KR101861716B1 - OsCYP21-4 gene from rice for increasing environmental stress resistance of plant and uses thereof - Google Patents

Abstract

The present invention relates to a rice-derived OsCYP21-4 gene and its use for increasing environmental stress tolerance of a plant, and the present invention allows the OsCYP21-4 gene to be involved in the environmental stress tolerance mechanism of oxidative stress and high concentration salt of plants And it can be used as a gene useful for the development of environmental stress tolerant crops.

Description

[0001] The present invention relates to an OsCYP21-4 gene derived from rice and to a use thereof for increasing environmental stress tolerance of a plant.

The present invention relates to rice-derived OsCYP21-4 gene and uses thereof for increasing environmental stress tolerance of plants.

Studies on the defense mechanism of plants with resistance to plant disease resistance or environmental stress have technological value to identify in vivo mechanisms not yet known. In addition, information on plant defense genes can be used not only as a direct material for molecular genetic breeding of crops, but also for traditional genetic breeding. In terms of environmentally friendly food production, various genes related to disease defenses, or crop breeding using these traits, can reduce food production costs by minimizing losses caused by economically important pathogens, weeds, and environmental stresses, Contributing to the recovery of agricultural products' competitiveness and contributing to the revitalization of various industries associated with the development of new varieties.

Most plants and other organisms are exposed to high temperatures, salting, pollution, pathogens, wounds, cold, extreme light conditions, ozone, excessive UV exposure, and osmosis caused by deterioration of the global environment as well as biological stresses such as germs, And various environmental stresses such as osmotic shock. Among them, high salt concentration causes toxic Na + ion accumulation, osmotic stress, oxidative stress, resulting in reduction of crop productivity and growth inhibition. It causes rapid changes in intracellular ion and water homeostasis, causing the plant to die. Dry stress due to lack of moisture is also a major factor in reducing crop productivity. Plants have a defense mechanism against such moisture, salt, or oxidative stress, and when they are in an environment unsuitable for growth, they tend to adjust to their environment or adjust their physiological metabolic processes to adapt to the environment and survive.

Korean Patent Laid-Open No. 1281072 discloses a method for producing a transgenic plant having enhanced resistance to environmental stress using the OsFKBP16-3 gene and a plant therefrom, and Korean Patent No. 0695072 discloses a method for producing a plant having an abiotic stress Stress-inducible OsAsrl gene and protein 'which promote tolerance to the plant, but the OsCYP21-4 gene derived from rice and its use for increasing the environmental stress tolerance of the plant as in the present invention have not been disclosed.

The present invention has been made in view of the above-described needs, and the present inventors have confirmed by qRT-PCR that expression of OsCYP21-4 gene is increased in response to various environmental stresses. OsCYP21-4 overexpressed transgenic rice plants were constructed to analyze OsCYP21-4 gene function against oxidative stress and salt stress during various environmental stresses. Through phenotypic analysis of the plants, we confirmed the strong resistance of OsCYP21-4 overexpression in oxidative stress and salt stress treatment in rice. Physiological analysis of biochemical, biomass, and oxidative stress related enzyme activity also revealed oxidative stress And resistance to salt stress. It was confirmed that overexpression of the OsCYP21-4 gene promoted tolerance to environmental stress of the plant, thereby completing the present invention.

In order to solve the above problems, the present invention relates to a method for producing a plant resistant to environmental stress, comprising the step of transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsCYP21-4 (cyclosporin A-binding protein) The method comprising the steps of:

The present invention also provides a method for producing a plant cell, which comprises transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsCYP21-4 protein; And

And regenerating the plant from the transformed plant cell. The present invention also provides a method of producing a transgenic plant having resistance to environmental stress.

In addition, the present invention provides transgenic plants and seeds thereof, which are prepared by the above method and whose tolerance to environmental stress is regulated.

In addition, the present invention provides a composition for controlling environmental stress tolerance of a plant, which comprises an amino acid sequence of SEQ ID NO: 2 and which contains a gene encoding rice-derived OsCYP21-4 protein as an active ingredient.

According to the present invention, the rice immunephylline OsCYP21-4 gene did not have a conserved amino acid residue of PPIase, and it was confirmed that PPIase activity was not observed in in vitro experiments using a synthetic substrate. In addition, we confirmed that gene expression was increased by various environmental stresses. Transgenic rice plants overexpressing OsCYP21-4 gene were constructed using the 35S promoter that induces normal expression. OsCYP21-4 overexpressed rice transgenic plants were used to determine whether the OsCYP21-4 gene responded to environmental stress of oxidative stress and high salt stress, and to contribute to the enhancement of environmental stress tolerance of rice. It can be seen from the present invention that the OsCYP21-4 gene is involved in the environmental stress tolerance mechanism of the oxidative stress and the high concentration salt of the plant and can be utilized as a gene useful for the development of environmental stress tolerant crops Expect.

FIG. 1 is a graph showing the expression of rice OsCYP21-4 gene in response to various stresses by semi-RT-PCR and qRT-PCR. A) Analysis of OsCYP21-4 gene expression by developmental stage and tissue of rice. 1W, 2W, 6W, 1 week, 2 weeks and 6 weeks after seed germination; En, endosperm; Ro, root; Sh, sheath; St, stem; Le, leaf. B) OsCYP21-4 gene expression analysis by time of various stresses showed that H ₂ O ₂ (Hydrogen peroxide) and MV (Methyl viologen), heat, and ABA plant hormone treatment, OsCYP21-4 gene expression analysis. qRT-PCR was calculated as the reference error value for 3 replicate experiments, and the cDNA value was quantified by correcting it with OsACT1 gene expression value.
FIG. 2 shows the sequence of the homologous gene of rice pseudomonofilin OsCYP21-4 gene in Brachypodium, corn, barley, Arabidopsis, Chinese cabbage, rapeseed, tea plantation and grape, It is confirmed that it does not have any amino acid residue.
FIG. 3 is a graph showing the results of measurement of PPIase activity of OsCYP21-4 protein by protease-coupled peptide assay. A) Escherichia coli transformed with pET28b recombinant plasmid in which His was fused at the C-terminus of OsCYP21-4, IPTG was treated to induce expression of recombinant protein, and OsCYP21-4 recombinant protein was expressed using Nickel-NTA agarose column The result of separation purification. Un (No induction), protein electrophoresis collected from Escherichia coli without IPTG-induced expression of recombinant protein; In (Induction), Protein electrophoresis collected from E. coli with IPTG for induction of recombinant protein expression; Pu (Purified fraction), Purified fraction fraction. B) Results of measurement of PPIase activity by time of purified OsCYP21-4 protein. PPIase activity was measured at a spectrophotometer absorbance of 390 nm. In the test tube, the OsCYP21-4 protein bound to the C-terminal of histone did not show activity. Using the CypD purified protein as a control.
FIG. 4 shows the results of RT-PCR analysis of the expression levels of recombinant genes in recombinant genes and overexpressed rice plant transformants for the production of overexpressed OsCYP21-4 gene. A) Schematic illustration of the recombination of the OsCYP21-4 ORF (1-235 aa) gene by the 35S promoter in the T-DNA region of the pCAMBIA1300 vector. LB, T-DNA left border; 35S-P, cauliflower mosaic virus 35S promoter; hptII, hygromycin resistance gene; T-NOS, nopaline synthetase terminator; RB, T-DNA right border. B) OsCYP21-4 gene expression analysis in the after OsCYP21-4 overexpressing recombinant gene transfected into rice crop production cost T ₃ generation transformant. Quantification of OsCYP21-4 mRNA expression level by calibrating with OsACT1 gene expression values. WT, wild-type (Dongjin); OE1-OE3, three independent line OsCYP21-4 overexpressed rice plant transformants.
FIG. 5 is a graph showing resistance to high salt stress of rice transgenic plants overexpressing OsCYP21-4 gene. FIG. High - salt stress tolerance phenotype of rice plants overexpressing OsCYP21-4 gene. Phenotype after 5 days after transferring to 1/2 MS medium containing high concentration salt after germination for 3 days in 1/2 MS medium containing hygromycin (50 mg / L) for transgenic plant selection. Photographs of rice plants grown for 5 days in 1/2 MS medium without normal, high concentration salt; Pictured rice plants grown for 5 days in 1/2 MS medium supplemented with 100 mM NaCl, 100 mM high salt; Photographs of rice plants grown for 5 days in 1/2 MS medium supplemented with 150 mM NaCl, 150 mM high salt. B, C) Relative root length measurement graph and plant biomass measurement graph for rice plant for quantitative analysis of salt stress resistance phenotype in A above. 30 individual plants were used for each independent line and calculated as the baseline error value for 3 replicates. D) Photographs of rice plants grown for 5 days in 1/2 MS medium supplemented with 200 mM NaCl, 200 mM high salt. E) Photographs showing the degree of DAB staining in the leaf of rice plants for the analysis of salt stress tolerance phenotype qualification of D above. F, G) Measuring the relative POD (peroxidase) and APX (ascorbate peroxidase) activity of rice plants under high salt stress conditions in D above. WT, wild-type (Dongjin); OE1-OE3, three independent line OsCYP21-4 overexpressed rice plant transformants. For each line, 30 plants were used and calculated as the baseline error for 3 replicates. *, p &lt;0.1; **, p <0.01, P-value by t-test.
FIG. 6 is a graph showing tolerance to H ₂ O ₂ oxidative stress of a rice plant transformant overexpressing the CYP21-4 gene. FIG. A) H ₂ O ₂ oxidative stress tolerance phenotype of OsCYP21-4 gene is overexpressed rice seedlings. Transgenic plants were transferred to 1/2 MS medium containing 10 mM and 30 mM H ₂ O ₂ after germination for 3 days in 1/2 MS medium containing hygromycin (50 mg / L) Phenotype. B) Shot length measurement graph of rice plants for quantitative analysis of oxidative stress tolerance with H ₂ O ₂ treatment for A above. WT, wild-type (Dongjin); OE1-OE3, three independent line OsCYP21-4 overexpressed rice plant transformants. For each line, 30 plants were used and calculated as the baseline error for 3 replicates. *, p &lt;0.1; **, p <0.05, P value by t-test. C, D) Measuring relative POD (peroxidase) and APX (ascorbate peroxidase) activity of rice plants under H ₂ O ₂ treatment oxidative stress condition in A above. WT, wild-type (Dongjin); OE1-OE3, three independent line OsCYP21-4 overexpressed rice plant transformants. For each line, 30 plants were used and calculated as the baseline error for 3 replicates. **, p <0.05, P value by t-test.

In order to accomplish the object of the present invention, the present invention relates to a method for producing a plant cell, which comprises transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsCYP21-4 (cyclosporin A-binding protein) Provides a method to control tolerance.

The method according to one embodiment of the present invention is characterized by overexpression of a recombinant vector comprising a gene coding for OsCYP21-4 protein from rice in a plant cell to increase tolerance to environmental stress of the plant compared to the non-transformant But is not limited thereto.

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 are typically pathogens. Abiotic stresses include high salt, drought (dry), low temperature, high temperature and oxidative stress. The term " environmental stress tolerance "refers to a trait that suppresses or delays plant growth or productivity deterioration due to environmental stress.

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

The OsCYP21-4 protein according to the present invention may be composed of the amino acid sequence of SEQ ID NO: 2, and the OsCYP21-4 gene may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The OsCYP21-4 protein of the present invention comprises a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous bioactivity" means an activity that regulates environmental stress tolerance of a plant.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

In the present invention, the OsCYP21-4 The gene sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host.

Expression vectors comprising the OsCYP21-4 gene sequences of the invention and appropriate transcription / translation control signals can be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

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

The expression vector will preferably comprise 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 phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant gene, aadA gene, and the like, but are not limited thereto.

In the recombinant vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, histone promoter, Clp 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 the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) Terminator of the Octopine gene, and the rrnB1 / B2 terminator of E. coli, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

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

The method of delivering the vector of the present invention into a host cell can be carried out by injecting a vector into a host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, can do.

The present invention also provides a method for producing a plant cell, which comprises transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsCYP21-4 protein; And regenerating the plant from the transformed plant cell. The present invention also provides a method for producing a transgenic plant having a tolerance to environmental stress.

The method according to one embodiment of the present invention is characterized by overexpressing a recombinant vector comprising a rice-derived OsCYP21-4 gene on a plant cell to increase tolerance to environmental stress of a plant compared to a non-transformant, It does not.

Preferably, the OsCYP21-4 protein comprises the amino acid sequence of SEQ ID NO: 2.

In the method according to one embodiment of the present invention, the environmental stress may be salt or oxidative 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.

In addition, the present invention provides transgenic plants and seeds thereof, which are prepared by the above method and whose tolerance to environmental stress is regulated.

Preferably, the transgenic plants and their seeds are transgenic plants and their seeds that have increased resistance to environmental stress compared to non-transformants.

The plant may be selected from the group consisting of Arabidopsis, potato, eggplant, tobacco, red pepper, tomato, burdock, cilantro, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, parsley, parsley, cabbage, cabbage, It may be a dicotyledonous plant such as squash, poultry, strawberry, soybean, mung bean, kidney bean or pea or a monocotyledon such as rice, barley, wheat, rye, maize, sorghum, oat, onion, Preferably a rice plant, but is not limited thereto.

In addition, the present invention provides a composition for controlling environmental stress tolerance of a plant, which comprises an amino acid sequence of SEQ ID NO: 2 and which contains a gene encoding rice-derived OsCYP21-4 protein as an active ingredient. The above composition contains a gene encoding OsCYP21-4 protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient. By transforming a plant with the gene or a recombinant vector containing the gene, Can increase salt or oxidative stress tolerance.

In addition, the present invention provides the use of a gene encoding a rice-derived OsCYP21-4 protein consisting of the amino acid sequence of SEQ ID NO: 2 for controlling environmental stress tolerance of a plant. The gene encoding the OsCYP21-4 protein can be preferably used to increase the salt or oxidative stress tolerance of the plant.

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

Example  1. Developmental timing and organization of rice OsCYP21 -4 gene expression and response to various stress treatments OsCYP21 4 gene expression

(En), roots (Ro), and roots (Ro) were observed from 1 week (1W), 2 weeks (2W) and 6 weeks (6W) Total RNA was extracted by dividing into shoot (Sh), stem (St), and leaf (Le). CDNA was synthesized from 2 μg of RNA extracted with MMLV Reverse Transcriptase (Invitrogen) and used for qRT-PCR analysis. (5'-ATG GCG AGG ATA AAG CCG AAG CAA TTG-3 ': SEQ ID NO: 3) and reverse (5'-TCA GCT CAA AGC TTG CTG TTT TAG as a specific primer for confirming the expression of OsCYP21-4 gene (5'-CAT GCT ATC CCT CGT CTC GAC CT-3 ': SEQ ID NO: 5) and reverse 5' (SEQ ID NO: 4) primers were used for expression correction. -CGC ACT TCA TGA TGG AGT TGT AT-3 ': SEQ ID NO: 6) primers were used. The OsCYP21-4 gene was expressed in all tissues, especially in the one - week - old plants. 2 weeks and 6 weeks showed lower expression in stem tissues than in other tissues, and expression in leaf tissues was higher in 6-week-old plants than in other tissues (FIG. 1A). To investigate the expression of OsCYP21-4 gene expression by various stress treatments, we measured the oxidative stress of 200 mM NaCl, 10 mM H ₂ O ₂ , 10 μM MV (methyl viologen) in rice seedlings grown 7 days after seed germination, High temperature stress at 42 ℃ and plant hormone ABA were treated to extract RNA at time intervals. CDNA was synthesized from 2 μg of RNA extracted with MMLV Reverse Transcriptase (Invitrogen) and used for qRT-PCR analysis. The expression of OsCYP21-4 gene was increased more than 12 times at 3 hours in high concentration salt treatment and increased more than 10 times in 24 hours in drought or ABA treatment. High-temperature stress and oxidative stress The expression of about 4-6 folds was also increased by treatment with 10 mM H ₂ O ₂ and 10 μM MV (methyl viologen), and then gradually decreased (FIG. 1B). Through analysis of gene expression, OsCYP21-4 gene was expressed in most tissues of plants and gene expression was induced and regulated by various stresses.

Example  2. OsCYP21 Sequence structure of the -4 gene

The OsCYP21-4 gene, which belongs to the rice immu- nophilin gene family, consists of 235 amino acids and has only one PPIase (peptidyl prolyl isomerase) domain but no conserved amino acid sequence for PPIase activity (Ahn et al. al., 2010 BMC Plant Biology 10: 253). As a result of sequence analysis of homologous genes of immu- nophilin OsCYP21-4 gene in rice species other than rice, Brachypodium, corn, barley, Arabidopsis, Chinese cabbage, rapeseed, tea plantation and grape, It was predicted that CYP21-4 protein of various species including OsCYP21-4 does not show PPIase activity because it has no amino acid residue (Fig. 2).

Example  3. OsCYP21 -4 protein PPIase  Verify Active

To determine the PPIase activity of OsCYP21-4 protein, protease-coupled peptide assay was used. The OsCYP21-4 ORF (1-235aa) portion was inserted into the pET28b vector and a recombinant plasmid fused with His (Histidine) at the C-terminus of the OsCYP21-4 gene was transformed into E. coli . IPTG was added to induce the expression of OsCYP21-4 protein, and the OsCYP21-4 protein was purified using a nickel-NTA agarose column (FIG. 3A). The PPIase activity of the OsCYP21-4 protein was determined by measuring the concentration of OsCYP21-4 protein in the reaction mixture at a concentration of 390 nm after measuring the concentration of OsCYP21-4 protein. . The concentration of OsCYP21-4 protein was increased to 50, 100, 200 nM, but the absorbance value was the same as that of the blank without the addition of OsCYP21-4 protein (Fig. 3B). At this time, the CypD protein was used as a positive control having PPIase activity (FIG. 3B). From the above examples, it was confirmed that OsCYP21-4 protein does not show activity as proline isomerase in vitro.

Example  4. OsCYP21 -4 gene overexpressed transgenic plants

(5'-GAA TTC ATG GCG AGG ATA AAG CCG AAG CAA TTG-3 ': SEQ ID NO: 7) which is an OsCYP21-4 specific primer was inserted into the pCAMBIA1300 vector into which the 35S promoter was introduced for the production of the overexpressed transformant of OsCYP21-4. PCAMBIA-OsCYP21-4 recombinant plasmid was prepared using primers (5'-GGA TCC TCA GCT CAA AGC TTG CTG TTT TAG CGT GAT G-3 ': SEQ ID NO: 8) (FIG. OsCYP21-4 genes were selected transformants was then high thereof OsCYP21-4 gene over-expressing transgenic T ₃ generation using the hygromycin antibiotic to be inserted into the genome of a rice plant. Total RNA was isolated from three independent overexpressing plants (OE1, OE2, and OE3) and a wild type WT (control) plant to determine the level of expression of foreign genes inserted in rice plants. As a result of PCR analysis, it was confirmed that OsCYP21-4 gene was expressed in all the transformants of three independent lines as compared with the control (Fig. 4B). (5'-CAT GCT ATC CCT CGT CTC GAC CT-3 ': SEQ ID NO: 5) and the reverse direction (5'-CGC ACT TCA TGA TGG AGT TGT AT- 3 ': SEQ ID NO: 6) primer was used. Therefore, each of three independent line transformants produced in rice was used as a material for OsCYP21-4 gene function analysis.

Example  5. OsCYP21 -4 Over-expressing  Identification of high salt stress tolerance using

OsCYP21-4 overexpressed transformants were used to analyze resistance to high salt stress. In order to analyze the salt stress tolerance phenotype of OsCYP21-4 overexpressing transformants in rice, 3 days old rice plants were treated with 100 mM, 150 mM, and 200 mM NaCl for 5 days. The control was germinated in 1/2 MS medium for 3 days and the overexpressed material was germinated in 1/2 MS medium containing hygromycin for 3 days and transferred to a medium containing high concentration salt and then observed for phenotype on day 5 Respectively. There was no difference in the growth of over - expressing plants and control plants when grown under normal conditions. In high salt stress treatments, plant growth was significantly decreased in control plants. In the overexpressed plants, the growth was inhibited as compared with the normal condition in which the salt was not treated, but showed a stress tolerance phenotype in which the root of the plant was better than the control (FIGS. 5A and 5D). The root length and biomass of the overexpressed and control plants were increased by at least 20% as compared to the control (FIG. 5B and 5C) after 100 mM and 150 mM high salt stress treatment, . In order to investigate the degree of ROS production, DAB staining was performed after high salt treatment of rice control and overgrown plant leaves. The stronger the staining during DAB treatment, the higher the concentration of ROS present in the leaves. DAB staining of the plant leaves of overexpressed and control plants resulted in the same degree of staining in the untreated steady state. However, when treated with 200 mM high salt, the overexpressed leaves were less stained than the control leaves It was confirmed that the leaves of the overexpressed plants had a smaller amount of ROS than the leaves of the control (Fig. 5E). In addition, the activity of POD (Peroxidase) or APX (Ascobate Peroxidase) enzymes involved in oxidative stress was measured and found to be higher in the overexpressed body than in the control group at the high salt stress treatment (FIGS. 5F and 5G). This confirmed the salt stress tolerance phenotype of the rice plant overexpressed OsCYP21-4 gene.

Example  6. OsCYP21 -4 Over-expressing  Used Oxidative  Confirm stress tolerance

OsCYP21-4 gene expression analysis revealed an increase in gene expression during oxidative stress (Fig. 1B). In addition, overexpression transformations at high salt treatment resulted in lower ROS than the control, and high activity of oxidation-related enzymes (FIG. 5E, 5F and 5G). Based on these results, resistance to oxidative stress was analyzed using OsCYP21-4 overexpressed transformants. The control was germinated in 1/2 MS medium for 3 days and the overexpressed material germinated in hygromycin-containing 1/2 MS medium for 3 days. The selected 3 day old rice seedlings were treated with 30 mM H ₂ O ₂ for 5 days and the phenotype was observed. When grown under normal conditions, the overexpressed plants grew slightly better than the control but did not show any significant difference. At high concentration of H ₂ O ₂ oxidative stress treatment, plant growth was significantly decreased at the control. The overexpressed plants also inhibited growth compared with the normal condition in which H ₂ O ₂ was not treated, but exhibited an oxidative stress tolerance phenotype in which shoot shoots of the plants grew better than the control (FIG. 6A). In order to quantitatively measure the phenotype of FIG. 6A, shoot lengths of the overexpressed and control plants were measured, and it was confirmed that the shoot length of the overexpressed plants was increased by about 20-30% as compared with the control (FIG. 6B). In addition, the activity of POD (Peroxidase) or APX (Ascobate Peroxidase) enzymes involved in oxidative stress was measured. As a result, high enzyme H ₂ O ₂ oxidative stress treatment showed higher enzyme activity in overexpressed body than control And 6D). This proved the oxidative stress tolerance phenotype of rice plants overexpressing OsCYP21-4 gene.

<110> Korea Research Institute of Bioscience and Biotechnology <120> OsCYP21-4 gene from rice for increasing environmental stress          resistance of plant and uses thereof <130> PN16003 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 708 <212> DNA <213> Oryza sativa <400> 1 atggcgagga taaagccgaa gcaattgcta attcaaagca agacgaagaa ggccccaaca 60 cggatcagtt actccactat cgttacttgg aacctaattg ttatcttggt tgcgctatcc 120 ctctatgcta cctacaggca ttggcatcac aggccaatgc tcgagactga aatggatctc 180 cctcgtgctg agcatgttgg aagatctgag gattccacga agactagtag accaagctat 240 gcggttattg atactgccaa aggctcaatt actatagaaa tttacaaaga tgcttctgct 300 gatgttgtgg atagatttgt tagcttgtgc aagagtaacc attttaaagg aatgccgttt 360 cgtcatgtca tcaaaaactt tgtaattcaa gggggtgatt tcgatttcaa tggtgctgcc 420 caggaatgga tactgaaagc caaagccagt ggagaaaacg ctctaagtcc aaagcacgag 480 gcattcatga tcgggactac gaagaatcct aataataaag gatttgatct ttttatcaca 540 actgctccaa ttcctgactt gaatgacaag cttgttgtat ttgggcaagt tatcaatgga 600 caagatattg ttcaggagat cgaagaagtt gatactgatg agcattacca gccaaaaacc 660 cctatcggca tactcaacat cacgctaaaa cagcaagctt tgagctga 708 <210> 2 <211> 235 <212> PRT <213> Oryza sativa <400> 2 Met Ala Arg Ile Lys Pro Lys Gln Leu Leu Ile Gln Ser Lys Thr Lys   1 5 10 15 Lys Ala Pro Thr Arg Ile Ser Tyr Ser Thr Ile Val Thr Trp Asn Leu              20 25 30 Ile Val Ile Leu Val Ala Leu Ser Leu Tyr Ala Thr Tyr Arg His Trp          35 40 45 His His Arg Pro Met Leu Glu Thr Glu Met Asp Leu Pro Arg Ala Glu      50 55 60 His Val Gly Arg Ser Glu Asp Ser Thr Lys Thr Ser Arg Pro Ser Tyr  65 70 75 80 Ala Val Ile Asp Thr Ala Lys Gly Ser Ile Thr Ile Glu Ile Tyr Lys                  85 90 95 Asp Ala Ser Ala Asp Val Val Asp Arg Phe Val Ser Leu Cys Lys Ser             100 105 110 Asn His Phe Lys Gly Met Pro Phe Arg His Val Ile Lys Asn Phe Val         115 120 125 Ile Gln Gly Gly Asp Phe Asp Phe Asn Gly Ala Ala Gln Glu Trp Ile     130 135 140 Leu Lys Ala Lys Ala Ser Gly Glu Asn Ala Leu Ser Pro Lys His Glu 145 150 155 160 Ala Phe Met Ile Gly Thr Thr Lys Asn Pro Asn Asn Lys Gly Phe Asp                 165 170 175 Leu Phe Ile Thr Thr Ala Pro Ile Pro Asp Leu Asn Asp Lys Leu Val             180 185 190 Val Phe Gly Gln Val Ile Asn Gly Gln Asp Ile Val Gln Glu Ile Glu         195 200 205 Glu Val Asp Thr Asp Glu His Tyr Gln Pro Lys Thr Pro Ile Gly Ile     210 215 220 Leu Asn Ile Thr Leu Lys Gln Gln Ala Leu Ser 225 230 235 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atggcgagga taaagccgaa gcaattg 27 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tcagctcaaa gcttgctgtt ttagcgtgat g 31 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 catgctatcc ctcgtctcga cct 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cgcacttcat gatggagttg tat 23 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gaattcatgg cgaggataaa gccgaagcaa ttg 33 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggatcctcag ctcaaagctt gctgttttag cgtgatg 37

Claims (10)

A plant comprising a step of overexpressing an OsCYP21-4 gene by transforming a plant cell with a recombinant vector comprising a nucleotide sequence of SEQ ID NO: 1 and encoding a rice-derived OsCYP21-4 (cyclosporin A-binding protein) protein Lt; RTI ID = 0.0 &gt; resistance to oxidative stress,
Wherein the shoot length of the transformant is increased by 20 to 30% as compared to the non-transformant under oxidative stress conditions. delete delete delete Transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsCYP21-4 (cyclosporin A-binding protein) protein comprising the nucleotide sequence of SEQ ID NO: 1; And
A method for producing a transgenic plant having increased resistance to oxidative stress comprising regenerating a plant from the transgenic plant cell,
Wherein the shoot length of the transformant is increased by 20 to 30% as compared to the non-transformant under oxidative stress conditions. delete A transgenic plant having increased tolerance to oxidative stress produced by the method of claim 5. delete A transformed seed of a plant according to claim 7. delete

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