玉米大斑病菌St3 HNR基因真核表达载体构建及表达分析

Expression analysis and construction of eukaryotic vector of St3 HNR gene in Setosphaeria turcica

本研究在克隆获得玉米大斑病菌1,3,8-三羟基萘还原酶基因(St3HNR)的基础上,拟对该基因编码产物进行异源表达分析,明确基因功能。试验利用毕赤酵母真核表达系统,构建了玉米大斑病菌St3HNR基因的真核表达载体pPIC9K-St3HNR,采用电击转化法将重组质粒转入表达宿主菌GS115中得到了重组酵母菌株,经甲醇诱导表达,SDS-PAGE检测发现,从诱导48 h开始有一个分子量约为28 kD的蛋白组分分泌,与St3HNR的编码产物分子量相近,且表达量随诱导时间的延长而逐渐增加,而对照及未诱导菌株中不存在该条带,推测St3hnr蛋白在宿主菌中分泌表达。该研究为进一步分析基因编码产物的催化活性,明确该基因在黑色素合成途径中的作用奠定了基础。

The object of this study was to analysis the gene function by heterogenous expression on the foundation of coloning 1,3,8-tri-HN reductase gene（St3HNR） of Setosphaeria turcica,Eukaryotic expression vector of St3HNR,pPIC9K-St3HNR,was constructed using Pichia pastoris in this study and expressed in host yeast strain GS115 through electroporation and methanol induction.SDS-PAGE detected a molecular weight of about 28 kDa protein components similar to St3HNR encoding products after inducted 48 h and the expression increased with the prolongation of induction time,while the control and not induced sample did not exist,suggesting St3hnr secreted in the host.It established the foundation for analyzing the property of the gene product of 1,3,8-tri-HN reductase and the role of St3HNR gene in the melanin biosynthesis.