作物学报Issue(12)：2081-2089,9.DOI:10.3724/SP.J.1006.2014.02081
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 Modi-fied Yeast Surface Display System
摘要
Abstract
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.关键词
酵母表面展示/Smart cDNA 文库/DNA 结合蛋白/酵母单杂交/PMT 基因Key words
Yeast surface display/Smart cDNA Library/DNA-binding protein/Yeast one-hybrid/PMT gene引用本文复制引用
陈红,牛海峡,王文静,马浩然,李加纳,柴友荣,张洪博..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[J].作物学报,2014,(12):2081-2089,9.基金项目
本研究由教育部博士点专项科研基金项目(20120182120031),高等学校学科创新引智计划(B12006)和西南大学中央高校基本科研业务费专项(XDJK2012C089,2362014xk09)资助。 （20120182120031）
