内生菌生物转化提高穿山龙中薯蓣皂苷元含量的关键酶及其基因过表达菌株构建的研究

Dioscorea nipponica has been listed on the second level of threatened medicinal plants in China. Its dried rhizome is the main medicinal material for extracting diosgenin. Diosgenin is not only the effective compound of many Chinese patent medicines, but also the main raw material for producing several hundreds of steroid hormones and contraceptive drugs due to its characters in chemical structure and pharmacological effects. The market demand for diosgenin is large, and the resource of it is shortage. An endophyte fungus was isolated from Dioscorea nipponica by our research group before. The fermentation of the endophyte and Dioscoreae Nipponicae Rhizoma increased 70%-90% diosgenin content in the later, which was granted invention patent in China. The biotransformation mechanism of the fermentation will be investigated from the way of key enzyme(s) and utilized it(them) to increase the amount of diosgenin. The methods of DDRT-PCR and Real-time PCR will be used to find out the enzyme genes of differential expression produced by the endophyte in the reaction system. The enzyme genes of differential expression will be cloned and their sequences will be determined. The biotransformation functions of the enzyme genes will be verified by constructing their RNA interference vectors and gene overexpression vectors, and compared with the wild endophyte with the diosgenin content for index. Finally, the key enzymes will be confirmed, and the biotransformation mechanism of increasing the amount of diosgenin by the endophyte will be discovered. At the meanwhile, obtaining the engineering strain of key enzyme gene overexpression will supply the technological method of increasing the diosgenin amount in Dioscoreae Nipponicae Rhizoma by utilizing the fermentation of the engineering fungus.

穿龙薯蓣为我国二级濒危药用植物，其干燥根茎穿山龙是提取薯蓣皂苷元的主要药材。由于化学结构和药理作用上的特性，薯蓣皂苷元不仅作为多种中成药的药效成分，而且是世界上生产数百种甾体激素和避孕药物的主要原料，市场需求量大，资源紧缺。本课题组前期从穿龙薯蓣中筛选出一种内生真菌，经过发酵可使穿山龙中薯蓣皂苷元含量提高70%-90%，已获得发明专利。本研究拟从关键酶的角度探讨并利用这一生物转化机制以提高薯蓣皂苷元的产量。采用基因差异表达和实时定量PCR技术寻找内生菌在反应体系中所产生的差异表达酶基因，对其进行克隆及基因序列分析，构建差异表达酶RNA干扰载体和基因过表达载体，以薯蓣皂苷元含量为指标，与野生型内生菌进行比较，验证差异表达酶基因的生物转化功能，以确定关键酶，揭示内生菌生物转化提高薯蓣皂苷元的作用机制。同时获得关键酶基因过表达工程菌株，为利用工程菌发酵提高穿山龙中薯蓣皂苷元产量提供技术方法。

穿龙薯蓣为我国二级濒危药用植物，其干燥根茎穿山龙是提取薯蓣皂苷元的主要药材。由于化学结构和药理作用上的特性，薯蓣皂苷元不仅作为多种中成药的药效成分，而且是世界上生产数百种甾体激素和避孕药物的主要原料，市场需求量大，资源紧缺。本课题组前期从穿龙薯蓣中筛选出一种内生真菌，经过发酵可使穿山龙中薯蓣皂苷元含量提高70%-90%，已获得发明专利。本研究从关键酶的角度探讨并利用这一生物转化机制以提高薯蓣皂苷元的产量。采用转录组测序和实时定量PCR技术找到了内生菌在反应体系中所产生的差异表达酶基因：β-葡糖苷酶基因、伯/铜胺氧化酶基因、α-α海藻糖酶基因，对其进行克隆及基因序列分析，成功构建了关键酶基因的过表达载体pLVX-puro-β-葡糖苷酶、pLVX-puro-伯/铜胺氧化酶和pLVX-puro-α,α-海藻糖酶以及干扰载体sh-β-葡糖苷酶-pGreenpuro、sh-伯/铜胺氧化酶-pGreenpuro和sh-α,α-海藻糖酶-pGreenpuro，以薯蓣皂苷元含量为指标，与野生型内生菌进行比较，验证差异表达酶基因的生物转化功能，揭示了内生菌生物转化提高薯蓣皂苷元的作用机制；进而构建了关键酶基因的过表达工程菌株，考察优化了其固体发酵培养条件，为利用工程菌发酵提高穿山龙中薯蓣皂苷元产量提供技术方法。