KR20130022561A - Use of mdhar gene from oryza sativa as regulator of yield and environmental stresses - Google Patents

Abstract

PURPOSE: A use of Oryza sativa-derived mdhar(monodehydroascorbate reductase) gene as a regulator of yield and environmental stress is provided to enhance the yield of rice plants and resistance to environmental stress. CONSTITUTION: mdhar gene is overexpressed by transforming a recombinant vector containing Oryza sativa-derived mdhar gene in a plant cell. The mdhar gene has a base sequence of sequence number 1.

Description

Use of mdhar gene from Oryza sativa as regulator of yield and environmental stresses

The present invention relates to the yield of rice-derived mdhar gene and its use as an environmental stress regulator, and more particularly to rice ( Oryza sativa ) mdhar (monodehydroascorbate reductase) gene to increase the yield of the plant, a method of producing a transgenic plant with an increased yield using the mdhar gene, a transgenic plant with an increased yield produced by the method and its seeds, a method, a method for producing the mdhar gene the environmental stress-resistant transformed increase conversion plant with increased environmental stress tolerance of a plant by using a for yield increase in plants containing the mdhar genetic composition, rice-derived mdhar gene In addition, the present invention relates to a composition for enhancing environmental stress resistance of a transgenic plant having increased environmental stress resistance and a seed thereof and a plant comprising the mdhar gene prepared by the above method.

Increasing incomes and improving cultural standards allow us to live healthier and more affluent lives. In response to these changes in the times, it is necessary to produce foods for various consumer preferences. In particular, the production of high-quality varieties with high taste, quality, and nutritional value should satisfy both farmers and consumers. Recently, the genome sequence of rice has been read, and the basis for analyzing numerous rice gene functions has been established. Using this, it is necessary to develop a technique for isolating useful genes and producing excellent rice varieties desired by the new era.

Fortunately, the recent translation of the rice genome sequence revealed most of the genes, and a population of insertional variants was established, a resource for easier analysis of their function. Therefore, there is a great need to secure the technology for producing useful rice varieties by analyzing useful gene functions by effectively utilizing these resources.

Rice gene sequencing was carried out with the Rice Genome Program established in Japan (International Rice Genome Sequencing Projects 2005). As sequencing and annotations are now over, competition for gene function is fierce. However, since rice is grown in the East, the center of rice research is taking place in the East. Particularly in Japan, systematic research at the national level has been conducted systematically to analyze gene function through the construction of a full-length cDNA library and genome wide full length cDNA ectopic expression. Competitive laboratories are conducting rice research in the US, Europe and Australia. This is because rice is used as a model system to study major traits such as disease and then apply it to wheat or corn. China lags behind Japan, but in terms of size, China is beginning a major research into rice gene function.

Early in Korea, rice gene function research was started, and a population of insertion variants was produced using T-DNA and Ac / Ds system. The T-DNA insert that has been developed so far is over 100,000, and the Ds insert, which is being constructed by the Gyeongsang National University / Rural Development Administration, has exceeded 50,000. Plant J 45: 123-132). These insertion variants facilitated separation of various gene variants through sequence analysis of T-DNA or Ds. Although many laboratories are making similar efforts abroad, they are not as good as Korea. On the other hand, rice transformation, cultivation, and breeding techniques are inferior to that of any other country in the world, but rather superior to most countries. In particular, compared with Japan, Europe, and the United States, where the regulation of transformants is very high, the basis for producing excellent rice varieties in Korea is considered to be relatively superior.

Korean Patent Publication No. 2009-0119884 discloses 'plants having improved yield-related traits and methods for producing the same', and Korean Patent Application Publication No. 2009-0027219 has a yield-related trait improved by regulating expression of NAC transcription factors. Plants and methods for their preparation.

The present invention was derived by the above-mentioned demands, and the present inventors completed the present invention by confirming that the yield of the transformed rice plants into which the mdhar gene derived from rice was increased and the resistance to environmental stress was increased. It became.

In order to solve the above problems, the present invention provides a method of increasing the yield of plants using the rice ( Oryza sativa ) -derived mdhar (monodehydroascorbate reductase) gene.

The present invention also provides a method for producing a transgenic plant having an increased yield using the mdhar gene.

The present invention also provides a transgenic plant with increased yield produced by the above method and its seeds.

In addition, the present invention provides a composition for increasing yield of a plant comprising the mdhar gene.

The present invention also provides a method of increasing the environmental stress resistance of plants using rice-derived mdhar gene.

In addition, the present invention provides a method for producing a transgenic plant having increased environmental stress resistance using the mdhar gene.

The present invention also provides a transgenic plant and its seed having increased environmental stress resistance produced by the above method.

The present invention also provides a composition for enhancing environmental stress resistance of a plant comprising the mdhar gene.

Rice plants transformed with mdhar genes derived from rice, which are developed through the present invention, are characterized by increased environmental stress resistance and increased yields, which can be very useful for increasing yields even in unfavorable conditions for growing rice crops. have.

1 shows PCR separation for homozygous lines selection of T ₂ transgenic rice plants.
2 shows RT-PCR analysis of transgenic rice plants overexpressing the BrMDHAR gene.
3 shows salt stress resistance analysis of transgenic rice plants.
4 shows phenotypic analysis in experimental packaging of transgenic rice plants.
Figure 5 shows the enzyme activity analysis of transformed rice plants.
Figure 6 shows cold stress resistance analysis of transgenic rice plants.
7 shows the growth rate of transgenic plants after sowing.
Fig. 8 shows the number of cereals directly related to the yield of the transformed rice plant.
9 shows enzyme activity analysis of transformed rice plants.
10 is T ₃ Transgenic rice plants are shown to measure growth length in cold stress.
11 shows salt stress resistance analysis of transgenic rice plants.
Figure 12 shows the growth rate of the transgenic plant from the seeding in June 2010 until the harvest season.
Figure 13 shows the transformed rice plant antioxidant activity and lipid oxidation analysis.
14 shows T ₃ and T ₄ Institutional characterization of transgenic rice plants is shown (TPW, total plants weight; CW, culm weight; RW, root weight; NP, number of panicles per hill; NSP, number of spikelets per panicle; FR, filling rate; TGW, total grain weight; 1000 GW, 1000 grain weight).
15 shows salt stress sensitivity confirmation of T-DNA insertion mutant rice plants.
Figure 16 shows the molecular biological function analysis of T-DNA insertion mutant rice plants (A: semi RT-PCR analysis of T-DNA insertion mutant rice plants # 1-4 ( OsMdhar ) and # 6-3 ( OsDhar ) Confirmation of expression of antioxidant genes through B, Enzyme activity analysis of T-DNA insertion mutant rice plant # 1-4 ( OsMdhar ).
Figure 17 shows the morphology analysis in T-DNA insert mutant rice plant experimental packaging (A: effective number of inserted mutant rice plants after sowing).
Figure 18 shows the growth rate of T-DNA insertion mutant rice plants from May 2010 sowing to harvesting.
Figure 19 shows the organ-specific characterization of experimental plants of T-DNA insert mutant rice plants (TPW, total plants weight; CW, culm weight; RW, root weight; NP, number of panicles per hill; NSP, number of spikelets per panicle; FR, filling rate; TGW, total grain weight; 1000 GW, 1000 grain weight).

In order to achieve the object of the present invention, the present invention is a rice ( Oryza sativa) was transformed with the recombinant vector containing the origin mdhar (monodehydroascorbate reductase) gene in the plant cell mdhar It provides a method for increasing the yield of a plant comprising overexpressing a gene.

Mdhar Genes may exist in a multigene family, preferably consisting of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto. In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a base 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, respectively. It may include. "% 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 >

In the method of the present invention, the increase in yield of the plant may be, but is not limited to, an increase in the number of plants.

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 mdhar 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 comprising the mdhar gene sequence and appropriate transcriptional / translational control signals can be constructed by methods well known to those of skill 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 properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance gene, aadA gene, and the like, but are not limited thereto.

In the recombinant vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS, histone promoter, SWPA2 promoter, Clp promoter. 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, the constitutive promoter does not limit the selection possibilities.

In the recombinant vectors of the present invention, conventional terminators can be used, for example nopalin 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 are 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.

Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

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 carrying the vector of the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

In addition, the present invention is a rice ( Oryza Provided is a method for producing a plant with increased yield, comprising transforming a plant cell with a recombinant vector comprising a sativa ) -derived mdhar (monodehydroascorbate reductase) gene and regenerating the plant from the transformed plant cell. Preferably, the mdhar The gene may consist of the nucleotide sequence of SEQ ID NO: 1.

In the method of producing a plant of the present invention, the yield increase may be an increase in the number of grains of the plant, but is not limited thereto.

The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tyumeo Pacific Enschede may be mediated by (Agrobacterium tumefiaciens). In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989, Momillan, N. Y.).

The present invention also provides a transgenic plant with increased yield produced by the above method and its seeds. Preferably, the plant may be a monocotyledonous plant, but is not limited thereto. Preferably, the monocotyledonous plants are Alismataceae, Hydrocharitaceae, Juncaginaceae, Schuchzeriaceae, Potamogetonaceae, Najadaceae, Zosteraceae, Liaceae ( Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae, Iridaceae, Burmanniaceae, Rupaceae, Juncaceae (Commelinaceae), Eriocaulaceae, Laminaceae (Gramineae, Poaceae), Araceae, Lemnaceae, Black Parrot (Sparganiaceae), Typhaceae, Cycloaceae, Cyperaceae It may be, but is not limited to, Musaceae, Zingiberaceae, Cannaceae, or Orchidaceae.

In addition, the present invention is a rice ( Oryza It provides a composition for increasing the yield of plants, including sativa ) derived mdhar (monodehydroascorbate reductase) gene. Preferably, the mdhar The gene may consist of the nucleotide sequence of SEQ ID NO: 1. The composition includes a rice-derived mdhar gene as an active ingredient, and by converting the gene into a plant, the yield of the plant can be increased.

In addition, the present invention is a rice ( Oryza sativa) was transformed with the recombinant vector containing the origin mdhar (monodehydroascorbate reductase) gene in the plant cell mdhar It provides a method of increasing the environmental stress tolerance of a plant comprising overexpressing a gene. Preferably, the mdhar The gene may consist of the nucleotide sequence of SEQ ID NO: 1.

In the method of the present invention, the environmental stress may be, but is not limited to, salt, low temperature or oxidative stress.

In addition, the present invention is a rice ( Oryza It provides a method for producing a plant with increased environmental stress resistance, comprising the step of transforming plant cells with a recombinant vector comprising a sativa ) derived mdhar (monodehydroascorbate reductase) gene and regenerating the plant from the transformed plant cells do. Preferably, the mdhar The gene may consist of the nucleotide sequence of SEQ ID NO: 1.

In the method for producing a plant of the present invention, the environmental stress may be salt, low temperature or oxidative stress, but is not limited thereto.

The present invention also provides a transgenic plant and its seed having increased environmental stress resistance produced by the above method. Preferably, the plant may be a monocotyledonous plant, but is not limited thereto.

In addition, the present invention is a rice ( Oryza Provided is a composition for enhancing environmental stress resistance of plants, including sativa ) -derived mdhar (monodehydroascorbate reductase) gene. Preferably, the mdhar The gene may consist of the nucleotide sequence of SEQ ID NO: 1. The composition includes a rice-derived mdhar gene as an active ingredient, by transforming the gene into a plant, it is possible to enhance the plant's resistance to environmental stress.

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.

 1. Materials and Methods

(One) Monodihydroascorbate Reductase  ( MDHAR ) And Dehydroascorbate  Reductase ( DHAR ) Overexpression vector construction of gene, transformed with the vector T _One  Generation of Rice Plants and Their Seeds

Rice cytoplasmic MDHAR CDNA coding for (NCBI Registration No. D85764.1) The total RNA of Oryza sativa cv.Ilmi was obtained by RT-PCR method. Inserted into TOPO-TA vector (Invitrogen) and confirmed by sequencing, to prepare constructs containing OsMDHAR gene, A gene OsMDHAR cut into Nco Ⅰ and Kpn Ⅰ SWPA2 was inserted into the promoter, nos terminator and the bar selectable marker gene that place match of the vector containing the insert pCAMBIA3300 plants. This construct (pCAMBIA3300 :: OsMDHAR ) was transduced into Agrobacterium strain LBA4404 by electroporation. Korean rice cultivar Ilmi was used as a host for the overexpression of pCAMBIA3300 :: OsMDHAR . Plant transformation, selection, re-differentiation of bar-resistant callus and plants were carried out by the methods described above (Nishimura et al., 2006 Nat protoc 1: 2796-2802). Osmdhar Place to match the pGA1611 plants inserted vectors with a OsMDHAR gene cut with Hind III and Kpn I to the Ubi promoter, nos terminator and selection marker hygromycin selection marker resistance genes in order to produce other structures that contain genes Inserted in This construct (pGA1611 :: OsMDHAR ) was inserted into the pCAMBIA3300 plant insertion vector containing the nos terminator and selection marker bar gene using BamH I and Sac II by blunt end ligation to construct (Ubi :: OsMDHAR ).

50 independent T ₀ expressing OsMDHAR Transformed rice plants (TP) were grown to maturity. T ₁ seed was harvested, and T ₁ progeny from 5 independent T ₀ rice plants were SWPA2-F (5'-caatcaagcattctacttctattgcagc-3 ': SEQ ID NO: 2) and OsMDHAR-R (5'-ttcagtgctcaggatcaactcaatgcc-3'): SEQ ID NO: 3) Segregation pattern analysis was performed using primers. About 1000 T ₂ harvested from T ₁ rice plants Seeds were germinated in rooting medium containing kanamycin. The kanamycin in rooting medium comprises screening for germination of seeds derived from T ₂ T ₁ is a rice plant germination T ₁ The T ₂ seeds harvested from the plants were repeatedly performed until analysis. To identify homozygous plants, PCR analysis, western blot and enzyme activity analysis were performed.

(2) MDHAR Wow DHAR  Gene overexpressed T ₂  Investigation of Environmental Stress Tolerance in Rice Plant Seeds

In February 2008, two pHY101 (Ubi :: BrMdhar) transformant homolines were selected, one pHY102 (SWPA2 :: BrMdhar) transformant homoline, and two pHY104 (SWPA2 :: OsMdhar) transformant homoline were selected. Seeds were examined for genotype and stress (salt and cold) resistance.

In June 2008, two pHY101 transformant homoline, one pHY102 transformant homoline, and two pHY104 transformant homoline were selected, and 12 seedlings were deployed to the experimental plant packaging plant of Kyungpook National University in Hyoyeong-myeon, Gyeongbuk-gun, Traits were observed and biological analysis was attempted.

T _{2 in the} greenhouse in December 2008 PHY101 three transgenic homoline 2 objects, pHY102 transgenic homoline of one object and the object pHY104 transgenic homoline 2 T ₂ Plants grown for 3 weeks after seeding were subjected to RNA expression, enzyme activity and microanalysis under stress conditions (salt, low temperature, oxidation).

After sowing for 6 days after sowing in a greenhouse, the leaves were harvested and the proteins were extracted to analyze the enzyme activity. Protein extraction was performed using an extraction buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM MgCl ₂ , 1 mM PMSF, protease inhibitor cocktails. RNAs of pHY101, pHY102, pHY104 and rice were extracted using RNeasy Plant Mini Kit (QIAGEN, USA).

(3) MDHAR Wow DHAR  Gene overexpressed T ₂ , T ₃  And T ₄ Environmental Stress Tolerance and Phenotypic Observation in Rice Plants

① Stress Tolerance Analysis, Biological Analysis, and Genetic Analysis after Deployment of T Generation ₂ Seeds

In February and June 2009, two pHY103 ( Ubi :: OsMDHAR ) transformant homoline, seven pHY105 ( Ubi :: OsDHAR ) transformant homoline and seven pHY106 ( SWPA2 : OsDHAR ) transformant homoline After selection, they were planted in greenhouses to investigate stress (salt and low temperature) resistance.

In June 2009, two pHY103 transformant homoline, seven pHY105 transformant homoline, and seven pHY106 transformant homoline were selected, and 12 seedlings were distributed to the experimental plant packaging plant of Kyungpook National University in Hyoyeong-myeon, Gyeongbuk-gun, Traits were observed and biological analysis was attempted.

Genotypes were analyzed to determine the isolation rates of two pHY103 transformant homoline, seven pHY105 transformant homoline, and seven pHY106 transformant homoline seeded in greenhouse in February 2009.

T ₂ About 15 seeds are spread in the rice paddy and then PCR is used to determine the genotype of the development line. PCR conditions were 95 ° C., 5 min; 94 ° C., 1 min; 57 ° C., 1 min; 72 ° C., 1 min (35 cycles); It carried out at 72 degreeC and 7 min.

② T ₃ Stress Tolerance Analysis, Biological Analysis, and Genetic Analysis after Generation Seed Development

In February and June 2009, pHY101 transformants, pHY102 transformants and pHY104 transformants were seeded in greenhouses to investigate stress (salt and cold) resistance.

The phenotype of the transformed rice plants sown in the greenhouse was observed. Phenotypic observation after 100 mM salt stress treatment and 10 ° C. low temperature treatment after seeding in a greenhouse was analyzed in comparison with ilmibap which is a control group according to the salt cold stress treatment period.

In June 2009, 12 seedlings of pHY101 transformant, pHY102 transformant and pHY104 transformant were developed in the experimental packaging plant of Kyungpook National University transgenic plant located in Hyoryong-myeon, Gyeongbuk-gun County, and traits were observed and biological analysis was attempted.

RNA, protein expression, and enzyme activity were analyzed in the salt and cold-resistant pHY101 transformants, pHY102 transformants, and pHY104 transformants sown in greenhouses and military experimental packaging in February and June 2009.

RNA was isolated using Tri-reagent solution (Molecular Research Center, INC.), And 1st strand cDNA synthesis was performed by RT-PCR using MMLV reverse transcriptase and oligo d (T) n (Promega).

After sowing for 6 days after sowing in a greenhouse, the leaves were harvested and the proteins were extracted to analyze the enzyme activity. Protein extraction was performed using an extraction buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM MgCl ₂ , 1 mM PMSF, protease inhibitor cocktails.

The activity of MDHAR was measured by measuring the absorbance change at 340 nm (ε = 6.2 mM-1 cm-1) in which the amount of MDHA was reduced to ascorbic acid (pH 7.2, 0.2 mM NADH, 2 mM ascorbate). , 1 unit ascorbic acid oxidase), and the protein content of each crude enzyme solution was measured by the Bradford method.

③ T ₂ , T ₃ Phenotypic Studies on Rice and T _4th Generation Rice Plants

The phenotype of transgenic rice plants sown in experimental packaging in June 2010 was observed. Phenotypic observation after sowing on the experimental package was compared with the control of Ilmi rice, which was the initial growth and the late growth, to analyze the effective number of cereals, weight and yield.

(4) T- DNA  Insert Mutations in Rice Plants Osmdhar  And Osdhar Investigation of gene expression

Knocked on the OsMdhar gene (knock-down) and OsDhar Knock-out Insertion Mutations for Genes Seeds of mutant rice plants were distributed at Pohang University of Science and Technology and deployed in experimental packaging to investigate T-DNA insertion genotypes and to observe traits.

(5) T- DNA  Specific of inserted rice plants Oxidative stress  Stress Sensitivity Analysis of Mutants Under

The T-DNA insertion mutant was developed in a greenhouse, and then genomic DNA of seedlings grown for about two weeks at about 30 ° C. was separated and genotyping was performed to confirm the separation ratio of the development line using PCR. Genomic DNA isolation was performed with homogenization buffer (0.1 M NaCl, 0.2 M sucrose, 0.01 M EDTA, 0.03 M Tris-HCl, pH 8.0) and lysis buffer (0.25 M EDTA, 2.5% (w / v) SDS, 0.5 M Tris- HCl, pH 9.2) was used to mix grind buffer 4: 1, and 0.2 g of plant tissue was frozen in liquid nitrogen and ground in a 1.5 ml tube. Thereafter, 400 μl grind buffer was added and the mixture was placed at 70 ° C. for 30 minutes, followed by centrifugation (12000 rpm, 10 min). The supernatant was washed with 70% EtOH, followed by centrifugation (12000 rpm, 10 min) to remove the supernatant. The precipitate was dried and then dissolved in distilled water at 50 ° C. to extract genomic DNA. PCR conditions were 93 ° C., 3 min; 93 ° C., 1 min; 54 ° C., 1 min; 72 ° C., 1 min (35 cycles); It carried out at 72 degreeC and 7 min.

RNA expression and enzymatic activity analysis of two OsMdhar knockdowns and four OsDhar knockouts sown in greenhouses and experimental packaging in February and June 2008 were performed.

After seeding the T-DNA insertion mutants into the greenhouse, the seedlings grown for about 3 weeks at around 30 ° C were treated with salt and cold stress, and after about 6 days, samples were taken to isolate proteins and to express protein and enzyme activity. It was confirmed. 0.2 g plant tissue was ground using a mortar and pestle. The ground tissue was divided into E-tubes using a baked spatula or all at once in a 15 ml conical tube. Protein extraction was performed by adding homogenization buffer (50 mM Tris-HCl (pH8.0), 1 mM EDTA, 1% NP-40, 1 mM PMSF, protease inhibitor cocktails). Vortex was performed after addition of the homogenization buffer. Thereafter, it was left on ice for 10 minutes, and then centrifuged at 12,000 rpm and 4 ° C. for 20 minutes, and the supernatant was collected and transferred to a new tube. The extracted protein was used for protein expression and enzyme activity measurement.

Homogeneous lines of the OsMdhar Knockdown and OsDhar Knockout lines, which were planted in greenhouses in February and June 2009, were subjected to sensitivity analysis for flame and cold resistance stress.

RNA, protein expression and enzyme activity assays were performed in OsMdhar knockdown and OsDhar knockout individuals sown in greenhouses in February and June 2009.

In June 2009, 12 OsMdhar Knockdown and OsDhar Knockout individuals were developed at the Kyungpook National University Transgenic Rice Plants in Hyoryong -myeon, Gyeongbuk-gun, Korea.

The phenotype of transgenic rice plants sown in experimental packaging in June 2010 was observed. Phenotypic observation after sowing on the experimental package was compared with the initial growth and late growth status of the Dongjin rice, the control group, to analyze the effective number of cereals, weight and yield.

Example  One. MDHAR And DHAR  Gene overexpressed T _One Derived from Seeds of Transgenic Rice Plants T ₂  Flame Tolerance of Transgenic Rice Plants

MDHAR in February 2008 And T ₁ overexpressed DHAR gene Genotyping was performed using PCR to extract the DNA of pHY101, pHY102 and pHY104 individuals seeded in the transformed rice plants in greenhouses to identify the inserted genes of the plants. Experimental results showed that there is a correlation with the insertion gene and the individuals having salt stress resistance (Fig. 1). As a result of extracting DNA from each individual of pHY102 and pHY104, the genotypes were analyzed. As a result, the inserted genes were identified in all individuals of the pHY102 and pHY104 strains except for the control group.

The RNAs of pHY101, pHY102 and pHY104 individuals sown in greenhouses in February 2008 were extracted and analyzed for semi-quantitative RT-PCR expression to determine whether transgenic rice plants had increased transgene expression. As a result of the experiment, transgenic rice plants with oxidative stress resistance were analyzed to have high expression level of transgene RNA. It was confirmed that the RNA expression level of the transgene was low in the transgenic plants grown in the salt stress-free state, and the RNA expression level gradually increased with the salt stress treatment period. The experimental results showed that the transgenic rice plants were resistant to salt stress (FIG. 2). The expression of antioxidant genes was higher in the transgenic rice plants than in WT (Ilmi).

The phenotype of the transformed rice plants sown in the greenhouse and the group was observed. Phenotypic observation after salt stress treatment after seeding in greenhouse was confirmed that transformed rice plants were more resistant to stress than ilmi rice compared to ilmi rice, which is a control group, according to the salt stress treatment period. The salt stress treatment period was treated 15 days after seeding, and treated with 100 mM for about 40 days after salt stress treatment (FIG. 3). Transgenic rice plants pHY102 and pHY104 show higher salt stress resistance than WT. 100 mM salt stress was treated for about 40 days and then regenerated for 10 days.

Phenotypic observation after seeding in the army was developed in June 2008 in the experimental packaging of transgenic plants in Kyungpook National University, Hyoyeong-myeon, Gunwi-gun, Gyeongbuk. After cultivation, the transformants were found to have early growth and late stage than the control group. Growth was confirmed to be good. As a result of the experiment, it was confirmed that the transgenic plants had better growth rate than rice in general paddy conditions (FIG. 4).

The growth rate of transgenic rice plants was investigated after sowing. The control group (ilmi rice), # 53-1 (pHY101), # 53-5 (pHY101), # 58-2 (pHY102), # 64-4 (pHY104) and # 65-2 (pHY104) were the initial growth and late stage In the growth status survey, the transformed rice plants showed faster growth than the control group Ilmi rice (Fig. 4A), and the transgenic rice plants were about 10-20 days earlier than the control group Ilmi rice (Fig. 4B). 4C shows that the number of dialects of transformants was higher than that of the control group as a result of examining the number of dialets directly related to the productivity of the transformants (Red, control; Green, transformants).

After sowing in a greenhouse, after the salt stress treatment, leaves were collected, protein was extracted, and enzyme activity was analyzed. As a result of analyzing the enzyme activity, it was confirmed that the transgenic plants expressed higher enzyme activity than ilmi rice, which is the control group, according to the salt stress treatment period. As a result of the experiment, the transgenic plants showed higher enzymatic activity than ilmi rice, which is the control group, under normal conditions, but increased the enzymatic activity under salt stress conditions. This can be confirmed that the transgenic plants are more resistant to salt stress conditions than ilmibyeong control (Fig. 5).

In enzyme activity analysis of control rice plant and pHY102 ( SWPA2 :: BrMDHAR ), the transformed rice plants showed higher enzymatic activity in salt stress than control rice plant. Experimental results using leaves after 12 days after 100 mM salt treatment.

Example  2. MDHAR And DHAR  Gene overexpressed T ₂ And T ₃ Investigation of Cold Resistance in Transgenic Rice Plants

The phenotype of the transformed rice plants sown in the greenhouse was observed. Phenotypic observations after cold soybean stress treatment after seeding in the greenhouse were confirmed that the transformed rice plants were more resistant to cold soybean stress than ilmi rice compared to ilmi rice, which is a control group, depending on cold soy stress treatment period. Cold thaw stress treatment period was treated after about 30 days after sowing seeds, was treated at 10 ℃ for about 50 days after cold thaw stress treatment (Fig. 6).

After treatment for 10 days cold stress for 50 days and then regenerated for 10 days, the transformed rice plants pHY102 and pHY104 showed higher cold stress resistance than WT (Ilmi rice).

T ₃ Phenotypic observations after sowing transgenic rice plants were carried out at the Kyungpook National University Transgenic Plants in Hyoyeong-myeon, Gyeongbuk-gun, Korea in June 2009. Early and late growth were found to be good. As a result of the experiment, it was confirmed that the transgenic rice plants had better growth rate than ilmi rice under general paddy conditions as in the T ₂ generation (FIG. 7). Early Growth Status and Late Stage of Control (Ilmi rice), # 53-1 (pHY101), # 53-5 (pHY101), # 58-2 (pHY102), # 64-4 (pHY104) and # 65-2 (pHY104) In the growth status survey, the transgenic plants showed better growth than the control group Ilmi rice (FIG. 7A), and the transgenic rice plants emerged about 10-20 days earlier than the control group Ilmi rice (FIG. 7B).

T ₃ Determination of the number of effective tillers after planting transgenic rice plants in the Gunwi was deployed to Kyungpook National University experimental transgenic plants located in North Gyeongsang packaging Gunwi County hyoryeongmyeon in June 2009, was observed to be consistently effective tillering after planting T ₃ The transformed rice plants were found to have a better initial fraction than the control (ilmi rice). The experimental results showed that the transgenic rice plants had better initial adaptability than rice in general paddy conditions (FIG. 8). In the initial number of irradiations of the control group (ilmi rice), # 53-1 (pHY101), # 53-5 (pHY101), # 58-2 (pHY102), # 64-4 (pHY104) and # 65-2 (pHY104) Transgenic rice plants showed better growth than ilmi rice, which is a control group.

After seeding in the greenhouse, the leaves were harvested after cold stress treatment, and proteins were extracted and analyzed for enzyme activity. As a result of analyzing the enzyme activity, it was confirmed that the transgenic rice plants expressed higher enzyme activity than ilmi rice, which is a control group, depending on the cold stress treatment period. The experimental results showed that the transgenic rice plants showed higher enzymatic activity than the ilmi rice, which is the control group, in general, but increased more in cold conditions. As a result, it was confirmed that the transformed rice plants had higher resistance to cold stress than the ilmi rice which is the control group (FIG. 9). Cold stress was grown for 6 days at 10 ° C. and then samples were collected. Enzyme activity analysis of control (ilmi rice), # 58-2 (pHY102) and # 65-2 (pHY104) showed that the transformed rice plants had higher enzymatic activity against cold stress than control.

The phenotype of the transgenic plants sown in the greenhouse was observed. Phenotypic observations after cold soybean stress treatment after planting in the greenhouse were confirmed that the transgenic rice plants showed longer resistance to cold freezing stress than ilmi rice compared to the control group, depending on the cold soybean stress treatment period. Cold thaw stress treatment period was treated after about 10 days after sowing seeds, was treated at 10 ℃ for about 2 weeks after cold thaw stress treatment (Fig. 10). Phenotypes were observed under normal conditions 10 days after sowing of control varieties ilmibap and # 58-2 (pHY102), and after treatment with cold thaw stress at 10 ° C. for about 2 weeks, phenotypes are the same as in FIGS. 10A and 10B. The control varieties ilmibap and # 58-2 (pHY102), and # 65-2 (pHY104) measured the growth length at 10 ℃, the transgenic rice plants were longer than the control variety (Fig. 10C).

Example  3. MDHAR And DHAR  Gene overexpressed T ₂ And T ₃ Transgenic rice plants Antioxidant Activity Investigation

The phenotype of the transformed rice plants sown in the greenhouse was observed. Phenotypic observation after salt stress treatment after seeding in greenhouse was confirmed that transgenic rice plants were more resistant to salt stress than ilmi rice compared to ilmi rice, which is a control group, according to the salt stress treatment period. The salt stress treatment period was treated about 30 days after seeding, and about 40 days after salt stress treatment. The recovery period was then treated for about 14 days (FIG. 11). The 100 mM salt stress was treated for about 40 days and then regenerated for 14 days. Transgenic rice plants pHY103, pHY105 and pHY106 showed higher salt stress resistance than WT.

T ₃ Phenotypic observation after sowing the transformed rice plants in the army was developed in June 2010 at the experimental packaging plant of Kyungpook National University, located in Hyoryong-myeon, Gunbuk-gun, Gyeongbuk, Korea. Early and late growth were found to be good. It bore transgenic rice plants with the result that the growth rate better than in the general non ilmibyeo conditions T ₂ As with the generation, it was confirmed (FIG. 12). The growth status of the control group (ilmi rice), pHY103 ( Ubi :: OsMDHAR ), pHY105 ( Ubi :: OsDHAR ), and pHY106 ( SWPA2 :: OsDHAR ) showed that the transformed rice plants had better growth than ilmi rice, which was the control group.

After planting in the greenhouse, after salt stress treatment, the leaves were harvested, and proteins were extracted, and antioxidant activity and lipid oxidation were analyzed. As a result of analyzing the antioxidant activity, it was confirmed that transgenic rice plants expressed higher antioxidant activity than ilmi rice, which is a control group, according to the salt stress treatment period. As a result of analysis of lipid oxidation, it was confirmed that transgenic rice plants had lower lipid oxidation than ilmi rice, which is a control group, according to the salt stress treatment period. The experimental results showed that transgenic rice plants showed higher antioxidant activity and lower lipid oxidation than ilmi rice, which is a control group, under stress. As a result, it was confirmed that the transformed rice plants had higher resistance to salt stress than ilmi rice, which was the control group (FIG. 13). Salt stress was collected for 12 days after growth at 100 mM, and the samples were collected, and the transformed rice plants were analyzed in the antioxidant activity and lipid oxidation assays of the control (one rice), # 58-2 (pHY102) and # 65-2 (pHY104). The salt stress showed higher antioxidant activity and lower lipid oxidation than ilmi rice.

T ₃ And T ₄ Transgenic rice plants after planting institutional characteristics Gunwi the survey was expanded to Kyungpook National University experimental transgenic plants located in North Gyeongsang packaging Gunwi County hyoryeongmyeon in June 2010, T ₃ October 2010 100000000000000000 years And T ₄ Transgenic rice plants results examining the properties by dividing the respective institution T ₃ Transgenic rice plants were confirmed to be superior to the organ-specific characteristics than the control (ilmi rice) (Fig. 14). The control group (ilmi rice), pHY101, pHY102, pHY103, pHY104, pHY105 and pHY106 were classified by each organ to investigate the properties. Institutional characterization of the rice plants grown in the experimental field showed that the transformed rice plants had higher growth and yield than ilmi rice.

Example  4. MDHAR  And DHAR  T- to Gene DNA Investigation of Flame Resistance and Molecular Biology of Rice Plants Inserted with

OsMdhar planted in greenhouses and troops in February and June 2008 Knockdown As a result of treating the salt stress by seeding the OsDh a r knockout individuals, it was confirmed that the T-DNA insert mutant rice plants were more sensitive to salt stress than the control dongjin rice. 100 mM salt stress was treated for about 30 days. This experiment confirmed that the antioxidant gene plays a very important role in the growth of plants (Fig. 15). T-DNA insertion mutant rice plants # 1-4 ( OsMdhar ) and # 6-11 ( OsDhar ) were treated with 100 mM salt stress for about 30 days. It was found that each T-DNA insertion mutant rice plant was more sensitive to salt stress than WT (Dongjin rice).

Planted in greenhouses and army in February and June 2008 Osmdhar Knockdown And OsDh a r a knockout by inoculating the object handle after salt stress to take a sample in accordance with the period by extracting the RNA and protein was subjected to RNA expression and enzyme activity assays. RNA expression was attempted for expression analysis dependent on semi-quantitative RT-PCR to see if the gene expression of T-DNA insertion mutant rice plants was reduced. As a result of this experiment, RNA expression of T-DNA inserted mutant rice plants was found to be lower than that of control (Dongjin rice) (Fig. 16A), and enzyme activity of T-DNA inserted mutant rice plants was decreased than that of control cultivated rice (Dongjin rice). Figure 16B). These results show that the antioxidant genes of rice plants play an important role in salt stress conditions.

OsMdhar seeded T-DNA insertion mutant rice plants Knockdown The measurement of the effective fraction of OsDh a r knockout individuals was carried out in June 2009 in the experimental plant of Kyungpook National University Transgenic Plants located in Hyoryong -myeon, Gyeongbuk-gun, Gyeonggi-gun, Korea. The plant was found to have a lower initial fraction than Dongjin rice, the control. It was also confirmed that the discharge time was later than Dongjin rice. The experimental results showed that T-DNA insertion mutant rice plants had a poor adaptability than Dongjin rice under normal paddy conditions (FIG. 17).

Investigation of effective number of control products of Dongjin rice, # 1-4 ( OsMdhar ), # 6-3 ( OsDhar ) and # 6-11 ( OsDhar ) showed that the inserted mutant rice plants had fewer effective numbers than that of control (FIG. 17A), the inserted mutant rice plants appeared to be about 10-20 days later than the control varieties Dongjin rice (Fig. 17B).

OsMdhar seeded T-DNA insertion mutant rice plants Knockdown and OsDhar knockout studies were conducted in June 2010 in the experimental packaging of Kyungpook National University Transgenic Plants located in Hyoryong -myeon, Gyeongbuk-gun, U.S. Army . Early growth and late growth were found to be worse. The experimental results showed that the T-DNA insertion mutant rice plants had a lower growth rate than Dongjin rice even in general paddy conditions (FIG. 18). The growth status of the control ( Dongjin rice), OsMdhar and OsDhar showed that the T-DNA insertion mutant rice plants were in poor growth than the control Dongdong rice.

After planting T-DNA inserted mutant rice plants in the army, the characteristics of each organ was developed in June 2010 at the experimental packaging plant of Kyungpook National University Transplanted Plant located in Hyoyeong-myeon, Gunbuk-gun, Gyeongbuk, Korea. As a result of dividing the plants by each organ and examining the characteristics, it was confirmed that the T-DNA insertion mutant rice plants had poorer characteristics by organ than the control (dongjin rice) (FIG. 19). The characteristics of WT ( Dongjin rice), # 1-4 ( OsMdhar ), # 6-3 ( OsDhar r), and # 6-11 ( OsDhar ) were investigated by each institution. DNA insertion mutant rice plants were found to be very poor in their time-dependent characteristics.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Use of mdhar gene from Oryza sativa as regulator of yield and          environmental stresses <130> PN11140 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1570 <212> DNA <213> Oryza sativa <400> 1 cggacgcgtg gcccccaaat cttctccaag tccaatcgac ctcatttcat tcgattcatc 60 gatcgctcgg cgcggcttag ggtttttggc ggcagcgatg gcgtcggaga agcacttcaa 120 gtacgtcatc ctcggcggcg gcgtcgcagc gggatatgcg gcacgggagt tcgccaagca 180 gggtgttaag ccaggggagc tcgccatcat ctccaaggag gccgtggctc cttatgagcg 240 ccctgctctc agcaagggat acctctttcc tcagaatgct gcaagactcc caggatttca 300 tgtgtgtgtc ggcagtggag gagagaggct tttgcccgaa tggtactcag agaaaggcat 360 tgagttgatc ctgagcactg aaattgtcaa agctgatctt gcctccaaga ctttgactag 420 tgcagttgga gcaaccttta catatgagat tttgatcatt gcaactggct cctcagtcat 480 caagctatca gattttggta ctcaaggagc tgattccaac aacattttat atctaaggga 540 agtagatgat gctgacaagc tggttgcagc tatccaagca aagaagggtg gaaaggcagt 600 tattgtagga ggaggttaca ttggtctaga acttagtgct gcattgaaga tcaatgattt 660 tgatgtcacg atggtgtttc ctgaaccttg gtgcatgcct cgtctcttca ctgctgatat 720 cgcggctttc tatgagagtt actacactaa caaaggagtt aagatcgtga agggtacagt 780 agctgttggt tttgatgctg atgctaatgg tgatgtcaca gcagttaacc tgaagaatgg 840 cagtgtgctt gaagctgata ttgttggtgt tggtgttggg ggcagaccgc tgactactct 900 ctttaaaggt caagttgctg aggagaaagg tggaattaag accgatgctt tctttgaaac 960 aagtgttcct ggagtctatg ctgtcggtga tgtggccacc ttccccatga agatgtacaa 1020 tgagttgagg agagtggaac atgttgacca tgctaggaag tctgcagagc aggctgtaaa 1080 ggcaatcaag ggaaaagagt ccggcgagtc cgttgtggag tatgactatc tgccatactt 1140 ctactcccgg tcattcgacc tgggatggca attctacggc gacaacgttg gcgacaccat 1200 cctgttcgga gacagtgacc cgacctctgc caagcccaag ttcgggtcct actggatcaa 1260 ggacggcaag gtgttgggtg ccttcctgga gggtgggtca cctgatgaga acaaggccat 1320 tgccaaggtc gcgaaaactc agccgcccgt tgccaacata gaggagctca agaaggaagg 1380 cctccagttc gccagcaaaa tatgagattt ttgtagtttt gatgtacatt tccacatggt 1440 actcccccta ttggttttct gattgtgatg cgtctgtact ctgtactcct cttaaatatg 1500 ggtacttgct atagctgctg agatttgaat aaagaagttt tctactacaa aaaaaaaaaa 1560 aaaaaaaaaa 1570 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 caatcaagca ttctacttct attgcagc 28 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttcagtgctc aggatcaact caatgcc 27

Claims (14)

Rice ( Oryza sativa) was transformed with the recombinant vector containing the origin mdhar (monodehydroascorbate reductase) gene in the plant cell mdhar A method of increasing the yield of a plant comprising overexpressing a gene. The method of claim 1, wherein the mdhar Gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. Rice ( Oryza transforming plant cells with a recombinant vector comprising a sativa ) -derived mdhar (monodehydroascorbate reductase) gene; And
Method for producing a plant with increased yield comprising the step of regenerating the plant from the transformed plant cells. A transgenic plant having an increased yield produced by the method of claim 3. A seed of a plant according to claim 4. Rice ( Oryza A composition for increasing yield of a plant, comprising a sativa ) -derived mdhar (monodehydroascorbate reductase) gene. Rice ( Oryza sativa) was transformed with the recombinant vector containing the origin mdhar (monodehydroascorbate reductase) gene in the plant cell mdhar A method of increasing the environmental stress tolerance of a plant comprising overexpressing a gene. 8. The system of claim 7, wherein the mdhar Gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. 8. The method of claim 7, wherein said environmental stress is salt, low temperature or oxidative stress. Rice ( Oryza transforming plant cells with a recombinant vector comprising a sativa ) -derived mdhar (monodehydroascorbate reductase) gene; And
Method for producing a plant with increased environmental stress resistance comprising the step of regenerating the plant from the transformed plant cells. The method of claim 10 wherein said environmental stress is salt, low temperature or oxidative stress. A transgenic plant with increased resistance to environmental stress produced by the method of claim 10. Seeds of plants according to claim 12. Rice ( Oryza A composition for enhancing environmental stress resistance of a plant, comprising a sativa ) -derived mdhar (monodehydroascorbate reductase) gene.

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