胶孢炭疽菌T-DNA插入突变体库的构建及石杉碱甲生物合成关键酶基因克隆

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.

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。

本项目以产石杉碱甲的蛇足石杉内生菌胶孢炭疽菌（Colletotrichum gloeosporioides）ES026为材料，研究建立了农杆菌介导的C. gloeosporioides ES026的高效遗传转化体系。在共培温度为25℃、共培时间为48h、400 μmol /l 的乙酰丁香酮（AS）、农杆菌浓度OD600 = 0.3-0.5、孢子浓度为1 ×106个/ml时，T-DNA 转化效率最高，达95个转化子/皿。目前利用此优化条件已建立了约含4000个转化子的胶孢炭疽菌ES026 T-DNA插入突变体库。对获得的部分突变菌株进行了石杉碱甲代谢突变筛选，筛选到1株石杉碱甲代谢增高菌株EST01044，其石杉碱甲含量7.73μg.g-1CDW显著高于出发菌株ES026的，约为1.83倍。以该突变菌株为材料，通过TAIL-PCR和Inverse-PCR克隆得到了该插入位点的基因序列，经过功能注释，该基因为甲基转移酶基因。同时本研究还构建了C. gloeosporioides ES026不同发育阶段的cDNA文库，运用第二代高通量测序技术进行了转录组测序。对测序结果进行生物信息学分析和试验验证，克隆得到了石杉碱甲生物合成途径中的CAO基因全长，并提交至NCBI数据库，序列号为KT001519。用荧光定量PCR对CAO基因在C. gloeosporioides ES026生长3、4、5、6、7、8天中的表达量进行分析，发现第5天的表达量最高。而C. gloeosporioides ES026菌丝中石杉碱甲的量在第6天最高。因此CAO基因可能在石杉碱甲的代谢途径中起到了关键的调节作用。通过项目的实施，本项目按期顺利完成了项目任务书中的所有研究内容，发表SCI研究论文4篇，培养博士1名、硕士2名，达到了任务书中的研究目标并为后期的进一步深入研究打下来坚实的基础。