粟弯孢霉叶斑病菌G蛋白β亚基基因克隆及表达

摘要

In order to make a foundation for illustrating the pathogenic mechanism of G protein beta-subunit in Curvularia lunata,this research focuses on acquiring the gene encoding Gβsubunit in C.lunata and exploring its expression pattern .The full-length cDNA of G-protein beta-subunit was cloned using SMART RACE RT-PCR,and the expression pattern of ClGβgene was analyzed under different growth time using Real-time PCR.The full-length cDNA of ClGβwas 1 056 bp and encoded 351 amino acids,while its DNA contained 4 introns and 5 extrons.The cDNA sequence of ClGβhad been deposited in GenBank with accession number JQ 768316 .The results of Real-time PCR indicated that the gene expression level of G-protein beta-subunit is lower at early stage and higher at later stage.We constructed the prokaryotic expression vector pET 28(a)-ClGβ,and E.coli BL21 was transformed by this recombinant construct and induced by IPTG .The molecular weight of the expression product was identical with the calculated molecular weight of ClGβ.%旨在获得粟弯孢霉叶斑病菌G蛋白β亚基基因，明确其表达模式，为阐明该基因对粟弯孢霉叶斑病菌致病性的调控机制奠定基础。运用SMART RACE RT-PCR技术，克隆粟弯孢霉叶斑病菌G蛋白β亚基基因ClGβ全长cD-NA序列，以qRT-PCR技术，对该基因在粟弯孢霉叶斑病菌不同生长时间的表达特征进行分析。结果表明，ClGβ基因cDNA编码区为1056 bp，DNA包含4个内含子和5个外显子，编码351个氨基酸。该基因的cDNA序列在GenBank中注册的登录号为JQ768316。 qRT-PCR结果表明，Gβ基因在菌株生长过程中表现出前期表达量较低而后期表达量明显升高的趋势。构建了pET28（a）-ClGβ原核表达载体，经IPTG诱导，目的蛋白在宿主菌E．coli BL21中获得表达，表达产物分子量与ClGβ蛋白计算分子量一致。