野三七与三七皂苷成分差异的分子机制研究

Panax notoginseng (Burk. ) F. H. Chen (PN) and P. vietnamensis var. fuscidiscus K. Komatsu, S. Zhu & S. Q. Cai (PVF) are important medicinal resources in Yunnan Province. They have been widely used for the treatment of bruises and cardiovascular diseases as Sanqi. However, there is not sufficient scientific evidence for their same constituents and function. Ginsenosides are the main active ingredients of panax. The difference of ginsenosides between PN and PVF is the basis of functional study. In this project, LC-MS would be used to systematically compare the saponins of PN and PVF and seek out the different composition between them, which could lay the theoretical foundation for pharmacological research and modern application of them. The candidate genes of oxidosqualene cyclase and cytochrome P450, which may be involved in the biosynthesis of ocotillol saponins, are screened from the constructed transcriptome data of PN and PVF at different growth stages by comparative transcriptome and co-expression analysis. In vitro enzyme reaction and synthetic biology will be used to characterize the candidate genes and figure out the biosynthetic pathway of ocotillol. Then, reveal the molecular mechanism of different saponin composition of PN and PVF. The research results of this project will provide the theoretical basis for the genetics and breeding of PN and PVF.

三七和野三七是云南省重要的药用资源，二者均被当作三七使用，但仍然缺乏足够的科学证据说明野三七和三七的物质基础和功效一致。人参皂苷是人参属植物的主要活性成分，故对比研究三七和野三七中皂苷成分是三七和野三七功效研究的基础。一方面，本项目将采用液质联用技术对三七和野三七中皂苷成分进行系统性比较，从而确定三七和野三七中皂苷成分差异，为三七和野三七的药理研究和现代化应用提供理论参考；另一方面，本项目将构建三七和野三七不同生长时期的转录组，用比较转录组和共表达分析方法筛选可能参与奥克梯隆型皂苷生物合成的氧鲨烯环化酶和细胞色素P450候选基因，利用酵母表达和合成生物学对其功能进行鉴定，解析奥克梯隆生物合成途径，揭示三七和野三七中皂苷成分差异的分子机制，为三七和野三七的遗传育种提供理论依据。

三七和野三七是云南省重要的药用资源，二者均被当作三七使用，但仍然缺乏足够的科学证据说明野三七和三七的物质基础和功效一致。本项目采用液质联用技术对三七和野三七根、根茎、茎和叶4个部位的化学成分进行代谢组学研究，建立了三七和野三七不同部位样品的UPLC/Q-TOF-MS指纹图谱，筛选获得三七和野三七4个部位在正、负离子模式下的42个潜在化学成分标志物，初步鉴定出38个潜在化学成分标志物，差异明显的化学成分以皂苷类成分为主。三七和野三七的成分研究为其药理研究和现代化应用提供了理论参考。构建了三七和野三七根、茎和叶的转录组文库，基于野三七转录组数据，筛选并验证了野三七不同组织部位适宜的内参基因ACT1和aTUB，为野三七中功能基因表达分析提供参考。基于野三七转录组数据库注释结果，进一步比对和ORF分析，筛选到46个与三萜生物合成的相关基因，克隆获得2条IDI基因、2条SS基因、4条SE基因、4条OSC和6条CYP716家族基因，并对其进行生物信息学分析及功能验证。PvfIDIs编码有功能的IDI，能促进番茄红素的积累。在大肠杆菌中成功表达PvfSS 、PvfSE1、PvfSE3和PvfSE4的融合蛋白。体外酶促证明，PvfSSs为有功能的鲨烯合酶，催化FPP生成鲨烯。通过酵母工程菌对PvfOSCs的功能进行研究，确定PvfOSC1为β-香树脂醇合酶，PvfOSC3为环阿屯醇合酶，PvfOSC4为达玛烯二醇合酶，并分别获得了生成β-香树脂醇、环阿屯醇和达玛烯二醇的酵母工程菌。野三七中OSC等基因的克隆及功能研究为解析野三七中人参皂苷生物合成途径奠定了基础，为野三七的遗传育种提供理论依据。