flat EST library. After analyzing the nucleotide sequence of the obtained gene, gene information was collected using the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). By sequencing, the entire sequencing of the RsERF1 gene was obtained (FIG. 1).

Sequencing shows that salt induced stressRsERF1 The cDNA of the gene was shown as a total of 1,086bp base sequence (SEQ ID NO: 1). 361 amino acids were deduced by the nucleotide sequence of SEQ ID NO: 1 (FIG. 2). The base sequence and deduced amino acid sequence was found to be a new gene induced by salinity stress by database search.RsERF1 Similarity analysis of the amino acid sequence encoded by the gene by database search showed that the protein sequence most similar to the protein encoded by the gene was Arabidopsis (Arabidopsis thalianaRAP2.2 (GenBank ID: AEE75492) showed 75% homology. remindRsERF1 The amino acids obtained from the gene are sequenced to GCC-boxcisIt can be seen that there is an AP2 domain having acting DNA element binding activity and a nuclear localization signal (NLS) domain that induces nuclear targeting (FIG. 3). AlsoRsERF1 Proteins determined to have high homology with amino acid sequences encoded by genes were analyzed by phylogenetic tree to examine evolutionary relationships (Fig. 4). remindRsERF1Of proteins and other plants that encodeERF (ethylene responsive transcription factor) Homology of the protein was about 38-75%. The genes used for comparative protein analysis and phylogenetic tree analysis are as follows:Arabidopsis thaliana , AEE75492); Telungiella halophilaThellungiella halophila,BAJ33606); Baby Pole (Arabidopsis thaliana, NP_175794); BeechFagus sylvatica, CAE54591); pepper(Capsicum annuum, AAS20427); cotton(Gossypium hirsutum, AAX07458); tobacco(Nicotiana tabacum, AAP40022); Plum TreePrunus salicina, ACM49847); Kraseninnicovia Aborescens (Krascheninnikovia arborescens, ACI62768); cassava(Manihot esculenta, AAX84670); Two legsActinidia deliciosa, ADJ67434); Petunia (Petunia x hybrida, ADP37418); tomato(Solanum lycopersicum, AAK95687); tomato(Solanum lycopersicum, AAQ91334); Gallega OrientalisGalega orientalis, ACI46677); pepper(Capsicum annuum, AAP72289); cotton(Gossypium barbadense, AAT77192); bean(Glycine max, ACD47129); rice plant(Oryza sativa Japonica, NP_001057044, Os06g0194000); corn(Zea mays, ACG38354); barley(Hordeum vulgare, AAZ14085).

Example  2: new RsERF1  Expression test according to salinity stress of gene

After treatment with high-salt (0.2M NaCl) stress on the mud cultivated for more than six weeks in growth, the leaves were sampled over time, and total RNA was isolated using an RNA extraction kit (Ambion). RT-PCR was performed using the isolated RNA as follows. Synthesis of cDNA with total RNA (2 μg), ubiquitin expression as a housekeeping gene using cDNA prepared for each sample as a template was performed by forward 5'-ubiquitin primer (5'-TCATCCGACACCATCGACAATG-3 ': SEQ ID NO: 3 ) And reverse 3'-ubiquitin primer (5'-ATGACGATCGAAATGACTCGCC-3 ': SEQ ID NO: 4) and forward 5'- RsERF1 primer (5'-ATGTGTGGAGGAGCTATAATCTC-3': SEQ ID NO: 5) to analyze RsERF1 expression And reverse 3'- RsERF1 primer (5'-AAAGAACTGATTGTTGAACATCTG-3 ': SEQ ID NO: 6). PCR was 95 ° C. for 5 minutes; 26 cycles of 95 ° C 1 minute, 55 ° C 1 minute, 72 ° C 1 minute; The last extension step was carried out at 72 ° C. for 7 minutes. As a result RsERF1 PCR products were found to increase gradually after saline stress treatment (FIG. 5A).

Northern blot analysis was also performed using the isolated RNA. The total RNA isolated was electrophoresed on a 1.0% formaldehyde gel, then the gel was transferred to a nylon membrane (AP Biotech) with 20 × SSC solution (3M NaCl, 0.3M sodium citrate) and radioisotope [ RsERF1 labeled with α- ³² P] dCTP Northern blot analysis was performed using the fragment 1,086bp cDNA of the gene as a probe. As a result, it was confirmed that the expression level of RsERF1 transcript gradually increased due to salinity stress, which was expressed at maximum at 6 hours, and then expressed at 24 hours afterward (FIG. 5B).

Example  3: RsERF1 - GFP  Using fusion protein expression RsERF1 of Intracellular  Location analysis

GFP was used in the plant expression binary vector to analyze the intracellular location of the RsERF1- GFP fusion protein expressed in the plant. Using the total length cDNA of the RsERF1 gene isolated in Example 1 as a template CACC- RsERF1 primer (5'-CACCATGTGTGGAGGAGCTAT-3 ': SEQ ID NO: 7) and 3'- RsERF1 primer (5'-TCAAAAGACACCACCATCCAG-3': SEQ ID No. 8) was amplified by PCR, and the amplified PCR product (1,081 bp) was cloned into a pENTR vector (Invitrogen), and the nucleotide sequence was confirmed by DNA sequencing. The nucleotide sequence identified pENTR- RsERF1 plasmid, GK targeting gateway vector pK7WFG2 (Plant Systems Biology) and LR recombinase (Invitrogen) were reacted to induce the RsERF1 coding region in the N-terminal portion of GFP. (in-frame) fusion. RsERF1- GFP fusion protein expression was carried out in tobacco plants. The intracellular location of the RsERF1- GFP fusion protein was expressed in tobacco epidermal cells and protoplasts. As a result, it was found that it was expressed around the nucleus (FIG. 6).

Example  4 : RsERF1  Recombinant protein isolation and DNA Binding Activity Assay

PDEST15 (Invitrogen), a GST (glutathione S-transferase) recombinant expression vector, was used to express in E. coli (FIG. 7). Using the entire RsERF1 gene cDNA isolated in Example 1 as a template CACC- RsERF1 primer (5'-CACCATGTGTGGAGGAGCTAT-3 ': SEQ ID NO: 7) and 3'- RsERF1 primer (5'-TCAAAAGACACCACCATCCAG-3': SEQ ID NO: 8) PCR amplification, and the amplified PCR product (1,081bp) was cloned into the pENTR vector (Invitrogen) and the base sequence was confirmed. PENTR- RsERF1 (Stop), a gateway sequence for expressing GST recombinant protein, pDEST15 (Invitrogen), and LR recombinase (Invitrogen) were reacted to clone the RsERF1 coding region after GST and DNA sequencing. The base sequence was confirmed through (FIG. 7). For recombinant protein expression, BL21-Al (Stratagene) competent cells were transformed and plated in LB (Epicillin, 100 μg / ml) selection medium and incubated overnight at 37 ° C. Using colonies obtained as a result of the culture, colony PCR was performed using the RsERF1 gene primer to confirm transformation. PGEX4T-1 (Amersham), BL21 (DE3), pLysS (Stratagene) and activating cells were transformed and plated in LB (Epicillin, 100 μg / ml) selection medium for use as a control experiment. The cells were incubated at 37 ° C. overnight, and colonies obtained as a result of the culture were used.

To isolate the GST fusion protein from Escherichia coli, the pGST- RSERF1 and pGEX4T-1 transformant Escherichia coli were incubated for 1 hour in LB ( Empicillin , 100 µg / ml) liquid medium, and then plated onto LB- Epicillin solid medium. The cells were incubated for 16 hours or longer. E. coli colonies obtained in solid medium were inoculated into LB (Ampicillin, 50 µg / ml) liquid medium, and after incubation to OD 0.6 (1.0 × 10 ⁶ cells), E. coli containing pGST- RSERF1 was treated with 0.2% arabinose and 1 mM IPTG. E. coli, including pGEX4T-1, was incubated at 37 ° C. for 3 hours with the addition of 0.1 mM IPTG. Thereafter, the cultures were centrifuged at 3,000 rpm for 5 minutes to collect the cells, and 1.7 ml of 50 mM Tris-HCl (pH 8.0) buffer was added thereto, and the cells were disrupted using a cell crusher and centrifuged at 5,000 rpm for 5 minutes. . After centrifugation, only the supernatant was centrifuged again at 12,000 rpm for 10 minutes to separate the GST fusion protein using a GST column (Fig. 8). The fusion protein was concentrated using Centricon (Millipore), and the fusion protein concentration was increased by 12% SDS-protein electrophoresis (Laemmli, 1970 Nature 227: 680-685) compared to the BSA sample having a concentration of 0.1 mg / ml. Analyzed.

The GST and GST- RsERF1 proteins were transferred to Hybond ECL nitrocellulose membrane (GE Healthcare Life Sciences), and Western blot was performed with GST antibody to confirm the protein (FIG. 9), and RsERF1 was relatively pure using GST-tag column. Protein was isolated (FIG. 10). Short-length oligonucleotides containing recombinant protein GST- RSERF1 expressed in E. coli and DNA binding recognition sites were artificially prepared, and protein electrophoresis (SDS-PAGE) was performed to confirm binding to DNA binding recognition sites. After electrophoresis, electrophoretic mobility shift assays (EMSA) experiments were performed using DNA labeled with radioisotopes (γ ³² P-ATP). GST- RsERF1 As a result of reacting (1 μg) with GCC and DRE DNA oligomers, it was found that specifically binding to GCC DNA (FIG. 11).

Example  5: RsERF1  Protein Self-Activity Ability Assay

The yeast expression vector pDEST15 (Invitrogen) was used to express in yeast (FIG. 12A). The cloned pENTR- RsERF1 plasmid in Example 3 and the yeast expression gateway pDEST15 vector (Invitrogen) were reacted and cloned to confirm the nucleotide sequence. The nucleotide sequence confirmed pENTR- RsERF1 plasmid, the yeast expression gateway vector pDEST15 (Invitrogen) and LR recombinase (Invitrogen) were reacted to clone the RsERF1 coding region behind the GAL promoter and confirmed the sequence through the DNA sequence. . The pDEST15- RsERF1 transforming each plasmid in MaV203 cells with LiAc method SD (Leu-) were incubated at 30 ℃ 2 days after plated on selective medium, the colonies obtained by culturing the result as the template, using gene primers RsERF1 Colony PCR was performed to confirm the transformation.

The identified colonies were scratched on SD (Leu-, Trp-, His-) medium and SD (Leu-, Trp-, His-, bX-gal) medium to confirm autobinding ability by color change. As a control, three kinds of plasmids provided by Invitrogene were used. WT (pXEP32 / Krev1- pXEP22 / RalGDS-wt) with strong binding force, m1 (pXEP32 / Krev1- pXEP22 / RalGDS-m1) with moderate binding force, and m2 (pXEP32 / Krev1- pXEP22 / RalGDS-m2 without binding force) ) Was used. As a result of comparing with the control group, it was confirmed that the binding force was stronger than the WT showing the strong binding force, but also in the change of color (FIG. 12B).

Example  6: RsERF1 Preparation and transformation of overexpressed transgenic plants

In Example 6, plant expression binary vectors were used to overexpress RsERF1 in Arabidopsis. RsERF1 isolated in Example 1 Using the entire gene cDNA as a template, PCR amplification was performed using CACC- RsERF1 primer (5'-CACCATGTGTGGAGGAGCTAT-3 ': SEQ ID NO: 7) and 3'- RsERF1 primer (5'-TCAAAAGACACCACCATCCAG-3': SEQ ID NO: 8). The PCR product (1,081bp) was cloned into the pENTR vector (Invitrogen) and the nucleotide sequence was confirmed. A 35S- RaERF1 plasmid was prepared by reacting the nucleotide sequence confirmed pENTR- RsERF1 plasmid, pB2GW7 (Plant Systems Biology) and LR recombinase (Invitrogen), which are gateways for overexpression transformation (FIG. 13A). Agrobacterium Tumer faciens C58c1 strain was transformed with freeze-thaw with 35S- RsERF1 and pCAMBIA1301, and transformed Agrobacterium was transformed into Arabidopsis by flower soaking method to transform the transformed seed into 1/2 MS (2 % Sucrose, Basta 25 ㎍ / ㎖) germinated medium to select transformed plants, seeds of each plant were harvested, germinated and genomic DNA was extracted from the growing plant leaves. PCR was performed on the genomic DNA to confirm RsERF1 expression (FIG. 13). RsERF1 gene expression was PCR amplified by 5'- RsERF1 primer (5'-ATGGCCTCTAAGCGGATTCTGAAG-3 ': SEQ ID NO: 9) and 3'- RsERF1 primer (5'-CTAGCCCATTGCATACTTCTGAGTCC-3': SEQ ID NO: 10). After electrophoresis on an agarose (1%) gel, the gel stained with ethidium bromide was photographed by UV irradiation (FIG. 13B). Control plants used wild type Arabidopsis (Columbia 0) plants.

Example  7: RsERF1 Using overexpressed transgenic plants High salt  And osmotic stress resistance functional analysis

Total RNA was isolated from wild-type and overexpressed RsERF1 transgenic Arabidopsis grown about 6 weeks in growth using RNA extraction kit (Ambion). RT-PCR was performed using the isolated RNA as follows. Synthesis of cDNA with total RNA (2 μg), the actin expression of the housekeeping gene Actin using the cDNA prepared for each sample as a template was 5'- actin primer (5'-TGGACTCTGGTGATGGTGTC-3 ': SEQ ID NO: 11) and reverse 3'-actin primer (5'-CCTCCAATCCAAACACTGTA-3 ': SEQ ID NO: 12) were used, and forward 5'- RsERF1 primer (5'-ATGTGTGGAGGAGCTATAATCTC-3': SEQ ID NO: 5) was used to analyze RsERF1 expression. ) And reverse 3'- RsERF1 primer (5'-AAAGAACTGATTGTTGAACATCTG-3 ': SEQ ID NO: 6). Osmotic stress marker gene expression in wild-type and overexpressing transgenic plants was PCR performed with the following primers; F-SOS3 (5'-TCAAGAACATGACTTTGCCATATC-3 ': SEQ ID NO: 13), R-SOS3 (5'-ATGGCTTATATTAGGAAGATACGT-3': SEQ ID NO: 14), F-AtPDF1.2 (5'-TTGCTTCCATCATCACCCTTATCTTC-3 ': SEQ ID NO: 15), R-AtPDF1.2 (5'-ACGTAACAGATACACTTGTGTGCT-3 ': SEQ ID NO: 16), F-Rd29b (5'-AAGCTACCTCTCTCCGGAGGTGGAAGTG-3': SEQ ID NO: 17), R-Rd29b (5'-TACGTGTAACAGCAAGGACGAGGGAG-3 ' : SEQ ID NO: 18), F-CHS (5'-TCACCAACAGTGAACACATGACC-3 ': SEQ ID NO: 19), R-CHS (5'-GAGTCAAGGTGGGTGTCAGAGG-3': SEQ ID NO: 20), F-ADH1 (5'-TCCACGTATCTTCGGCCATG-3 ': SEQ ID NO: 21), R-ADH (5'-TAGCACCTTCTGCAGCGCC-3': SEQ ID NO: 22), F-SUS1 (5'-ACGGTCAGTTCAGGTGGATCTCCTC-3 ': SEQ ID NO: 23), R-SUS1 (5'-AAGGTAACGACGGGCCTCAAGACG- 3 ': SEQ ID NO: 24), F-ICK1 (5'-ACGCACACGTAACCTAAATCG-3': SEQ ID NO: 25), R-ICK1 (5'-GCATCTCCGTCATCAATTTCG-3 ': SEQ ID NO: 26), F-HXK1 (5'-GTAAAGTAGCTGTTGGAGCGAC -3 ': SEQ ID NO: 27), R-HXK I (5'-CATTCTGTAGCGACAAACTTCGC-3': SEQ ID NO: 28), F-COR47 (5'-AAGGTAACGACGGGCCTCAAGACG-3 ': SEQ ID NO: 29), R-COR47 (5'-ACAAGCCTAGTGTCATCGAAAAGC-3 ': SEQ ID NO: 30), F-RAB18 (5'-TGATGTGACACGAAGAGACACGTTCAC-3': SEQ ID NO: 31) and R-RAB18 (5'-AAGACGAGCAGCAGTAGCTCGACGAGG-3 ': SEQ ID NO: 32) . PCR conditions were 95 ° C. 5 minutes; 26 cycles of 95 ° C 1 minute, 55 ° C 1 minute, 72 ° C 1 minute; The final elongation step is 72 ° C for 7 minutes. As a result, it was found that the expression of genes such as SOS3 , AtPDF1.2 , Rd29b , CHS , ADH1 , SUS1 and ICK1 increased in RsERF1 overexpressing transgenic plants than in wild type (FIG. 14).

RsERF1 To determine whether the overexpressing transgenic plants were resistant to saline (NaCl, 0.15M) and mannitol (0.2M) media, seedlings 10 days after germination were transferred to the medium without saline, saline and mannitol. Incubation for 9 days compared plant root growth and live weight (FIG. 15A). The result is RsERF1 in salt and mannitol stress Overexpressing transgenic plants grew better than controls but were less effective against mannitol stress than salt stress. Apparently, transgenic plants overexpressing RsERF1 were found to have superior root and leaf growth, especially in the salt stress treatment group, than control plants (FIG. 15B). Therefore, RsERF1 in plants Gene overexpression is believed to confer salinity and osmotic stress resistance.

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Claims (14)

Consisting of the amino acid sequence of SEQ ID NO: 2, gaetmu (Raphanus sativus ) -related RsERF1 ( Raphanus) sativus ethylene responsive transcription factor 1 ) protein. A gene encoding the protein of claim 1. 3. The gene according to claim 2, wherein the gene consists of the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. Arabidopsis plant cell transformed with the recombinant vector of claim 4. delete A method of enhancing salt or osmotic stress resistance of Arabidopsis plants comprising overexpressing the RsERF1 gene by transforming Arabidopsis plant cells with the recombinant vector of claim 4. delete Transforming Arabidopsis plant cells with the recombinant vector of claim 4 to overexpress the RsERF1 gene; And
A method of producing a transgenic Arabidopsis plant with enhanced salt or osmotic stress resistance comprising the step of regenerating Arabidopsis plants from the transformed plant cell. delete A transgenic Arabidopsis plant with enhanced salt or osmotic stress resistance prepared by the method of claim 9. delete Seed of a transgenic Arabidopsis plant with increased salt or osmotic stress resistance according to claim 11. A composition for enhancing salt resistance or osmotic stress resistance of Arabidopsis plants comprising RsERF1 (Raphanus sativus ethylene responsive transcription factor 1) gene derived from Raphanus sativus, consisting of the nucleotide sequence of SEQ ID NO: 1.

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