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

doi:10.3724/SP.J.1006.2014.02081

Abstract

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 endogenous 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 experimental 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.

Key words: Yeast surface display, Smart cDNA Library, DNA-binding protein, Yeast one-hybrid, PMT gene

CHEN Hong, NIU Hai-Xia, WANG Wen-Jing, MA Hao-Ran,LI Jia-Na,CHAI You-Rong,ZHANG Hong-Bo. Screening of Promoter-Binding Factors of Tobacco PMT Gene Using a Modified Yeast Surface Display System[J].Acta Agron Sin, 2014, 40(12): 2081-2089.

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

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