KR101412555B1 - Method for producing transgenic plant with increased resistance to environmental stresses using OsCYP19-4 gene - Google Patents

Abstract

The present invention relates to: a method for increasing the environment stress resistance of a plant in comparison with a non-transformant by including a step of overexpressing an OsCYP19-4 gene by transforming a recombinant vector, which includes a rice-derived Oryza sativa cyclosporin A-binding protein (OsCYP19-4) coding gene, on a plant cell; a method for producing a transformant plant with increased environment stress resistance in comparison with a non-transformant by including a step of transforming a recombination vector, which includes the rice-derived OsCYP19-4 protein coding gene, on a plant cell; and a composition for increasing the environment stress resistance of a plant which includes a transformant plant with an increased environment stress resistance in comparison with a non-transformant produced by the method, a seed thereof, and the OsCYP19-4 gene as effective components. In the present invention, the gene is expected to increase a resistance to the low-temperature stress of rice as an OsCYP19-4 gene is approved to have an important function in the low-temperature stress resistance.

Description

[0001] The present invention relates to a method for producing transgenic plants having increased tolerance to environmental stress using OsCYP19-4 gene derived from rice,

The present invention relates to a method for producing a transgenic plant in which resistance to environmental stress is enhanced using a rice-derived OsCYP19-4 gene, and more particularly, to a method for producing a transgenic plant having OsCYP19-4 gene (Oryza sativa cyclosporin A-binding protein) A method for increasing environmental stress tolerance of a plant in comparison with a non-transformant comprising the step of overexpressing OsCYP19-4 gene by transforming a recombinant vector containing the gene into a plant cell, a method of increasing OsCYP19-4 protein coding gene , A method for producing a transgenic plant having increased resistance to environmental stress compared to a non-transformant comprising transforming a plant cell with a recombinant vector comprising the plant, The increased transgenic plants and their seeds and plants containing the OsCYP19-4 gene as an active ingredient And a composition for increasing environmental stress tolerance.

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.

Environmental stresses are water stress, cold stress, salt stress, and oxidative stress, which inhibit plant growth and limit the productivity of crops in many important agricultural fields. Particularly, water stress is caused by external environment such as dehydration, high saltiness and low temperature, and is one of the most severe stresses in environmental stress which inhibits plant growth.

Cold weather, one of the environmental stresses, is a disaster that affects the growth and harvesting of crops for a long period of time, which is low enough to cause the crops to grow during the growth period of the crops. The extent to which cold weather is affected depends on the type of crop and the degree of growth. The cold weather of rice can be divided into various types of cold weather depending on the time of receiving cold weather. Typical examples are delayed cold weather, obstructive cold weather and mixed cold weather. Differences are also widely recognized. Delayed cold weather is a type of cold weather in which rice grows from the early stage of growth to heading during cold weather, delaying heading and maturing, and causing poor roasting. In the rice cold weather, rice from cold water formation to flowering, It is a cold form in which normal reproductive organs are not formed or infertility occurs due to obstruction of water and fertilization. Although the weather is good in the vegetative growth period, the pollen cell is temporarily damaged by the low-temperature invasion in the transgenic shooter or the outbreak period, which is an important period of the reproductive growth period, and the pollen blisters, the scattering of the pollen, It is a cold-weather type that causes infertility by inhibiting its action. Mixed cold weather is a cold weather caused by overlapping of delayed cold weather and obstructive cold weather in rice, and the degree of water reduction is extremely high.

The years of severe cold weather in Korea have been occurred in 1971, 1980, 1988 and 1993 with a period of about 10 years. In 2004, the cold weather in the southern middle mountainous area was about 4,000ha. In the summer of 1980, the Sea of Okhotsk was strongly expanded to affect the lower reaches of the Yangtze River in China, resulting in a cold weather throughout the peninsula. At that time, rice paddies and other crops suffered a great deal of cold weather, especially in the highlands of more than 300 meters and the East Coast. In addition, if the summer rainy season lasts for a long time and the low temperature temperate is continued, it will often suffer cold weather. Especially in the mountains and highlands, even if the temperature is lower than normal, the damage is great. The low temperature phenomena in 1980 and 1993 accompanied delayed and obstructed cold weather, and cold weather in Taebaek quadrangle in mid August 1988 was a typical obstacle cold weather. The low temperature phenomenon that appears in summer in Korea is the East Sea type cold water which is mainly caused by the expansion of the high pressure of Okhotsk, and the cold period is long and the damage is great. The cold weather in the East Sea is characterized by the end of Korea and the damage to the east.

Cold weather due to anthropogenic causes is a case where breeds, seedlings, rice cultivation, fertilization, water management, pest control are inappropriate for the local climatic conditions. Recently, a large number of new varieties have been developed with the rapid increase in yields, but in the case of abnormal cold accidents, the damage is often much worse than other conventional varieties.

Korean Patent Registration No. 0440725 discloses a method for increasing the tolerance of monocotyledonous plants against abiotic stress, and Korean Patent No. 101154323 discloses a method for increasing the tolerance of monocotyledonous plants against abiotic stress by using a gene encoding a OsGRP6 protein derived from rice Or frozen-tolerance. 'However, the method for producing an environmental stress-tolerant plant using the rice-derived OsCYP19-4 gene as described in the present invention and its use have not been disclosed.

The present invention has been made in view of the above-mentioned demand, and it is confirmed that the gene expression of OsCYP19-4 ( Oryza sativa cyclosporin A-binding protein) derived from rice is increased 10 times or more under various stress conditions under low temperature stress by qRT-PCR Respectively. In order to identify the function of OsCYP19-4 gene against cold stress, transgenic plants expressing OsCYP19-4 gene by 35S promoter were produced by reverse genetics to analyze cold stress tolerance. OsCYP19-4 overexpressing plants showed strong resistance and high survival rate compared with wild type rice plants in control at 4 ℃ under low temperature stress condition and the cell membrane damage was more than 2 times lower than that of the control by the measurement of ion leakage And it was found that the physiological condition of plant was better at low temperature stress. Therefore, the present invention has been accomplished by demonstrating that the OsCYP19-4 gene performs an important function involved in cold stress tolerance and has utility in environmental tolerance of major crop sensitive to low temperatures such as rice.

In order to solve the above problems, the present invention includes a step of overexpressing the OsCYP19-4 gene by transforming a plant cell with a recombinant vector containing a gene coding for OsCYP19-4 (Oryza sativa cyclosporin A-binding protein) The present invention provides a method for increasing environmental stress tolerance of a plant compared to a non-transformed plant.

The present invention also provides a method for producing a transgenic plant having increased resistance to environmental stress compared to a non-transformant comprising transforming plant cells with a recombinant vector comprising the rice-derived OsCYP19-4 protein coding gene to provide.

The present invention also provides a transgenic plant having increased environmental stress tolerance compared to the non-transformant produced by the above method.

The present invention also provides a seed of the plant.

Also, the present invention provides a composition for increasing environmental stress tolerance of a plant, which comprises the OsCYP19-4 (Oryza sativa cyclosporin A-binding protein) protein coding gene as an active ingredient.

The rice-derived OsCYP19-4 ( Oryza sativa cyclosporin A-binding protein gene under various environmental stress conditions including low temperature, drought, and oxidation, and the gene expression is increased more than 10 times, especially at low temperature stress. Phenotypic analysis using OsCYP19-4 overexpressed transgenic plants showed that the OsCYP19-4 gene plays an important role in the cold stress tolerance mechanism and the availability of genes that can be introduced into the major useful crops has been proved, It is expected to be useful for increasing the resistance to low temperature stress.

Fig. 1 is a graph showing the results of rice- This is the result of qRT-PCR analysis of gene expression changes in response to various environmental stresses of genes. A) Analysis of OsCYP19-4 gene expression by environmental stress. Gene expression at 0, 1, 3, 6, 12, 24 and 48 hours at 4 ℃ (cold stress, cold), dry (drought stress, Drought) and 10 mM hydrogen peroxide (oxidative stress, H ₂ O _2, hydrogen peroxide) B) Analysis of OsCYP19-4 gene expression by environmental stress. Gene expression analysis at 0, 1, 3, 6, 12, 24, and 48 hours by 200 mM NaCl (salt stress), 100 μM methyl jasmonic acid, and 100 μM cadumium.
Figure 2 is a recombinant construct with T ₃ generation transgenic expression of PCR and RT-PCR analysis of the extent of the inserted gene and inserted in chromosome of a recombinant gene from the body for gene overexpression OsCYP19-4 by reverse genetics (reverse genetics) . A) Schematic diagram of the recombination of the OsCYP19-4 open reading frame (1-208 aa) gene by the 35S promoter in the T-DNA region of the pCAMBIA1300 vector. CaMV 35S, cauliflower mosaic virus 35S promoter; NOS, nopaline synthase terminator. B) Results of OsCYP19-4 gene insertion and insertion gene expression analysis. WT1 and WT2, wild type rice lines; OE1-OE4, independent OsCYP19-4 over-transformed line; RT-PCR, reverse transcription-polymerase chain reaction; 35S-OsCYP19-4, 35S promoter forward and OsCYP19-4 gene specific reverse primer set; OsCYP19-4, OsCYP19-4 gene specific forward and reverse primer sets; OsACT1, OsActin1 gene specific forward and reverse primer sets for cDNA quantification.
FIG. 3 is a phenotype result showing that the transformant overexpressing OsCYP19-4 gene is resistant to cold stress. A) Left and right figure, phenotype of rice plants 2 weeks prior to cold stress. WTs, wild type Dongjin rice lines; OEs, T ₃ Generation-independent OsCYP19-4 overexpression lines. B) On the left, 2-week-old rice plants were subjected to cold stress for 4 days at 4 ° C, followed by phenotype; On the right, 2-week-old rice plants were subjected to cold stress for 4 days at 4 ° C for 5 days. C) Phenotypes of rice plants after restoration to normal temperature after cold stress. On the left, 2-week-old rice plants were subjected to cold stress for 4 days at 4 ° C and then restored for 2 days at 28 ° C. On the right side, a phenotype in which the 2-week-old rice plants were restored at 4 ° C for 5 days under cold stress and then for 28 days at normal temperature for 1 day.
FIG. 4 shows the survival rate of OsCYP19-4 overexpressed transformants against cold stress. A) Phenotypic results after 2 weeks of rice plants under cold stress for 5 days at 4 ° C and then for 10 days at 28 ° C in normal temperature condition to measure survival rate for cold stress. Phenotypic Short - Cycle of 2 - week Rice Plant Growth at 28 ℃ Normal Temperature Before Cold Stress; Phenotypic results after 2 days of recovery from cold stress, after restoring the plants at 28 ℃ for 10 days after cold stress for 5 days at 4 ℃. B) Percentage value indicating the survival rate of wild type and OsCYP19-4 overexpressed after recovery in FIG. 4A. For each line, 20 plants were used and calculated as the standard error for 3 repetitions.
FIG. 5 is a result of analyzing the degree of cell membrane damage caused by low-temperature stress as an ion leak measurement value. A) A graph showing the ion leakage value obtained from the upper second leaf from the 1st day to the 5th day after 2 weeks of rice plants under cold stress at 4 ℃. B) Phenotypic resistance phenotype on day 5 of OsCYP19-4 overexpressed transformants used for ion leakage analysis. Phenotypic Short - Cycle of 2 - week Rice Plant Growth at 28 ℃ Normal Temperature Before Cold Stress; After 5 days of low-temperature treatment, 2-week-old rice plants grown at 28 ℃ normal temperature condition were subjected to cold stress for 5 days at 4 ℃ and then phenotype.

In order to accomplish the object of the present invention, the present invention provides a method for producing a recombinant vector comprising the step of overexpressing OsCYP19-4 gene by transforming plant cells with a recombinant vector comprising a gene coding for OsCYP19-4 (Oryza sativa cyclosporin A-binding protein) The present invention provides a method for increasing the environmental stress tolerance of a plant compared to a non-transformant comprising the plant.

In the method according to an embodiment of the present invention, the OsCYP19-4 protein may be an amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The OsCYP19-4 The range of the protein includes 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 increases the environmental stress tolerance of a plant.

In addition, the present invention provides a gene encoding the OsCYP19-4 protein. The gene of the present invention includes all genomic DNAs or cDNAs encoding the OsCYP19-4 protein. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are 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 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 OsCYP19-4 gene sequence can 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. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors containing OsCYP19-4 gene sequences 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, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cell may be used. The host cell is preferably a plant cell.

The method of 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 and gene bombardment .

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. Examples of biological stresses include pathogens. Abiotic stresses include high concentrations of salt, drying, 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 an embodiment of the present invention, the environmental stress may be low temperature stress, but is not limited thereto.

In addition,

Transforming a plant cell with a recombinant vector comprising OscYP19-4 (Oryza sativa cyclosporin A-binding protein) protein coding gene derived from rice; And

The present invention provides a method for producing a transgenic plant having increased environmental stress tolerance compared to a non-transformant comprising the step of regenerating a plant from the transformed plant cell.

Preferably, the OsCYP19-4 protein comprises the amino acid sequence of SEQ ID NO: 2.

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 the method according to an embodiment of the present invention, the environmental stress may be low temperature stress, but is not limited thereto.

The present invention also provides a transgenic plant having increased environmental stress tolerance and seeds thereof compared to the non-transformant produced by the above method.

Preferably, the transgenic plants and their seeds are transgenic plants and their seeds with increased tolerance to cold, drought or oxidative stress.

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, It is preferably rice.

The present invention also provides a composition for increasing the environmental stress tolerance of a plant, which comprises, as an active ingredient, a gene encoding the OsCYP19-4 protein comprising the amino acid sequence of SEQ ID NO: 2. The composition comprises a gene coding for the OsCYP19-4 protein consisting of the amino acid sequence of SEQ ID NO: 2, and the gene can be transformed into a plant to increase cold stress tolerance of the plant.

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  1: derived from rice by various environmental stress treatment OsCYP19 4 gene expression

The rice-derived OsCYP19-4 protein coding gene (Os06g49470) was classified as an immunophilin group gene having one peptidyl prolyl isomerase domain composed of 208 amino acids, (Ahn et al., 2010 BMC Plant Biology, 10: 253). So far, no study has been conducted on the characteristics and functions of genes. To investigate whether the function of OsCYP19-4 is related to environmental stress defense mechanism, gene expression response to environmental stress treatment was first confirmed by qRT-PCR. Oxidative stress, high concentration salt, methyl jasmonic acid and cadium heavy metals were studied at low temperature, drying, H ₂ O ₂ treatment. For each stress treatment, 2 weeks after seed germination, rice plants were treated with 4 ℃ low temperature stress, dried, 10 mM H ₂ O ₂ , 200 mM NaCl, 10 μM methyl jasmonic acid and 100 μM cadumium Total RNA was extracted at time 0, 1, 3, 6, 12, 24, and 48 hours, and cDNA was synthesized from 2 RNA RNA by MMLV reverse transcriptase (Invitrogen, USA) and used for qRT-PCR. The OsCYP19-4-specific primers were sequenced in the forward direction (SEQ ID NO: 3, 5'-GAA TTC ATG GCG GCG AGG GAG ACG TCG CGC CAC-3 ') (SEQ ID NO: 4, 5'- CTC GAG TCA CTT CAG TTC GCC GCT GTC (SEQ ID NO: 5, 5'-CAT GCT ATC CCT CGT CTC GAC CT-3 ') and the reverse direction (SEQ ID NO: 6, 5'-CGC ACT TCA TGA TGG AGT TGT AT-3 '), respectively. The expression of OsCYP19-4 gene was most strongly increased in low temperature stress among various environmental stresses and started to increase from 3 hours after treatment and showed a very large increase of 13 times or more after 48 hours. After 24 hours of treatment, the cells showed a slight decrease after the increase, and it was confirmed that the cells tended to decrease specifically after 24 hours through three biological repeats. OsCYP19-4 gene expression increased from 3 hours after treatment to 24 hours, and increased to 10 mM H ₂ O ₂ , which induces reactive oxygen species (ROS) In the treatment, it rapidly increased after 1 hour of treatment and continued for 6 hours. After 24 hours, It was confirmed that OsCYP19-4 gene expression was increased rapidly in response to oxidative stress as compared with environmental stress (Fig. 1A). It was also confirmed that the expression of methyl jasmonic acid phytohormone was slightly increased after 3 hours of treatment with high concentration of salt and methyl jasmonic acid phytohormone. For the cadmium treatment of heavy metals, the expression of OsCYP19-4 gene was reduced more than 2-fold from 3 hours after treatment and continued to decrease until 48 hours (Fig. 1B). These results suggest that the expression of OsCYP19-4 gene may play a very important role in response to various environmental stresses, especially at low temperature stress.

Example  2: OsCYP19 -4 overexpressed transformant strain mountain

(SEQ ID NO: 3, 5'-GAA TTC ATG GCG GCG AGG GAG ACG TCG CGC CAC-3 ') and reverse direction (SEQ ID NO: 4, SEQ ID NO: 3) for the production of overexpressed OsCYP19-4 using the 35S promoter, 5'-CTC GAG TCA CTT CAG TTC GCC GCT GTC TGA TAT G-3 ') (FIG. 2A). Using the hygromycin antibiotic from the T ₃ generation selected transformants over-expressing transformant and a 35S genomic DNA that is inserted into the gene OsCYP19-4 secure the rice chromosome (genomic DNA) as the template in a PCR analysis promoter forward primer (SEQ ID NO: 7 , 5'-ATG ACG CAC AAT CCC ACT AT-3 ') and OsCYP19-4 gene-specific reverse primer (SEQ ID NO: 4, 5'-CTC GAG TCA CTT CAG TTC GCC GCT GTC TGA TAT G-3' , All four independent OsCYP19-4 over-transformants were proven to be able to safely insert the T-DNA site into the rice chromosome (FIG. 2B). In order to confirm the level of expression of the inserted foreign gene, total RNA was isolated from four independent overexpressants, and cDNA was synthesized and analyzed by RT-PCR. As a result, all of the four transformants showed a stronger (Fig. 2B). Therefore, these four independent line transformants will be used as reverse genetics materials for functional analysis.

Example  3: OsCYP19 -4 Over-expressing  Identification of cold stress tolerance using

The expression of OsCYP19-4 overexpression showed no resistance phenotype for high salt, dry, H ₂ O ₂ , and methyl jasmonic acid stress, and cadmium (cadium) It was rather sensitive during stress treatment. The OsCYP19-4 gene showed the strongest increase in gene expression during cold stress and resistance to cold stress. Resistance phenotypes were observed after 4 or 5 days of low temperature stress treatment at 2 weeks in soil condition. 2 days before the cold stress treatment, the control group, Dongjin wild type and OsCYP19-4 transformant showed almost similar phenotype (Fig. 3A). When 4 캜 low temperature stress was treated for 4 days or 5 days, wild type rice plants, While the severely dry and twisted typical low-temperature stress phenotype was strongly observed, a slight wilting phenomenon was observed in the over-expressing OsCYP19-4 strain, but showed a very strong resistance compared to the control (Fig. 3B). When restored to the normal temperature condition after the cold stress treatment, it was observed that the wild type plants were almost necrotic by the large temperature difference, whereas the overexpressed plants overcome the large temperature difference well in the recovery process at the normal temperature condition than the control (FIG.

Example  4: OsCYP19 -4 Overexpression Increases Chilling Stress Survival Rate

In order to analyze the resistance to cold stress more precisely, the degree of recovery at normal temperature condition after cold stress was investigated by the survival rate. As shown in FIG. 3C, when the wild type plants are restored to the normal temperature after the low temperature treatment at 4 ° C, most of the plants exhibit necrotic phenotype when the stress is maximized and about 10 days after the recovery period. However, OsCYP19-4 (Fig. 4A). As shown in Fig. Survival rates for 20 plants were calculated by repeating the biological experiment four times for five plants per line for four independent lines of wild type and OsCYP19-4 overexpressed plants. 2 weeks old plants were treated with cold stress at 4 ℃ for 5 days in dark condition and cultured for 10 days under 12 hours light / 12 hours dark condition in plant growth period at 28 ℃ temperature condition. After plant condition was completely dry and no chlorophyll Cases were calculated as mortality. On the average, about 20% of the plants survived in the recovery process after cold stress on the four lines in the wild type, but OsCYP19-4 overexpressed plants showed a high survival rate of about 70-90% 4B). From these results, the transgenic overexpressing OsCYP19-4 gene could reaffirm strong tolerance to cold stress.

Example  5: OsCYP19 -4 Overexpressing  Low ion leakage during cold stress ( ion  leakage value

To analyze the physiological level of OsCYP19-4 overexpression at low temperature stress tolerance, ion leakage was measured. The ion leakage values at 0, 1, 2, 3, 4, and 5 days after cold stress were sampled from the top of the second leaf from the top of each plant, The values were analyzed. After 1 day and 2 days of low temperature treatment, there was no difference in ion leakage values between control and wild type plants, but from 3 days after the low temperature treatment, there was a difference in the ion values released from the cell membrane in the control wild type plants 20%, and after 4 and 5 days, the ion leakage value was rapidly increased to about 40% to 70%, and the phenotype on day 5 was also visually distinguished as a cold stress phenotype . On the other hand, in the overexpressed OsCYP19-4, the difference in ion leakage values between the control and the control began to appear on the 3rd day, and after 4 and 5 days, the ion leakage value was significantly lower than that of the control 5A). As for the phenotype, OsCYP19-4 overexpressant showed stronger resistance than control (Fig. 5B) after 5 days. From the above results, it was confirmed that the cell membrane damage caused by overexpression of OsCYP19-4 was reduced at low temperature stress through analysis of ion leakage value, and the resistance phenotype was confirmed by physiological method.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for producing transgenic plant with increased resistance          to environmental stresses using OsCYP19-4 gene <130> PN13426 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 627 <212> DNA <213> Oryza sativa <400> 1 atggcggcga gggagacgtc gcgccacgcg tcgctctgcc tgtggctcgc gctcgtcgcc 60 gccaccctct ctctcgccca ggcggtagaa tcagaagccg aattgaccaa ggtcaccacc 120 aaggtcttct tcgacatcac aatcaatggc aagccggcag gtcggattgt catggggctt 180 tttggaaaca ctgttcctaa aacagcagag aacttccgag caatttgcac aggtgagaaa 240 ggacttggca agagtggcaa acctctctcc tacaaaggaa cgccattcca caggatcatc 300 ccaggcttca tgatccaagg aggcgacacc gtgagcggca atggaaccgg ctgtgattcg 360 atctatggtg gaatgttccc tgatgagaac ttcaagatca accactctgc accagggttg 420 ctgtccatgg ccaactatgc gaaagataca aacggatccc agttcttcat caccaccgta 480 aagctaaccc ggttggatgg gaagcatgtt gtgtttggca aggtgctttc tggaatggac 540 gtcgtctaca agattgaagc tgaaggcagt cagagtggta ctccacggtc caaagtcctc 600 atatcagaca gcggcgaact gaagtga 627 <210> 2 <211> 208 <212> PRT <213> Oryza sativa <400> 2 Met Ala Ala Arg Glu Thr Ser Arg His Ala Ser Leu Cys Leu Trp Leu   1 5 10 15 Ala Leu Val Ala Ala Thr Leu Ala Gl Ala Val Glu Ser Glu              20 25 30 Ala Glu Leu Thr Lys Val Thr Thr Lys Val Phe Phe Asp Ile Thr Ile          35 40 45 Asn Gly Lys Pro Ala Gly Arg Ile Val Met Gly Leu Phe Gly Asn Thr      50 55 60 Val Pro Lys Thr Ala Glu Asn Phe Arg Ala Ile Cys Thr Gly Glu Lys  65 70 75 80 Gly Leu Gly Lys Ser Gly Lys Pro Leu Ser Tyr Lys Gly Thr Pro Phe                  85 90 95 His Arg Ile Ile Pro Gly Phe Met Ile Gln Gly Gly Asp Thr Val Ser             100 105 110 Gly Asn Gly Thr Gly Cys Asp Ser Ile Tyr Gly Gly Met Phe Pro Asp         115 120 125 Glu Asn Phe Lys Ile Asn His Ser Ala Pro Gly Leu Leu Ser Met Ala     130 135 140 Asn Tyr Ala Lys Asp Thr Asn Gly Ser Gln Phe Phe Ile Thr Thr Val 145 150 155 160 Lys Leu Thr Arg Leu Asp Gly Lys His Val Val Phe Gly Lys Val Leu                 165 170 175 Ser Gly Met Asp Val Val Tyr Lys Ile Glu Ala Glu Gly Ser Gln Ser             180 185 190 Gly Thr Pro Arg Ser Ser Lys Val Leu Ile Ser Asp Ser Gly Glu Leu Lys         195 200 205 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gaattcatgg cggcgaggga gacgtcgcgc cac 33 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 ctcgagtcac ttcagttcgc cgctgtctga tatg 34 <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> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 atgacgcaca atcccactat 20

Claims (10)

A step of transfecting a plant cell with a recombinant vector comprising a gene encoding an OsCYP19-4 (Oryza sativa cyclosporin A-binding protein) protein derived from rice comprising the amino acid sequence of SEQ ID NO: 2 and overexpressing the OsCYP19-4 gene The method of increasing cold stress tolerance of a plant compared to a non-transformant. delete delete Transforming a plant cell with a recombinant vector comprising a gene encoding a rice-derived OsCYP19-4 (Oryza sativa cyclosporin A-binding protein) protein comprising the amino acid sequence of SEQ ID NO: 2; And
Wherein the low temperature stress tolerance is increased compared to the non-transformant comprising regenerating the plant from the transformed plant cell. delete delete A transgenic plant having increased cold stress tolerance compared to the non-transformant produced by the method of claim 4. [8] The transgenic plant according to claim 7, wherein the plant is a monocotyledonous plant. A seed of a plant according to claim 7. A composition for increasing the low-temperature stress tolerance of a plant, which comprises, as an active ingredient, a gene encoding an OsCYP19-4 (Oryza sativa cyclosporin A-binding protein) protein consisting of the amino acid sequence of SEQ ID NO: 2.

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