酵母表面展示系统的改进及其在筛选烟草PMT基因启动子结合蛋白中的应用

Screening of Promoter-Binding Factors of Tobacco PMT Gene Using a Modified Yeast Surface Display System

筛选DNA结合蛋白常用的酵母单杂交系统在应用中有时会因内源干扰而影响筛选结果。与之相比,酵母表面展示系统(yeast surface display system)将外源蛋白展示在细胞表面,可通过接近体外实验的方法筛选DNA结合蛋白,能在一定程度上避免酵母内源干扰,但是,该系统在DNA结合蛋白筛选研究中的应用还很有限。本研究对酵母表面展示系统常用载体p YD1进行改造,使其与Clontech公司的Smart cDNA文库构建系统相匹配,提高了cDNA酵母表面展示文库的构建效率,并通过比较试验建立了一个以酵母表面展示系统筛选DNA结合蛋白的试验体系。在以酵母单杂交筛选烟草腐胺甲基转移酶基因PMT(putrescine N-methyltransferase)启动子结合蛋白的研究中,其茉莉素信号应答元件GAG片段是一段筛选效率极低的DNA片段。本研究利用改进的酵母表面展示系统对GAG片段的DNA结合蛋白进行了筛选,并获得若干烟草蛋白基因,其中包括2个可能结合GAG片段的ERF(ethylene responsive factor)转录因子。进一步研究发现,这2个ERF转录因子可与GAG片段在体外结合,但不能在酵母单杂交系统中激活由GAG片段操纵的报告基因表达。本研究也证明酵母表面展示系统可有效克服酵母内源干扰,弥补酵母单杂交系统在筛选易受酵母内源干扰DNA结合蛋白方面的不足。

Yeast surface display system is an important tool for studying molecular interaction of proteins, however, its application in DNA-binding-protein screening is relatively limited. Yeast surface display system secretes exogenous proteins onto cell surface, thus, it could be applied to determine protein interaction under in-vitro-like experiment conditions. Therefore, it may be more efficient than yeast one-hybrid system in screening the DNA-binding proteins whose DNA-binding capability is affected by en-dogenous yeast factors. In this study, we modified the pYD1 vector in yeast surface display system to make it compatible with the Smart cDNA library construction kit from Clontech, which will be helpful to increase the library construction efficiency. An ex-perimental procedure for DNA-binding protein isolation was established using the modified yeast surface display system. Then, it was successfully applied in screening DNA-binding proteins of a jasmonate （JA） responsive element in the promoter of tobacco PMT （putrescine N-methyltransferase） gene. Among the isolated genes, two encoded ERF transcription factors, which were found to bind the JA responsive element in PMT promoter in vitro but unable to activate the expression of reporters in yeast-one-hybrid system. Our study suggests that yeast surface display system is efficient in screening the DNA-binding proteins whose DNA-binding capability in yeast-one-hybrid system is disrupted by endogenous factors.