水稻高盐胁迫下的酵母双杂交文库构建及OsRPK1胞内互作蛋白质的筛选  

Construction of yeast two-hybrid cDNA library of rice under high salinity stress and screening of intracellular interacting protein of OsRPK1

作　　者：邹禹[1] 刘园园[2] 钱宝云[1] 占新春[1] 郑乐娅[1] 张炜[2] 张培江[1] ZOU Yu;LIU Yuan-yuan;QIAN Bao-yun;ZHAN Xin-chun;ZHENG Le-ya;ZHANG Wei;ZHANG Pei-jiang(Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 210031, China;College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China)

机构地区：[1]安徽省农业科学院水稻研究所,安徽合肥210031 [2]南京农业大学生命科学学院,江苏南京210095

出　　处：《江苏农业学报》2019年第4期753-763,共11页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31701409);安徽省重点研究和开发计划项目(1804a07020111、1804h07020156);安徽省自然科学基金项目(1408085MKL63);安徽省农业科学院院长青年创新基金(17B0101)

摘　　要：为了挖掘水稻OsRPK1胞内互作蛋白质,阐明OsRPK1参与高盐胁迫的分子机制,本研究利用SMART技术,构建水稻高盐胁迫下根尖的酵母双杂交文库。PCR扩增获得OsRPK1基因编码胞内区域的碱基序列,构建诱饵表达载体(pGBKT7-OsRPK1-CD),检测诱饵表达载体在酵母中的毒性和自激活活性,筛选OsRPK1胞内互作蛋白,进一步分析高盐胁迫下候选基因的表达模式。结果表明,构建的cDNA文库库容量为1.11×10~7 CFU,文库重组率为96%,文库插入片段多态性较好。成功构建了诱饵表达载体(pGBKT7-OsRPK1-CD),经检测诱饵表达载体无毒性,无自激活活性。诱饵表达载体与cDNA文库进行双杂交筛选,经测序和比对分析获得了11个重要的候选基因,检测候选基因在高盐处理下的表达情况,其中8个候选基因受高盐诱导表达,2个候选基因受高盐胁迫抑制表达,1个候选基因受高盐胁迫瞬时诱导表达后表达量又受到显著抑制。In order to explore the intracellular interacting protein of OsRPK1 in rice, and describe the molecular mechanism of OsRPK1 involved in high-salinity stress, a yeast two-hybrid cDNA library of rice root tip under high-salinity stress was constructed by switching mechanism at 5′ end of the RNA transcript (SMART) technique. The base sequence of OsRPK1 gene encoding intracellular region was obtained by PCR amplification, and the bait expression vector (pGBKT7 -OsRPK1-CD ) was constructed. Then the toxicity and self-activation activity of pGBKT7 -OsRPK1-CD in yeast were detected. The intracellular interacting proteins of OsRPK1 were screened and the expression patterns of candidate genes were analyzed under high-salinity stress. The results showed that the capacity of the cDNA library was 1.11 ×10 7 CFU, the recombinant rate was 96%, and the polymorphism of the cDNA fragments was good. The bait vector pGBKT7 -OsRPK1-CD was constructed successfully and tested to be no toxicity and no self-activation. The cDNA library was screened by bait vector, and 11 important candidate genes were found by sequencing and alignment analysis. Under high-salinity stress, eight candidate genes were up-regulated. Inversely, two candidate genes were down-regulated. Besides, one candidate gene was up-regulated transiently, and then the expression was inhibited significantly.

关 键 词：水稻 OsRPK1 高盐胁迫 酵母双杂交cDNA文库 

分 类 号：Q943.2[生物学—植物学] S511[农业科学—作物学]