基于分子谱系地理学与谱-效相关联方法研究竹节参资源品质差异形成机制

Panax japonicas (PJ) belongs to Araliaceae Panax family in the plant kingdom and grows wildly from the west region of China Yunnan province to east Japan, which has been used as a normal herbal medicine in minority ethnic group. From 2003, we have done a systematic study on PJ including pharmacochemical and pharmacological efficacy. The saponins from PJ were the main and effective ingredients which have anti-inflammatory, antioxidant activity. The quality differences of PJ were found in different origin through resource survey and quality evaluation in Yunnan, Sichuan, Hubei province, etc. But the mechanism of quality differences has not yet been understood. Recently, many researches showed that there were relationship between quality differences and genetic variation in the medicinal plants, so we hypothesized that the quality differences of PJ were induced by genetic variation. Our project intends to study formation mechanism of quality difference of Panax japonicas in different region using molecular phylogeography and spectrum-activity methods. Firstly, we establish the evolutionary spectrum diagram of PJ and analyze the relevance of its genetic variation and the environmental factors; secondly, we establish the relationship between pharmacodynamic fingerprint and DNA sequence polymorphism of PJ in different region, then analysis the variant characteristics of the gene sequence correlated to the pharmacodynamic differences, and furthermore investigate the molecular mechanism of the formation on its resource quality differences. It will establish the foundation for core collections and the quality control of Panax japonicus.

竹节参系五加科人参属植物，分布在西起我国云南东至日本的广大区域，是我国西南地区民间常用中草药。自2003年以来，我们对竹节参化学成分与药理作用进行了系统研究，分离鉴定了其主要化学成分，并初步确认其抗炎、抗氧化活性；同时对竹节参生长区域进行了资源调查与品质评价，发现不同产地竹节参存在显著品质差异，但对其形成机制目前仍不明确。近期相关研究显示，中药品质差异与其遗传变异有关，那么竹节参的品质差异是否与其遗传变异相关？基于此，本项目拟采用分子谱系地理学与谱效关系相结合方法，首先检测不同地理环境生长的竹节参基因多态性，通过谱系地理学分析，构建竹节参进化谱系地理图，分析其遗传分化情况及相关环境因素的影响；另外建立竹节参药效指纹图谱，将不同居群竹节参药效指纹图谱与其谱系地理图谱相关联，分析形成药效差异的相关基因特征，进而探讨其资源品质差异形成的分子机制，为有效控制竹节参质量、建立其核心种质资源奠定基础。

竹节参（Rhizoma of Panax japonicus）系五加科人参属植物，生长于海拔1500米以上的常绿阔叶与落叶阔叶混交林带，在我国主要分布于云南、四川、湖北、贵州等省。竹节参具有抗炎等多种生物学活性，但其分布区域的广泛，变异品种繁多、野生和栽培品的质量都可能影响其药材的使用。因此本项目 (1) 采用分子生物学技术，建立了ITS2序列条形码技术进行物种鉴定，对不同居群竹节参从分子水平上进一步佐证植物样本物种为五加科人参属竹节参。(2) 从竹节参及其变种（狭叶竹节参、羽叶三七、珠子参）样本中的基因组DNA中分别克隆得到4条β-AS基因的gDNA序列，并成功预测和分析了四者的cDNA序列、氨基酸序列及各自蛋白结构，发现竹节参变种的β-AS基因的gDNA具有明显差异。(3) 克隆了野生和栽培品竹节参的β-AS基因cDNA编码区序列，cDNAs全长均为2286bp,编码761个氨基酸。两者之间存在8个氨基酸差异，生物信息学分析和比较发现两个品种的β-AS蛋白在理化性质、蛋白质二级、三级结构以及磷酸化位点出现差异，可能会导致两者的催化活性存在差异。(4) 在药理活性及机制研究中发现竹节参皂苷Ⅳa具有良好的抗炎效果，其抗炎作用机制可能与抑制miR-155的表达有关，miR-155通过抑制其靶基因GSK-3β的活性进而调控NF-κB信号通路从而促进炎症反应。