应用酵母双杂交系统筛选橡胶延长因子REF的互作蛋白  

Screening Interaction Proteins of Rubber Elongation Factor(REF)by Yeast Two-hybrid System

作　　者：曾日中[1] 李先昆[1] 聂智毅[1] 代龙军[1] 黎瑜[1] 段翠芳[1] 

机构地区：[1]中国热带农业科学院橡胶研究所,农业部橡胶树生物学重点开放实验室,省部共建国家重点实验室培育基地,海南儋州571737

出　　处：《热带亚热带植物学报》2011年第5期400-406,共7页Journal of Tropical and Subtropical Botany

基　　金：国家重点基础研究发展计划项目(2007CB116203);国家自然科学基金项目(30860232);中央级公益性科研院所基本科研业务费项目(XJSYWFZX2009-13)资助

摘　　要：利用酵母双杂交系统,以橡胶树(Hevea brasiliensis)橡胶延长因子基因REF的开放阅读框(ORF)构建无自激活性的诱饵表达载体pBD-GAL4-REF,并筛选以pAD-GAL4-2.1载体构建的橡胶树胶乳cDNA文库,对阳性克隆的cDNA插入片段进行测序及生物学功能分析。通过酵母双杂交筛选,共获得5种可能与REF互作的候选蛋白质,它们分别为与诱饵蛋白REF高度同源的REF家族成员、小橡胶粒子蛋白(SRPP)、翻译控制肿瘤蛋白(TCTP)、激发子响应蛋白和泛素耦联酶E2,这表明橡胶延长因子REF除了与自身高度同源蛋白质可能存在相互作用之外,还可能与TCTP和激发子响应蛋白等其它蛋白质发生相互作用。这些结果有助于揭示橡胶粒子的生物学功能。The interaction proteins of rubber elongation factor（REF） were screened using yeast two-hybrid system.A bait vector pBD-GAL4-REF without auto-activation was constructed by subcloning ORF cDNA of REF gene into the yeast two-hybrid vector pBD-GAL4,and the pAD-GAL4-2.1 cDNA prey library was prepared with mRNA extracted from the latex of rubber tree（Hevea brasiliensis）.The bait and prey vectors were then co-transformed into YRG-2 yeast competent cells by two-step method.Five proteins interacted with REF were identified by yeast two-hybrid screening method,including two of REF family proteins,and small rubber particle protein（SRPP）,translation control of tumor protein（TCTP）,elicitor-responsive protein and ubiquitin-conjugating enzyme E2.The data suggested that REF could not only interact with high homologous proteins,but also with other proteins,such as TCTP,and E2,etc.These protein-protein interactions might help to revealing the biological function of H.brasiliensis rubber particles.

关 键 词：橡胶树 橡胶延长因子REF 橡胶生物合成 酵母双杂交系统 

分 类 号：S794.1[农业科学—林木遗传育种]