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

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。

Abstract:

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.