KR102608324B1 - OsSIRP4 gene from Oryza sativa for controlling salt stress tolerance of plant and uses thereof - Google Patents

Abstract

The present invention relates to the OsSIRP4 gene derived from rice, which regulates the salt stress tolerance of plants, and its use. The present invention can regulate the tolerance to salt stress of plants by regulating the expression of the OsSIRP4 gene, thereby increasing tolerance to environmental stress. It is expected that it will be useful in developing new transgenic plants.

Description

OsSIRP4 gene from rice for controlling salt stress tolerance of plants and uses thereof {OsSIRP4 gene from Oryza sativa for controlling salt stress tolerance of plant and uses thereof}

The present invention relates to the OsSIRP4 gene derived from rice, which regulates salt stress tolerance in plants, and its use.

Plants live by constantly exchanging signals under biological stress, such as pathogen infection and attack by herbivores and insects, and environmental stress, such as drought, heat, cold, and nutritional deficiencies. Plants with low resistance to environmental stress require a lot of energy when adapting to an isolated environment and consume a lot of water and fertilizer, which can place a large burden on the environment. Among environmental stresses, high salt, drought, and temperature changes have a significant impact on the growth and development of sessile plants. If salt concentration increases in the soil, it can affect plant growth, flowering time, etc., and an imbalance in intracellular ion concentration and osmotic pressure can cause nutritional, metabolic or photosynthetic disorders, which can reduce plant production. there is. In this way, plants that live a sessile life face various environmental stresses. In stressed environments, plants regulate protein metabolism and gene expression for ion and plant resistance and cell stability.

Meanwhile, RING (Really Interesting New Gene) E3 ligase has a common sequence consisting of cysteine and histidine residues (Cys-X2-Cys-X9-Cys-X1-3-His-X2-3-Cys/His-X2-Cys- X4-48-Cys-X2-Cys, where X can be any amino acid), and these structures are bonded to two zinc atoms. This structure is generally known that RING-finger proteins are classified into two basic types, including RING-H2 and RING-HC, based on cysteine or histidine positions. Additionally, several modified forms, such as RING-D, RING-v, RING-S/T, RING-G, and RING-C2, have been found in the genomes of higher plants, including Arabidopsis or rice.

Recently, many researchers are interested in and actively researching the functions of genes involved in environmental stress, and breeders are attempting to develop crops that are resistant to environmental stress through cultivar improvement using these genes. Existing breed improvement technology involves selecting only the desired breed from hybrids created by crossing similar species with desired characteristics, and it takes a lot of trial and error and time to develop one breed. In comparison, genetic recombination technology can obtain a desired variety by directly inserting a gene with desired characteristics into another organism. In addition, since the gene to be inserted can be obtained not only from the same species but also from different species, the range of breed improvement is wide and the time required for breed improvement is shorter than that of conventional breed improvement.

Meanwhile, Korean Patent No. 2231136 discloses 'OsCP1 gene derived from rice that regulates salt stress tolerance in plants and its uses', and Korean Patent No. 1962751 discloses 'OsCP1 gene derived from rice that regulates salt stress tolerance in plants'. 'OsMAR1 gene and its use' is disclosed, but there is no description about the rice-derived OsSIRP4 gene that regulates salt stress tolerance of the plant of the present invention and its use.

The present invention was developed in response to the above-mentioned needs. The present inventors confirmed that the expression of the OsSIRP4 gene increased in the stems and roots of rice when treated with salt, and prepared and analyzed Arabidopsis transformants overexpressing the OsSIRP4 gene. As a result, the root growth, survival rate, chlorophyll content, and antioxidant enzyme activity of the overexpression transformant decreased compared to the control plant, and the cotyledon decolorization rate and hydrogen peroxide content increased, indicating sensitivity to salt stress through overexpression of the OsSIRP4 gene. The present invention was completed by confirming that it is possible to increase .

In order to solve the above problem, the present invention transforms plant cells with a recombinant vector containing a gene encoding the rice-derived OsSIRP4 ( Oryza sativa salt-induced RING finger protein 4) protein consisting of the amino acid sequence of SEQ ID NO: 2. A method for controlling salt stress tolerance in plants is provided, which includes regulating the expression of the OsSIRP4 gene.

In addition, the present invention includes the steps of transforming plant cells with a recombinant vector containing a gene encoding the rice-derived OsSIRP4 protein consisting of the amino acid sequence of SEQ ID NO: 2; and redifferentiating plants from the transformed plant cells. A method for producing a transformed plant with controlled salt stress tolerance is provided.

Additionally, the present invention provides transgenic plants with controlled salt stress tolerance prepared by the above method and their transformed seeds.

In addition, the present invention provides a composition for regulating salt stress tolerance in plants, which contains as an active ingredient the gene encoding the OsSIRP4 protein derived from rice consisting of the amino acid sequence of SEQ ID NO: 2.

Since the OsSIRP4 gene of the present invention can regulate the salt stress tolerance of plants, it is expected to improve the productivity of crops by developing plants that are resistant to environmental stress.

Figure 1 shows the results of treating rice with salt (NaCl) and measuring the relative expression level of the OsSIRP4 gene in the shoots and roots of rice.
Figure 2A shows the results of multiple alignment analysis of the ring finger domain of the OsSIRP4 protein and the other Poaceae plants, Bradi5g02290 from wild grass ( Brachypodium distachyon ), GRMZM2G131611 from corn ( Zea mays ), and Sb06g000460 from sorghum ( Sorghum bicolor ). , and Figure 2B is the result of an in vitro ubiquitination assay confirming the E3 ligase activity of OsSIRP4 protein. MBP; maltose binding protein, Poly-Ub; poly-ubiquitin chain, OsSIRP4 ^C269A ; A mutant OsSIRP4 protein in which the cysteine (C) at residue 269 in the ring finger domain of the OsSIRP4 protein is replaced with alanine (A).
Figure 3 is a photograph (A) and root growth observed in OsSIRP4 gene overexpressor (OE-OsSIRP4), mutant OsSIRP4 gene overexpressor (Mutated OE-OsSIRP4), and control plant (WT) treated with salt (NaCl). This is the result of measurement (B).
Figure 4 shows the results (A) and decolorization of cotyledons of OsSIRP4 gene overexpressor (OE-OsSIRP4), mutant OsSIRP4 gene overexpressor (Mutated OE-OsSIRP4), and control plant (WT) treated with salt (NaCl). This is a photo (B) showing the formed cotyledons.
Figure 5 shows the survival rate (A) and chlorophyll content (B) of OsSIRP4 gene overexpressor (OE-OsSIRP4), mutant OsSIRP4 gene overexpressor (Mutated OE-OsSIRP4), and control plant (WT) treated with salt (NaCl). This is one result. Ca; Chlorophyll a, Cb; Chlorophyll b, C(a+b); total chlorophyll
Figure 6 shows hydrogen peroxide content (A) and superoxide dismutase activity (B) of OsSIRP4 gene overexpressor (OE-OsSIRP4), mutant OsSIRP4 gene overexpressor (Mutated OE-OsSIRP4), and control plant (WT) treated with salt (NaCl). , catalase activity (C), peroxidase activity (D), proline content (E), and soluble sugar content (F) were measured.
Figure 7 shows AtSOS1 (A) , AtAKT1 (B), AtNHX1 ( This is the result of measuring the relative expression levels of C) and AtHKT1;1 (D) genes.

In order to achieve the purpose of the present invention, the present invention is to transform plant cells with a recombinant vector containing a gene encoding the rice-derived OsSIRP4 ( Oryza sativa salt-induced RING finger protein 4) protein consisting of the amino acid sequence of SEQ ID NO: 2. Provided is a method for controlling salt stress tolerance in plants, which includes the step of controlling the expression of the OsSIRP4 gene by switching.

The method of controlling salt stress tolerance of plants according to the present invention may be, but is not limited to, increasing the sensitivity of plants to salt stress by overexpressing the OsSIRP4 gene in plant cells.

The scope of the OsSIRP4 protein according to the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 2 isolated from rice and functional equivalents of the protein. In the present invention, the term "functional equivalent" means at least 70%, preferably 80%, more preferably 90% of the amino acid sequence represented by SEQ ID NO: 2 as a result of addition, substitution, or deletion of amino acids. More preferably, it refers to a protein that has a sequence homology of 95% or more and exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 2. “Substantially homogeneous physiological activity” refers to the activity that regulates the salt stress tolerance of plants.

The present invention also includes a gene encoding the OsSIRP4 protein of the present invention, and the scope of the gene includes all of genomic DNA, cDNA, and synthetic DNA encoding the OsSIRP4 protein. Preferably, the gene encoding the OsSIRP4 protein of the present invention may include the base sequence represented by SEQ ID NO: 1. Additionally, homologs of the above base sequence are included within the scope of the present invention. Specifically, the gene includes a base sequence having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the base sequence of SEQ ID NO: 1. can do. The “% sequence homology” for a polynucleotide is determined by comparing two optimally aligned sequences, wherein a portion of the polynucleotide sequence in the region of comparison is a reference sequence (not containing additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps).

As used herein, the term “recombinant” refers to a cell that replicates a heterologous nucleic acid, expresses a heterologous nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the natural form of the cell, either in sense or antisense form. Additionally, recombinant cells can express genes found in cells in their natural state, but the genes have been modified and reintroduced into the cells by artificial means.

As used herein, the term “vector” is used to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. Vectors replicate DNA and can reproduce independently in host cells. The term “vector” is often used interchangeably with “vector”.

Vectors of the present invention can typically be constructed as vectors for expression or cloning. Additionally, the vector of the present invention can be constructed 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 advancing transcription (e.g., pLλ 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 Escherichia coli is used as a host cell, the promoter and operator regions of the E. coli tryptophan biosynthetic pathway and the left-handed promoter of phage λ (pLλ promoter) can be used as control regions.

In the recombinant vector of the present invention, the promoter is a promoter suitable for transformation, preferably the CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter, or histone promoter, and preferably the CaMV 35S promoter. However, it is not limited to this.

As used herein, the term “promoter” refers to the region of DNA upstream from a structural gene and refers to the DNA molecule to which 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 states or cell differentiation. A constitutive promoter may be preferred in the present invention because selection of transformants can be accomplished at various stages and by various tissues. Therefore, constitutive promoters do not limit selection possibilities.

The recombinant vector of the present invention can be constructed by methods well known to those skilled in the art. The methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be effectively linked to an appropriate promoter within an expression vector to drive mRNA synthesis. The vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is the Ti-plasmid vector, which can transfer a part of itself, the so-called T-region, into plant cells when present in a suitable host such as Agrobacterium tumefaciens . Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences into plant cells or protoplasts from which new plants can be produced by properly inserting the hybrid DNA into the plant's genome. there is. A particularly preferred form of Ti-plasmid vectors are the so-called binary vectors as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host include viral vectors, such as those that may be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses, etc. For example, it may be selected from non-intact plant virus vectors. The use of such vectors can be particularly advantageous when it is difficult to properly transform plant hosts.

The recombinant expression vector may preferably contain one or more selectable markers. The marker is a nucleic acid sequence that has characteristics that can be generally selected by chemical methods, and includes all genes that can distinguish transformed cells from non-transformed cells. The marker gene may be an antibiotic resistance gene, but is not limited thereto.

In the recombinant vector of the present invention, common terminators can be used, examples of which include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ), but is not limited to the terminator of the Octopine gene. Regarding the necessity of terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

Any host cell known in the art can be used as a host cell capable of stably and continuously cloning and expressing the vector of the present invention, and examples of prokaryotic cells include E. coli JM109, E. coli BL21, and E. coli. Bacillus strains such as RR1, E. coli LE392, E. coli B, E. coli Enterobacteriaceae strains such as Salmonella typhimurium , Serratia marcescens , and various Pseudomonas species.

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 are used as host cells. , 3T3, RIN and MDCK cell lines) and plant cells can be used. The host cell is preferably a plant cell.

Methods for transporting the vector of the present invention into a host cell include, when the host cell is a prokaryotic cell, the CaCl ₂ method, the Hanahan method (Hanahan, D., 1983 J. Mol. Biol. 166, 557-580), and electroporation. It can be carried out by methods, etc. In addition, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment. You can.

The present invention also includes the steps of transforming a plant cell with a recombinant vector containing a gene encoding the rice-derived OsSIRP4 protein consisting of the amino acid sequence of SEQ ID NO: 2; and

A method for producing a transgenic plant with controlled salt stress tolerance is provided, comprising the step of redifferentiating the plant from the transformed plant cell.

In the method for producing transgenic plants with controlled salt stress tolerance of the present invention, the range of the OsSIRP4 protein is the same as described above.

Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily require a regeneration and/or tissue culture period. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the hybrid DNA according to the invention into suitable progenitor cells. The method is the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373). electroporation (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185) ), particle bombardment (DNA or RNA-coated) of various plant elements (Klein T.M. et al., 1987, Nature 327, 70), Agrobacterium tumefacosis by infiltration of plants or transformation of mature pollen or spores. It can be appropriately selected from ens-mediated gene transfer, infection by a (non-complete) virus (EP 0 301 316), etc. A preferred method according to the invention involves Agrobacterium mediated DNA transfer. Particular preference is given to using the so-called binary vector technique as described in EP A 120 516 and US Pat. No. 4,940,838.

Additionally, any method known in the art can be used to redifferentiate a transformed plant from the transformed plant cell.

The present invention also provides transgenic plants with controlled salt stress tolerance prepared by the above method and their transformed seeds.

The plants include monocots such as rice, barley, wheat, rye, corn, sugarcane, oats, and onions, or Arabidopsis thaliana, potatoes, eggplants, tobacco, peppers, tomatoes, burdocks, mugwort, lettuce, bellflower root, spinach, Swiss chard, and sweet potatoes. , carrots, water parsley, Chinese cabbage, cabbage, mustard radish, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, pea, etc., preferably a monocot, and more preferably a monocot. It may be rice, but is not limited thereto.

The present invention also provides a composition for regulating salt stress tolerance in plants, which contains as an active ingredient the gene encoding OsSIRP4 protein derived from rice, which has the amino acid sequence of SEQ ID NO: 2. The composition of the present invention contains the rice-derived OsSIRP4 protein coding gene as an active ingredient, which can regulate the plant's tolerance to salt stress. When the expression of the gene is increased, the plant's sensitivity to salt stress can be increased.

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1. In rice plants following salt treatment OsSIRP4 Gene expression pattern analysis

Rice ( Oryza sativa cv. Donganbye) seeds were cultured in a growth chamber at 30°C and 70% relative humidity, and 14-day-old rice plants were incubated hourly with MS (Murashige and Skoog) medium containing 150 mM sodium chloride (NaCl). After treatment (6, 12, 24, and 48 hours), qPCR analysis was performed using forward (5'-TCTATGCGACAATTCAGT-3', SEQ ID NO: 3) and reverse (5'-CCTCCACCGTATAATCTC-3', SEQ ID NO: 4) primers. The expression level of OsSIRP4 gene was measured.

As a result, it was confirmed that the expression of the OsSIRP4 gene significantly increased in rice stems and roots upon salt treatment (Figure 1).

Example 2. Structure and E3 ligase activity analysis of OsSIRP4 protein

2-1. Structural analysis of OsSIRP4 protein

To analyze the structure of the OsSIRP4 protein, the Conserved Domain Database (CDD; http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) of the National Center for Biotechnology Information (NCBI) was used. As a result of performing sequence similarity analysis, it was confirmed that the OsSIRP4 protein has a C ₄ HC ₃ type RING domain (C ₄ HC ₃ -type RING domain).

In addition, multiple alignment analysis was performed using the OsSIRP4 amino acid sequence of the present invention and the amino acid sequence of other rice plants having a C ₄ HC ₃ type ring domain (Clustalw2 software, http://ebi.ac.uk/clustalw/) was carried out.

As a result, it was confirmed that the OsSIRP4 protein has a similar amino acid sequence to Bradi5g02290 from wild grass ( Brachypodium distachyon ), GRMZM2G131611 from corn ( Zea mays ), and Sb06g000460 from sorghum ( Sorghum bicolor ) (Figure 2A).

2-2. E3 ligase activity assay of OsSIRP4 protein

The fact that the OsSIRP4 protein has a C ₄ HC ₃ type ring domain means that it has E3 ligase activity, so the E3 ligase activity of the OsSIRP4 protein was measured by performing an in vitro ubiquitination assay. did. The OsSIRP4 gene was cloned into the pMAL-c5x vector (New England BioLabs, USA) containing maltose binding protein (MBP), and the mutant-OsSIRP4 (mutant-OsSIRP4) was cloned through site-directed mutagenesis using mutagenic primers. OsSIRP4, C269A) was prepared. The recombinant protein was expressed in E. coli and purified using amylose resin (New England BioLabs), and the purified MBP-OsSIRP4 protein was mixed with human E1, Arabidopsis E2 (AtUBC10), ubicutin (Ub), and ubiquitination buffer ( After reacting for 2 hours at 30°C (50mM Tris-HCl, PH 7.5; 40mM ATP; 100mM MgCl ₂ ; 40mM dithiothreitol), Western blot was performed. Anti-MBP antibody or anti-Ub antibody was used as the primary antibody, goat anti-mouse IgG or goat anti-rabbit IgG antibody was used as the secondary antibody, and the ChemiDoc ^TM XRS+ imaging system (Bio-Rad, USA) was used. The band was confirmed.

As a result, when the MBP-OsSIRP4 protein was reacted with E1 and AtUBC10 (lane 4), poly-ubiquitein chain (Poly-Ub) was detected, whereas the mutant-OsSIRP4(C269A) protein was detected with E1 and AtUBC10. It was confirmed that poly-ubiquitin chains were not detected when reacted (lane 5) (Figure 2B). Through the above results, it was found that OsSIRP4 protein has E3 ligase activity.

Example 3. OsSIRP4 Analysis of response to salt stress in Arabidopsis transgenic plants with overexpressed genes

To analyze the effect of the OsSIRP4 gene on the regulation of salt stress tolerance in plants, 35S:OE-OsSIRP4-YFP (overexpression) , 35S:mutated OE-OsSIRP4-YFP (mutant) , and 35S:YFP (control) were introduced, respectively. Arabidopsis thaliana plants were transformed using the Agrobacterium GV3101 strain. Transformed seeds (T ₃ ) were cultured in half MS (Murashige and Skoog) medium for 3 days in a growth chamber at 22°C for 16 hours/8 hours (light/dark), and 150 and 200 mM sodium chloride was added to the medium. Each was processed.

The transcript expression levels of each T ₃ line (#1, #2, and #3) of the transformant overexpressing the OsSIRP4 gene, the transformant overexpressing the mutant OsSIRP4 gene, and the control plant were measured using q-PCR (quantitative real time polymerase). chain reaction). As a result, the expression of the OsSIRP4 gene increased in the OsSIRP4 gene overexpressor compared to the control plant, and this confirmed that the OsSIRP4 gene overexpressor was properly produced.

3-1. Root growth analysis

OsSIRP4 gene overexpressor, mutant OsSIRP4 gene overexpressor, and control plants were each treated with sodium chloride for 10 days to measure root growth. As a result, when salt was not treated, there was a significant difference between the OsSIRP4 gene overexpressor, mutant OsSIRP4 gene overexpressor, and control plants. It was confirmed that there was no significant difference in root growth. However, as the concentration of treated salt increased, the root growth of the OsSIRP4 gene overexpressor was confirmed to be significantly reduced compared to the control plant, and the root growth of the mutant OsSIRP4 gene overexpressor was confirmed to be no significant difference from the control plant (Figure 3).

3-2. Cotyledon decolorization rate analysis

As a result of checking the cotyledon decolorization rate by treating OsSIRP4 gene overexpressor, mutant OsSIRP4 gene overexpressor, and control plants with sodium chloride for 10 and 24 days, respectively, it was confirmed that the cotyledon decolorization rate of OsSIRP4 gene overexpressor was significantly increased compared to the control plant. It was confirmed that the cotyledon decolorization rate of the mutant OsSIRP4 gene overexpressor was not significantly different from that of the control plant (Figure 4).

3-3. Survival rate and chlorophyll content analysis

OsSIRP4 gene overexpressor, mutant OsSIRP4 gene overexpressor, and control plants were each treated with 150 mM sodium chloride for 24 days to check the survival rate. As a result, the survival rate of the OsSIRP4 gene overexpressor was confirmed to be reduced by more than 50% compared to the control plant. It was confirmed that the survival rate of the mutant OsSIRP4 gene overexpressor was not significantly different from that of the control plants (Figure 5A).

In addition, the chlorophyll content of the OsSIRP4 gene overexpressor was confirmed to be significantly reduced compared to the control plant, and the chlorophyll content of the mutant OsSIRP4 gene overexpressor was confirmed to be not significantly different from that of the control plant (Figure 5B).

3-4. Antioxidant activity analysis

OsSIRP4 gene overexpressor, mutant OsSIRP4 gene overexpressor, and control plants were each treated with 150 mM sodium chloride for 24 days to determine hydrogen peroxide (H ₂ O ₂ ) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, and peroxidase (POD). ) Activity, proline content, and soluble sugar content were measured.

Add 0.1% (w/v) trichloroacetic acid to Arabidopsis plants, react on ice for 1 hour, centrifuge at 12,000 g for 30 minutes, take the supernatant, and add 25 mM ferric ammonium sulfate, 0.5 M sulfuric acid, 0.1 M sorbitol and 0.125 mM xylenol orange were added and reacted for 30 minutes in dark conditions. Afterwards, the hydrogen peroxide content was analyzed by measuring the absorbance at 560 nm. SOD, CAT and POD activities were determined by Beauchamp C et al. (1971, Anal Biochem. 44:276-287), Aebi H et al. (1984, Methods Enzymol. 105:121-126) and Chance B et al. (1955, Methods enzymol. 2: 764-775), and proline and soluble sugar contents were measured according to Bates LS et al. (2013, Plant Soil. 39:205-207) and Ci DW et al. (2009. Chemosphere. 77:1620-1625). Each was measured according to the method described in .

As a result, it was confirmed that the hydrogen peroxide content of the OsSIRP4 gene overexpressor was significantly increased compared to the control plant, and the hydrogen peroxide content of the mutant OsSIRP4 gene overexpressor was confirmed to be not significantly different from that of the control plant. In addition, the SOD, CAT, and POD activities and proline and soluble sugar contents of the OsSIRP4 gene overexpressor were confirmed to be decreased compared to the control plants, and the hydrogen peroxide content of the mutant OsSIRP4 gene overexpressor was confirmed to be not significantly different from that of the control plants ( Figure 6).

3-5. Analysis of gene expression involved in salt stress response

OsSIRP4 gene overexpressor, mutant OsSIRP4 gene overexpressor, and control plants were each treated with 150 mM sodium chloride for 24 days. AtSOS1 (K+ uptake and transport), AtAKT1 (K+ uptake), and AtNHX1 (Na+/ The expression levels of H+ antiporter) and AtHKT1;1 (K+ uptake and transport) genes were measured.

As a result, it was confirmed that the expression level of genes involved in the salt stress response of the OsSIRP4 gene overexpressor was significantly reduced compared to the control plant, and the expression level of the gene involved in the salt stress response of the mutant OsSIRP4 gene overexpressor was significantly different from that of the control plant. It was confirmed that there was no such thing (Figure 7).

Through the above results, it was confirmed that when the OsSIRP4 gene is overexpressed in plants, the sensitivity to salt stress increases, and it was found that tolerance to salt stress in plants can be controlled by regulating the expression of the OsSIRP4 gene.

<110> KNU-Industry Cooperation Foundation <120> OsSIRP4 gene from Oryza sativa for controlling salt stress tolerance of plant and uses it <130> PN20258 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 1488 <212> DNA <213> Oryza sativa <400> 1 atgaccgtga tttttctaaa taattctttt agctacaaat tgtccatgat ttcgggattg 60 tttgcccttc accagtttga gattgattcc tcctcatacc ctaccctacc ctctctgctg 120 aatgaaatta atgggactag tactgctaca actgttccat tgcaaggtga ccctgcttct 180 gatcaccacc cacatctctc aatcgacata ccaccagctg cagcaagcat gtcacctgca 240 ccgacacaag ctgctgcaga tatcacaccc acaccgacca cttcgatttt gagcaccaaa 300 gcgagcacac ctgcaggttc atgttccagc agaagcacca gcgttgcccc aaagccgcaa 360 cgatcgtcat ccttcatgct gaggcagact gtcaagagcc tcttgccagt gggaagcttc 420 aagtcgtcag tcaagttctt caacgccaga atttcgagga catcgtcgct cccggtgacc 480 gatgtctcgc aggaacaagc cgataaaact tcgactactc atgctgttga taaagcaggt 540 cacatgtacc ggtcacagtc gcttcccatg aacatgaaga aattgaataa tggaaagagc 600 ttcaagagaa tgaattcact cggcggtgtc taccgcgtag ttccttcgac accatcagtc 660 cccgtgacaa gcagcaatgt catcccagat atagtcccat ctgaaccagg tgatgaagat 720 ggagaggaca tagcagagga agaggcagta tgcaggatct gcatggttga gctgtcagaa 780 gggagtgaca ctctgaagct ggagtgttca tgcaagggcg agctcgctct agctcacaag 840 cactgcgcca tgaagtggtt caccatgaaa ggtaccagga catgcgaggt ctgcaaggaa 900 gatgttcaga acctccctgt cacccttgtt cgtgttcaga gcatgcagca gcctgagctt 960 cagaccaacc ctgccaacgc atcaagatac gatcgcctca ggatgtggca gggagcgcca 1020 atccttgtga tcgtcagcat cctcgcctac ttctgcttct tggaacagct gctggttgcc 1080 cgtgatggta ttgcagcgct ggcaatatcg ctgcccttct catgtatcct tggcctcttc 1140 tcatccctca ccacaacaag catggtggca aggagatacg tgtggatcta tgcgacaatt 1200 cagtttctgt tcgtcgtctt cttcacccat ctcttctaca gatatctcca tttgcaagcg 1260 gtgatatcaa tcatcctggc cacgtttgcc gggttcggcg tagggatgac cggcaactcc 1320 atcatcgtgg agattatacg gtggagggcg gcgagggcgg cggcggcgcc gccggctcaa 1380 acccgtcatc gtcgtcgtcg tcatggtcgc aggcaacaac agccaccacc tgcacaacca 1440 gctgcttctt cagctgccgt cgccgacgtt gagaacccac ctgtataa 1488 <210> 2 <211> 495 <212> PRT <213> Oryza sativa <400> 2 Met Thr Val Ile Phe Leu Asn Asn Ser Phe Ser Tyr Lys Leu Ser Met 1 5 10 15 Ile Ser Gly Leu Phe Ala Leu His Gln Phe Glu Ile Asp Ser Ser Ser 20 25 30 Tyr Pro Thr Leu Pro Ser Leu Leu Asn Glu Ile Asn Gly Thr Ser Thr 35 40 45 Ala Thr Thr Val Pro Leu Gln Gly Asp Pro Ala Ser Asp His His Pro 50 55 60 His Leu Ser Ile Asp Ile Pro Pro Ala Ala Ala Ser Met Ser Pro Ala 65 70 75 80 Pro Thr Gln Ala Ala Ala Asp Ile Thr Pro Thr Pro Thr Thr Ser Ile 85 90 95 Leu Ser Thr Lys Ala Ser Thr Pro Ala Gly Ser Cys Ser Ser Arg Ser 100 105 110 Thr Ser Val Ala Pro Lys Pro Gln Arg Ser Ser Ser Phe Met Leu Arg 115 120 125 Gln Thr Val Lys Ser Leu Leu Pro Val Gly Ser Phe Lys Ser Ser Val 130 135 140 Lys Phe Phe Asn Ala Arg Ile Ser Arg Thr Ser Ser Leu Pro Val Thr 145 150 155 160 Asp Val Ser Gln Glu Gln Ala Asp Lys Thr Ser Thr Thr His Ala Val 165 170 175 Asp Lys Ala Gly His Met Tyr Arg Ser Gln Ser Leu Pro Met Asn Met 180 185 190 Lys Lys Leu Asn Asn Gly Lys Ser Phe Lys Arg Met Asn Ser Leu Gly 195 200 205 Gly Val Tyr Arg Val Val Pro Ser Thr Pro Ser Val Pro Val Thr Ser 210 215 220 Ser Asn Val Ile Pro Asp Ile Val Pro Ser Glu Pro Gly Asp Glu Asp 225 230 235 240 Gly Glu Asp Ile Ala Glu Glu Glu Ala Val Cys Arg Ile Cys Met Val 245 250 255 Glu Leu Ser Glu Gly Ser Asp Thr Leu Lys Leu Glu Cys Ser Cys Lys 260 265 270 Gly Glu Leu Ala Leu Ala His Lys His Cys Ala Met Lys Trp Phe Thr 275 280 285 Met Lys Gly Thr Arg Thr Cys Glu Val Cys Lys Glu Asp Val Gln Asn 290 295 300 Leu Pro Val Thr Leu Val Arg Val Gln Ser Met Gln Gln Pro Glu Leu 305 310 315 320 Gln Thr Asn Pro Ala Asn Ala Ser Arg Tyr Asp Arg Leu Arg Met Trp 325 330 335 Gln Gly Ala Pro Ile Leu Val Ile Val Ser Ile Leu Ala Tyr Phe Cys 340 345 350 Phe Leu Glu Gln Leu Leu Val Ala Arg Asp Gly Ile Ala Ala Leu Ala 355 360 365 Ile Ser Leu Pro Phe Ser Cys Ile Leu Gly Leu Phe Ser Ser Leu Thr 370 375 380 Thr Thr Ser Met Val Ala Arg Arg Tyr Val Trp Ile Tyr Ala Thr Ile 385 390 395 400 Gln Phe Leu Phe Val Val Phe Phe Thr His Leu Phe Tyr Arg Tyr Leu 405 410 415 His Leu Gln Ala Val Ile Ser Ile Ile Leu Ala Thr Phe Ala Gly Phe 420 425 430 Gly Val Gly Met Thr Gly Asn Ser Ile Ile Val Glu Ile Ile Arg Trp 435 440 445 Arg Ala Ala Arg Ala Ala Ala Ala Pro Pro Ala Gln Thr Arg His Arg 450 455 460 Arg Arg Arg His Gly Arg Arg Gln Gln Gln Pro Pro Pro Ala Gln Pro 465 470 475 480 Ala Ala Ser Ser Ala Ala Val Ala Asp Val Glu Asn Pro Pro Val 485 490 495 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tctatgcgac aattcagt 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cctccaccgt ataatctc 18

Claims (6)

Including the step of controlling the expression of the OsSIRP4 gene by transforming plant cells with a recombinant vector containing a gene encoding the rice-derived OsSIRP4 ( Oryza sativa salt-induced RING finger protein 4) protein consisting of the amino acid sequence of SEQ ID NO: 2. A method of controlling salt stress tolerance in plants. The method according to claim 1, wherein the sensitivity of the plant to salt stress is increased by overexpressing the OsSIRP4 gene. Transforming a plant cell with a recombinant vector containing a gene encoding the rice-derived OsSIRP4 protein consisting of the amino acid sequence of SEQ ID NO: 2; and
A method of producing a transgenic plant with controlled salt stress tolerance, comprising the step of redifferentiating a plant from the transformed plant cell. A transgenic plant with controlled salt stress tolerance prepared by the method of claim 3. A transformed seed of the plant according to claim 4. A composition for regulating salt stress tolerance in plants, containing as an active ingredient the gene encoding OsSIRP4 protein derived from rice, which consists of the amino acid sequence of SEQ ID NO: 2.