紫杉醇合成相关候选基因A02725的克隆及转基因菌株的构建

真菌紫杉醇合成相关基因的功能研究是揭示真菌紫杉醇合成机制的关键。为了揭示产紫杉醇内生真菌枝状枝孢霉MD2的候选基因A02725对宿主紫杉烷类合成的作用,本研究克隆了候选基因A02725的DNA全长序列(1 524 bp);生物信息学预测结果表明该基因编码蛋白为亲水蛋白,含508个氨基酸,无信号肽和跨膜结构,与牻牛儿基牻牛儿基焦磷酸合成酶(geranylgeranyl pyrophosphate synthase,GGPPS)具有79%的一致性;进一步构建该基因的超表达载体并转化枝状枝孢霉MD2,通过抗性筛选以及Southern blot检测,共获得27株转基因菌株,其中5株转基因菌株的外源基因是以单拷贝方式整合。该结果为深入分析超表达候选基因A02725对宿主紫杉烷类合成的影响奠定了基础。

Cloning of Candidate Gene A02725 Related to Taxol Biosynthesis and Construction of A02725-Transgenic Strains

Functional research of fungal taxol-related genes is key to reveal the biosynthesis mechanism of fungal taxol.In order to uncover the function of the candidate gene A02725 in the taxane biosynthesis of the taxol-producing endophytic fungus Cladosporium cladosporioides MD2,the full-length DNA sequence(1 524 bp)of one candidate gene A02725 was cloned from C.cladosporioides MD2 in this study.Bioinformatics analysis showed that the encoding protein of A02725 was hydrophilic protein,contained 508 amino acids,had no signal peptide and transmembrane structure,and had 79% identity to geranylgeranyl pyrophosphate synthase(GGPPS).Overexpression vector of A02725 was further constructed and transformed into C.cladosporioides MD2.Through resistance screening and Southern blot detection,a total of 27 A02725-transgenic strains were obtained,and 5 of them harbored single copy of the exogenous genes.These results laid a foundation for further investigating the effect of A02725-overexpression on the taxane biosynthesis of C.cladosporioides MD2.