盾叶薯蓣环阿屯醇合酶基因克隆与表达

摘要

In present study,a cDNA encoding cycloartenol synthase (CAS) was cloned from Dioscorea zingiberensis by nested PCR and RACE,designated as DZCAS.DZCAS consists of a 2 280 bp open reading frame encoding a 759 amino acid polypeptide with a molecular weight of 86 924 Da and isoelectric point of 6.39.Sequences comparison showed that the D.zingiberensis CAS shared more than 70% identity with other plant CASs and contained all the conserved regions existing in all CASs.The cycloartenol production in the oxidosqualene cyclase gene mutant yeast cells expressing DZCAS was confirmed by both high performance liquid chromatography and liquid chromatographymass,which proved that DZCAS encoded CAS.RT PCR analysis demonstrated that DZCAS expression is the highest in young leaves,next in young tubers and the lowest in young stems of D.zingiberensis.%利用巢式PCR和RACE技术从盾叶薯蓣中克隆了阿屯醇合酶的cDNA(定名为DZCAS).结果表明,该cDNA的开放读码框为2 280 bp,编码759个氨基酸的多肽,分子量为86 924 Da,等电点为6.39.氨基酸序列比对表明盾叶薯蓣阿屯醇合酶与其他植物阿屯醇合酶的相似性超过70%,含有环阿屯醇合酶共有的保守序列.利用氧化鲨烯环化酶基因缺陷型酵母表达DZCAS,并用高效液相色谱和液质联用仪检测酵母细胞中环阿屯醇合成情况,证明DZCAS编码环阿屯醇合酶.RT-PCR分析表明,DZCAS在盾叶薯蓣幼叶中表达量最高,其次是在地下幼嫩块茎中,而地上幼茎表达量最低.