稻瘟菌蛋白激发子在酵母双杂交系统中的自激活检测

Testing for Self-activation of Magnaporthe Grisea Protein Elicitor in Yeast Two-Hybrid System

利用PCR方法扩增稻瘟菌蛋白激发子基因pemG1,并在其上下游分别引入酶切位点EcoRI和XhoI,经双酶切后与诱饵质粒载体pLexA连接构建重组诱饵质粒pLexA-PEMG1,将该重组诱饵质粒转入酵母菌株EGY48[p8op-lacZ]中进行β-半乳糖苷酶整板分析检测自激活。结果表明,成功构建了重组诱饵质粒pLexA-PEMG1,并且其无自激活报告基因作用,对酵母菌株也无毒性作用。这说明该重组诱饵质粒可用于酵母双杂交系统,为筛选番茄cDNA文库获得诱饵蛋白PemG1的相互作用蛋白奠定了基础。

The Magnaporthe grisea protein elicitor pemG1 gene was amplified by PCR with the EcoR I and Xho I restriction sites incorporated into the primers,digested by restriction enzymes and ligated with the vector pLexA to construct recombinant bait plasmid pLexA-PEMG1.After the transform of recombinant plasmid into yeast strain EGY48[p8op-lacZ],the autonomous reporter gene activation was then observed with β-galactosidase whole plate assay.The results suggested that the recombinant plasmid pLexA-PEMG1 was constructed. which neither had the ability of self-activation, nor yeast cell toxicity.Therefore, the reeombinant plasmid can be used in ）,east two-hybrid system to screen a tomato cDNA library for proteins interacted with the bait protein PemGl.