水稻OsVDAC4诱饵蛋白载体的构建及鉴定
Construction and identification of OsVDAC4 bait protein vector in rice
投稿时间:2018-04-05  修订日期:2018-04-05
DOI:
中文关键词: OsVDAC4  诱饵蛋白载体  膜蛋白酵母双杂交
英文关键词: OsVDAC4  bait protein vector  Membrane-based yeast two-hybrid
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目)
作者单位E-mail
王春台 中南民族大学大学生命科学学院 3052607@scuec.edu.cn 
白雪琪 中南民族大学大学生命科学学院  
谭艳平 中南民族大学大学生命科学学院  
覃永华 中南民族大学大学生命科学学院  
王亚楠 中南民族大学大学生命科学学院  
杨武 中南民族大学大学生命科学学院  
龚秋涵 中南民族大学大学生命科学学院  
刘学群 中南民族大学大学生命科学学院  
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中文摘要:
      电压依赖性阴离子通道(voltage-dependent anion channels, VDACs)是广泛存在于线粒体外膜的一类孔道蛋白, OsVDAC4是本实验室前期鉴定出的1种水稻VDAC蛋白,为了探索OsVDAC4作用的信号通路,确定可用于筛选膜蛋白酵母双杂交文库的诱饵蛋白载体。本实验在对OsVDAC4进行生物信息学分析的基础上,将OsVDAC4全长ORF分别与pBT3-N和pBT3-SUC连接,构建2个诱饵蛋白载体pBT3-OsVDAC4-N-Cub和pBT3-SUC-OsVDAC4-C-Cub,利用膜蛋白酵母双杂交技术鉴定出可用于筛库的诱饵蛋白载体。结果表明pBT3-OsVDAC4-N-Cub和pBT3-SUC-OsVDAC4-C-Cub都可以与目标蛋白互作,但由于OsVDAC4的N端约50个可自由伸展的氨基酸残基导致pBT3-OsVDAC4-N-Cub本底反应非常强, OsVDAC4的C端存在于膜中而没有可自由伸展的氨基酸残基,pBT3-SUC-OsVDAC4-C-Cub的本底反应很弱,因此pBT3-SUC-OsVDAC4-C-Cub可用于文库筛选,这为获取OsVDAC4的互作蛋白提供了基础。
英文摘要:
      The voltage-dependent anion channel (voltage-dependent anion channels, VDACs) is a kind of pore protein that exists extensively in the outer membrane of the mitochondria, and OsVDAC4 is 1 kind of rice VDAC proteins identified in the prophase of this laboratory. In order to explore the signaling pathway of OsVDAC4 participated, the bait protein vector that suited for screening of the membrane protein yeast two-hybrid library is need to be identified. In this experiment, on the basis of bioinformatics analysis of OsVDAC4, 2 bait protein vectors, pBT3-OsVDAC4-N-Cub and pBT3-SUC-OsVDAC4-C-Cub, were constructed through connecting the full length ORF with PBT3-N and PBT3-SUC respectively. The bait protein vector best suited to screen the library was identified with membrane protein yeast two-hybrid technique. The results show that there were interactions between both pBT3-OsVDAC4-N-Cub and pBT3-SUC-OsVDAC4-C-Cub with the target protein. However, because of about 50 free-stretching amino acid residues in the N-terminal of OsVDAC4, there was a very strong background reaction between pBT3-OsVDAC4-N-Cub and other proteins. And the C-terminal of OsVDAC4 exists in the membrane without free-stretching amino acid residues, pBT3-SUC-OsVDAC4-C-Cub"s background reaction is very weak. These demonstrated that pBT3-SUC-OsVDAC4-C-Cub can be used for library screening, which provides a basis for acquiring proteins interacted with OsVDAC4.
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