Gene related to dehydration isolated from potato

专利摘要:
PURPOSE: A gene related to dehydration tolerance of potato and dehydration-tolerant plant using the same are provided, thereby amplifying the expression of the gene when the plant is faced with dehydration, so that the plant can grow under dehydration condition. CONSTITUTION: The gene StRD22 related to dehydration tolerance of potato is isolated from potato and has the nucleotide sequence set forth in SEQ ID NO: 1, wherein the gene StRD22 is isolated by the steps of: freezing potato treated at 4 deg. C for 8 hours and untreated potato and pulverizing them; extracting RNA from the pulverized potatoes; isolating poly A+RNA from the extracted total RNA; subjecting the poly A+RNA to RNase H-reverse transcriptase to synthesize first strand cDNA; removing RNA using RNase and synthesizing second strand cDNA using DNA polymerase I; inserting the synthesized cDNA into Uni-zap XR vector; in vitro packaging the cDNA inserted vector; infecting E. coli with the cDNA inserted vector to determine Phage titer and amplify the genes; and sequencing the genes.
公开号:KR20040050634A
申请号:KR1020020078495
申请日:2002-12-10
公开日:2004-06-16
发明作者:변명옥；송혜숙；배신철；박재복；김둘이；이기완；이승범；정미정；이부영；권혁빈
申请人:대한민국(관리부서:농촌진흥청)；
IPC主号:

专利说明:

Potato drone adaptation gene and cold-resistant crop using the same {GEN RELATED TO DEHYDRATION ISOLATED FROM POTATO}
[9] The present invention relates to a gene that is specifically expressed in potato, and is intended to be used in developing a poor environmentally-adaptive crop by analyzing the function of the expressed gene.
[10] When a general plant encounters an environment that is unsuitable for plant growth such as drought, salt, low temperature, and high temperature, changes in plant metabolism process or material biosynthesis occur, and it tries to survive by adapting to the poor environment. The RD (Related to Dehydration) gene is the first in the Arabidopsis. An isolated gene is known as a drone related gene.
[11] However, it was very doubtful how much environmental resistance would actually be increased when a gene with amplified gene expression was stressed in plants.
[12] An object of the present invention is to find a drought adaptation gene in a plant and to isolate a gene that is specifically induced during poor environmental stress and to utilize the drought adaptation plant for development.
[1] 1 is a nucleotide sequence of the drought adaptation gene StRD22 gene (1.6kb, 348aa) isolated from the potato of the present invention.
[2] 2 is a Northern expression analysis of the StRD22 gene of the present invention,
[3] 2a is the expression level at low temperature treatment.
[4] 2b is the expression level upon Nacl treatment.
[5] 2c is the expression level in the drought treatment.
[6] Figure 3 is a schematic diagram of the transformation vector and expression of the StRD22 gene of the present invention.
[7] Figure 4 is a transformant expression of the StRD22 gene of the present invention.
[8] Figure 5 is a comparison of bioassay regeneration of the present invention StRD22 transgenic tobacco.
[13] Hereinafter, a specific embodiment of the present invention will be described.
[14] In the present invention, the StRD22 gene was put into a plant transformation vector to measure the change in drought adaptability in tobacco.
[15] In other words, clones were isolated from cDNA library prepared by cold treatment on potato through reverse northern analysis. The nucleotide sequence of the isolated clone was confirmed to be homologous to the StRD22 clone, and the expression characteristics of this gene were compared by Northern analysis. The StRD22 gene was cloned into a plant transformation vector to transform the tobacco under the control of an environmental stress-specific expression promoter. The transgenic tobacco was dried for 20 days with a non-GMO control, a 35S promoter and a pBI121 vector transgenic cigarette containing only a selection marker, allowing the plant to almost dry out, and then immersed again in water to regenerate the plant. Confirmed.
[16] <Step 1> Gene isolation and sequencing for selection of environmentally specific clones from cDNA library
[17] RNA was recovered from pot and untreated ground part of potato treated at 4 ℃ for 8 hours, ground with liquid nitrogen, RNA was extracted with BRL's Trizol reagent, and cDNA gene bank was prepared by Stratagene's Lambda ZAP II. cDNA synthesis kit and Gigapack II gold packaging extract were prepared according to the method suggested by the manufacturer. Total RNA isolated from trizol was isolated from poly A + RNA by Qiagen's oligotex, poly A + RNA was used as a template, RNase H- reverse transcriptase was used to synthesize first strand cDNA, and RNA was removed using RNase H. Second strand cDNA was synthesized using DNA polymerase I. EcoRI adapters were ligation into the synthesized cDNA and treated with XhoI to create EcoRI and XhoI sites and ligation to EcoRI and XhoI treated Uni-zap XR vectors. The ligation solution was packaged using an in vitro packaging kit (Stratagene), infected with E. coli , measured for phage titers, amplified, and used as a preservation stock. The number of plaques was about 700,000.
[18] The sequencing of the selected gene was performed using ABI's big dye terminator, and the homology analysis was confirmed to belong to rd22 using the US NCBI database.
[19] <Step 2> Northern Expression of StRD22 Gene
[20] After 8 hours treatment at 4 ℃ and 250mM NaCl in the pollen, after the time indicated for each sample in Figure 2, the leaves were collected and extracted RNA with BRL company's Trizol reagent and in agarose containing formaldehyde A total of 30 µg of RNA was electrophoresed to transfer the membrane to the membrane by capillary transfer, and Northern hybridization was performed by labeling EcoRI and XhoI cut fragments of StRD22 cDNA clone with Takara's labeling method. As a result, the expression of StRD22 was amplified from 12 hours of low temperature treatment, 3 hours of NaCl treatment, and 8 hours after drought treatment, respectively, through FIGS. 2A to 2C.
[21] <Step 3> Tobacco and potato transformation of StRD22 gene
[22] To introduce the isolated StRD22 gene into tobacco, the chimeric gene produced by linking the structural gene of the StRD22 gene to the rd29A promoter isolated from Arabidopsis was cloned into pBI121, a plant transformation vector, and infected with Agrobacterium (LB4404).
[23] Agrobacterium infected with StRD22 gene was incubated overnight in YEP (bacto-peptone, l0g; bacto-yeast extract.10g; NaC1.5g; phytagar, l5g; DW, 1L) in liquid medium, and then diluted 1/40 times in sterile water. . Meanwhile, Agrobacterium was diluted by cutting the leaves of the plant (stock name: Xanthi) plants grown for 3-4 weeks in MSO medium (MS, 4.4 g; sucrose, 30 g; phytagar, 8 g; DW, 1 L) to 5-l0 mm2. Incubate in 50 ml of culture medium in 7 ml of MSO medium (MS salt 4.3 g, sucrose 30 g, MES 0.5 g / L pH5.6-5.8) and incubate for 3-4 days at 28 ° C dark conditions. After washing 3-4 times with MS selection medium containing antibiotics and hormones (MSO medium, 500µg / ml carbenicillin, l00µg / ml kanamycin, NAA (1mg / ml) l00µl, BA (1mg / ml)) 1, phyto agar 8g / L) was incubated for 2-3 weeks. When the shoot was re-differentiated from the callus of MS selection medium, the shoot was transferred to MSO solid medium to regenerate roots (FIG. 3).
[24] StRD22 gene transformed plants, control plants transformed with the pBI121 vector without the StRD22 gene, and ground parts were taken from untransformed plants, ground in liquid nitrogen, RNA was extracted with BRL Trizol reagent, and extracted from agarose containing formaldehyde. A total of 30 ㎍ of RNA was electrophoresed and transferred to the membrane by capillary transfer, and Northern hybridization was performed by labeling StRD22 gene with Takara's labeling method.
[25] As a result, as shown in Figure 4, the StRD22 gene is not transformed in the tobacco (C; Control) and pBI121 vector transformed plants, but the transformed tobacco (# 1, # 3, # 4; transgenic plants) It was confirmed that only expressed.
[26] <Step 4> Potato Transformation of StRD22 Gene and Northern Expression Analysis of Transformant
[27] To introduce the isolated StRD22 gene into potatoes, the chimeric gene produced by connecting the structural gene of the StRD22 gene to the rd29A promoter isolated from Arabidopsis was cloned into pBI121, a plant transformation vector, and infected with Agrobacterium (LB4404).
[28] Agrobacterium (LBA 4404) infected with StRD22 gene was cultured overnight in YEP (bacto-peptone, l0g; bacto-yeast extract.l0g; NaC1.5g; phytagar, 15g; DW, 1L) in liquid medium and then l / Diluted 40 times.
[29] Meanwhile, the internodes of the stock potatoes (cultiform name: Sumi superior) plants grown in storage medium PM (MS, 4.4 g; sucrose, 30 g; phytagar, 8 g DW, 1 L) for 3-4 weeks were diluted to a size of 3-5 mm and diluted. After soaking in Agrobacterium culture medium for about 5 minutes, it was transferred to a plate made of Whatman No.1 filter paper sterilized on the surface of callus organic solid medium PM and co-cultured in the dark for 48 hours. After 48 hours transfer to PC medium containing (MS, 4.4 g; sucrose, 30 g; phytagar, 8 g; NAA, 0.1 mg; BA, 0.5 mg; carbenicillin, 500 mg; kanamycin, 50 mg; DW, 1 L) After 4 weeks of incubation, it was again taken with kanamycin containing organic medium PS (MS, 4.4 g; sucrose, 30 g; phytagar, 8 g; carbenicillin, 500 mg; BA, 5 mg; GA₃, 0.3 mg; DW, 1 L). Transfer shoots were incubated for 4-8 weeks and transferred to PM medium to be incubated and stored.
[30] StRD22 gene transgenic plants, control plants transformed with the pBI121 vector without the StRD22 gene, and ground parts were collected from untransformed plants, ground in liquid nitrogen, and RNA was extracted with BRL's Trizol reagent, and agarose containing formaldehyde. A total of 30µg of RNA was electrophoresed, transferred to the membrane by capillary transfer, and StRD22 gene was labeled by Takara's labeling method to perform northern hybridization.
[31] As a result, the StRD22 gene was not expressed in the non-transformed tobacco and pBI121 vector transgenic plants, but only in transgenic potatoes.
[32] <Step 5> Analysis of drought stress of StRD22 transgenic tobacco
[33] Transgenic plant confirmed the expression of StRD22 gene, control transformed only pBI121 vector except StRD22 gene, and untransformed plants were transferred to Jippy pot, purified for 5 days, and then watered once every 2 days for 1 week. Growing in the chamber. In the drought treatment, the samples were dried in the growth chamber without irrigation for 20 days, and then all the plants were almost dried. At this time, irrigation was performed to compare the regeneration of the plants.
[34] As a result, after 1 day, the transformants (# 1, # 4) were regenerated as shown in Fig. 5, but the plants were revived, but the control transformed only with the pBI121 vector and the untransformed plants were not recovered.
[35] As described above, the StRD22 gene, which is a potato drone adaptation gene of the present invention, is a gene whose expression is amplified when the plant is in a drought state. As it is confirmed that it is greatly improved, it can be seen that it can be utilized for the development of drought resistant crops.

权利要求:
Claims (2)
[1" claim-type="Currently amended] Potato drone adaptation gene, characterized in that it has been isolated from potato and has the nucleotide sequence of SEQ ID NO: 1.
[2" claim-type="Currently amended] Cold-resistant crop using the gene of claim 1.

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同族专利:

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公开号 | 公开日

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KR100514433B1|2005-09-14|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2002-12-10|Application filed by 대한민국(관리부서:농촌진흥청)

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2002-12-10|Priority to KR10-2002-0078495A

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2004-06-16|Publication of KR20040050634A

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2005-09-14|Application granted

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2005-09-14|Publication of KR100514433B1

优先权:

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申请号 | 申请日 | 专利标题

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KR10-2002-0078495A|KR100514433B1|2002-12-10|2002-12-10|Gene related to dehydration isolated from potato|