石杉碱甲是当前治疗老年痴呆症最有效的药物之一，但其自然资源日益匮乏，而化学合成成本高、活性低、毒副作用大，远远不能满足临床需求。运用生物技术工业化生产天然石杉碱甲是未来发展的主要方向。目前石杉碱甲生物合成途径已成为研究热点，但均以蛇足石杉为材料，研究进展不大。申请者从蛇足石杉体内分离、筛选得到一株产石杉碱甲的内生菌胶孢炭疽菌。以此为材料，通过农杆菌介导转入标记DNA，构建T-DNA插入突变体库。采用HPLC法筛选石杉碱甲代谢突变菌株，选取单拷贝插入突变体菌株，T-DNA插入位点即为石杉碱甲代谢关键酶基因或调控因子。以插入的标记DNA设计引物，通过TAIL-PCR、RACE等技术获得石杉碱甲生物合成关键酶基因，并运用实时荧光定量PCR、RNAi等技术验证获得的基因功能。本研究将为进一步阐明石杉碱甲的生物合成机理提供理论依据，为运用生物技术构造产石杉碱甲的工程菌奠定理论基础。

Huperzine A is a chemical isolated from traditional Chinese medicinal plant Huperzia serrata. As a acetylcholinesterase inhibitor, Huperzine A has been used for the treatment of AD disease own to the advantages of low toxity, long effection time. However, the resource of Huperzine A from plant is limited and syntheticl Huperzine A comes with low activity and high toxicity.Application of modern molecular biology and genetic engineering technology is becoming mainstraim of production of natural huperzine A. Previously, we successfully screened a strain of endophyte from Huperzia serrata which produces huperzine A. The endophyte was named ES026 and identified as Colletotrichum gloeosporioides by morphological characteristics and ITS sequence. The purpose of this study is to dissect the synthetic map of Huperzine A from Huperzia serrata by screening of random T-DNA insertional mutation library. The mutational library will be constructed by agrobacterium mediated transformation. After screening, the key genes responsible for the biosynthesis will be obtained through the thermal asymmetric interlaced PCR and rapid-amplification of cDNA ends. Real-time fluorescence quantitative PCR and RNAi will be further used for verification. This study will provide theory basis for further genetic engineering and production of huperzine A by microbial fermentation.