CAJ | 학술논문

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。
지재획득속만포매협반병균G단백β아기기인，명학기표체모식，위천명해기인대속만포매협반병균치병성적조공궤제전정기출。운용SMART RACE RT-PCR기술，극륭속만포매협반병균G단백β아기기인ClGβ전장cD-NA서렬，이qRT-PCR기술，대해기인재속만포매협반병균불동생장시간적표체특정진행분석。결과표명，ClGβ기인cDNA편마구위1056 bp，DNA포함4개내함자화5개외현자，편마351개안기산。해기인적cDNA서렬재GenBank중주책적등록호위JQ768316。 qRT-PCR결과표명，Gβ기인재균주생장과정중표현출전기표체량교저이후기표체량명현승고적추세。구건료pET28（a）-ClGβ원핵표체재체，경IPTG유도，목적단백재숙주균E．coli BL21중획득표체，표체산물분자량여ClGβ단백계산분자량일치。
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.