野生与种植人参次生代谢物差异机制的定量蛋白质组学研究

It has been reported that the substantial differences of the medical efficacies in wild ginseng and cultivated ginseng were due to the different composition and content of the secondary metabolites such as ginsenosides and phenols. Whereas, few reports were shown the reasons on which kinds of protein and enzymes resulted in these differences on generating those secondary metabolites. In our previous projects being supported by Chinese National Science Foundation, the difference mechanisms of glycometabolism and amino acid metabolism in which processes providing the precursors for the biosynthesis of those secondary metabolites were analyzed by two-dimensional gel electrophoresis (2DE). In addition, the differentially expressed proteins such as squalene synthase and isoflavone reductase, which involved in the formation of ginsenosides were obtained too. It should be pointed out that most of enzymes involving in the processes of secondary metabolites biosynthesis were membrane proteins, which were poor stability, low content and dynamic changes, so it was necessary to find more powerful analysis methods to detected those enzymes. Basing on the preliminary work, more and more differentially expressed proteins related to secondary metabolism will be obtained by 2D-DIGE and iTRAQ, which are quantitative proteomics technologies with a wider analysis scope and higher sensitivity and accuracy. And combined with those results on the contents of secondary metabolites, the activities of key enzymes in biosynthetic pathway and the interaction molecules with key enzymes, the differences mechanism of secondary metabolites could be elucidated at protein levels clearly. Furthermore, the experimental basis for the efficacy differences between wild ginseng and cultivated ginseng will be clarified. After studying mechanism of the changes in the secondary metabolites during the processes of ginseng development and by stresses resistance, the medical efficacy and resistance of cultivated ginseng will be regulated too. Meanwhile, this project will also provide a reference for the other kinds of wild and cultivated medicinal plants.

野生与种植人参（野山参与园参）功效差异主要是其皂苷、酚类等次生代谢物组成及含量不同造成的，但对于是哪些酶和蛋白相互作用及调控导致二者差异，尚未见报道。课题组在前期完成基金等项目中已利用双向电泳技术阐述了在野山参与园参中为次生代谢物合成提供前体及能量的糖代谢和氨基酸代谢差异的机制，同时找到鲨烯合成酶、异黄酮还原酶等与皂苷和黄酮合成相关的差异表达酶。但与次生代谢合成相关酶大多为膜蛋白，含量极低、稳定性差、反应动态，因此需利用灵敏度和准确度更高的定量蛋白质组学方法--2D-DIGE和iTRAQ，寻找更多同野山参与园参次生代谢相关的差异蛋白，再结合次生代谢物含量、合成途径关键酶活性及与关键酶相互作用蛋白等结果，在分子水平上阐明野山参与园参次生代谢物差异机制，解释二者药用功效差异的原因，进而通过了解次生代谢物在生长和抗胁迫中的变化规律来调控人参药效及抗胁迫能力，并为其他野生与种植药材研究提供参考。

野生与种植人参（野山参与园参）功效差异可能是其皂苷、酚类等次生代谢物组成及含量不同造成的。本项目利用灵敏度和准确度更高的定量蛋白质组学方法--2D-DIGE和iTRAQ，寻找更多同野山参与园参次生代谢相关的差异蛋白，再结合次生代谢物含量、合成途径关键酶活性等结果，在分子水平上阐明野山参与园参次生代谢物差异机制。首先糖酵解和三羧酸循环相关酶及其中间产物在野生人参有高水平的积累，为次生代谢物合成提供前体及能量；氨基酸代谢相关酶及其衍生物,如谷氨酸脱羧酶和SAM在野山参中高积累，说明野山参的氨基酸代谢比园参更活跃；在野山参中皂苷合成关键酶、苯丙烷代谢关键酶高表达，促进皂苷与酚酸等次生代谢产物合成，可能是野山参中药效活性成分含量高的原因之一；AsA-GSH 循环中关键抗氧化酶活性及抗氧化物量均为野山参高于园参，导致野山参抗氧化能力强于园参，为野山参与园参功效差异研究提供了理论依据。