基于代谢组方法研究内生真菌影响蛇足石杉生根生理机制

Huperzine A is an efficient, reversible and selectable inhibitor against acetyl cholinesterase (AchE). It is extracted from the Huperzia serrata, and is at present a successful medicine for the curing of senile dementia in China. Huperzia serrata is a type of scarce natural resource with a low production of Huperzine A. Artificial cultivation has not been reported, and as a result, the mass production and application thereof is limited. The research group has obtained certain partial knowledge of the commensal technique of the endophytic fungi and Huperzine A, as well as the effects of endophytic infestation on the physiology of the Huperzia serrata. From the above, we attempt to concentrate on how the endophytic fungi affect the physiological mechanism of the adventitious root formation, through the advantages of Metabolomics on aspects of the test, quantization and editing of the endogenous metabolin from organisms. To begin with, the Metabolomic Method is founded, based on the GC-MSLC-MS test and PCA\PLS-DA analysis. Then we analyze the metabolic variation of the Huperzia serrata infested by the fungi. The key metabolic materials are identified as well. Meanwhile the metabolic pathway in which the characterized compounds took part is demonstrated, along with the relation between such pathways and the rootage process of the Huperzia serrata. Some related databases offered a check service during the experiments. Finally, the commensal mechanism of endophytic fungi and the Huperzia serrata may further delve into, which is the main purpose of this paper. In the meantime, the effect of the endophytic fungi on hosts' alkaloid metabolin pathway is discussed, and the metabolic outline of alkaloid in the Huperzia serrata is also studied.

从蛇足石杉中提取的石杉碱甲是一种高效、可逆、高选择性的乙胆碱酯酶抑制剂，是目前国内开发最为成功的治疗老年性痴呆症的药物。蛇足石杉自然资源稀缺、无人工栽培史及石杉碱甲含量低等原因限制了该药物的大规模生产和应用。本课题组已进行了内生真菌与蛇足石杉共生培养技术研究、内生真菌侵染对蛇足石杉的生理影响研究。在此基础上， 本课题计划利用代谢组学在检测、量化和编录生物内源性代谢物方面的优越性，研究内生真菌影响蛇足石杉不定根形成的生理机制。首先建立起基于GC-MS,LC-MS 检测和PCA、PLS-DA 分析的代谢组研究方法，分析真菌侵染后蛇足石杉发生变化的代谢物质，对关键代谢物质进行指认，通过检索数据库，分析特征性化合物参与的代谢通路，以及该通路与蛇足石杉生根过程的关系。另一方面，通过描述蛇足石杉生物碱代谢轮廓研究内生真菌对寄主生物碱代谢途径的影响，本课题研究工作旨在深入探讨蛇足石杉与内生真菌的共生机制

本课题阐述了蛇足石杉的生根与真菌的侵染有密切联系，原生境带菌土壤、接种了内生真菌的土壤、灭菌土壤上蛇足石杉生根率不同。灭菌土壤的插条生根率明显低于其它两种处理。报告建立了GC-MS的代谢组学分析方法，能够分析出糖类、生物碱等化合物在蛇足石杉生长发育中的动态变化。利用主成分分析法(PDA)分析出，由于内生真菌的侵染情况不同，三种处理的植物在代谢产物上表现出明显差异。进一步监督性分析方法（OPLS-DA）分析出，发生代谢差异化的标识物。发现了灭菌土壤与接种内生真菌的土壤培养的植株的代谢差异物35个，灭菌土壤与原生境土壤培养的植株的代谢差异物29个，并对化合物进行了鉴定。报告将可检测到差异的化合物进行了KEGG compound名称注释，大部分化合物可以获得代谢途径。与这些化合物相关的生物代谢途径120条，在其中进行筛选，Compounds Counts数目大于5的，植物代谢途径进行解析。包括生物碱合成途径、次生产物形成途径，ABC蛋白调控途径，酰胺合成途径。说明真菌的侵染从多条途径影响生根，特别对生物碱的合成有重要意义。