多基源民族药岩陀DNA条形码识别系统构建

Rodgersiae Rhizoma comes from the botanical of Saxifragaceae Rodgersia, due to has a good effect on a variety of common diseases, it becomes the traditional medicine among Bai, Miao, Lisu tribe. Nowadays,the three kinds of Rodgersia podophylla are all used as Rodgersiae Rhizoma in clinic and there is a large variation on the clinical effects,however, conventional methods fail to distinguish the three kinds of Rodgersia podophylla effectively..DNA barcoding molecular identification technology is the latest development of modern molecular biology identification,which focus on the work of biological classification and demarcation in recent years.What’s more, DNA barcoding molecular identification technology is widely used in the aspect of identification of traditional Chinese medicine and original species, which is especially suitable for the identification of closely related species, confused and rare species..Based on the previous studies, DNA barcoding technology is first applied for the differentiation and identification of the three kinds of Rodgersiae Rhizoma in the present issue, in order to provide experimental gist of the species identification and novel method and technology for the quality control and to lay the foundation for further study of the relevance of different plants and biological activities.It not only offers theoretical reference for alleviating the shortage of resources and for selecting the fine varieties to inoculate culture ,but also plays a promotion function on the rational development and utilization of natural herbal medicine in Yunnan province, which has important reference value for the related research of other TCM.

岩陀来源于虎耳草科鬼灯檠属多基源植物，对多种疾病有较好疗效，为白、苗、傈僳族等民间习用药材。中医临床把岩陀所包含的三种基源植物混乱使用，其疗效差异较大，但传统鉴定方法未能有效区分鉴别此多基源植物。.DNA条形码分子鉴定是现代分子生物学中分子鉴定的最新发展，是近年来生物学分类和界定工作的研究热点，因具有简单、快速、准确，重复性高和稳定性强等特点，现广泛应用于中药材及基源物种的鉴定，尤其适用于近缘种、易混淆品种以及珍稀品种等的植物鉴别。.本课题在前期研究基础上，首次采用DNA条形码分子鉴定技术对三种岩陀植物进行区分鉴别研究，为其品种鉴别提供实验依据；为相关质量控制提供新方法和新技术；为深入研究不同岩陀原植物特征性与生物活性的关联性奠定基础；为择优品种进行驯化培养以缓解资源短缺等问题提供理论参考，对云南省植物药的合理开发利用也具有一定促进作用，同时也对其它中药的相关性研究提供重要参考价值。

课题采用ITS、ITS2、rbcL、matK和psbA-trnH这五种DNA候选条形码探讨不同序列及其组合序列在多基源民族药岩陀物种鉴定方面的有效性，筛选出适合该物种区分鉴定的条形码单片段或组合序列；并运用UPLC-Q-TOF-MS/MS技术，对岩陀所含三种鬼灯檠的化学成分进行研究，明确三者化学成分的相似性和差异性。研究结果为:（1）以各序列扩增和测序成功率、种内种间遗传距离、Neighbor-joining建树图、Barcoding gap和鉴定效率为主要考察指标进行综合考虑，发现psbA-trnH与其它候选条形码相比具有明显优势，因此，我们推荐psbA-trnH序列作为理想的DNA条形码用于多基源民族药岩陀的物种区分鉴定研究；（2）采用UPLC-Q-TOF-MS/MS法，分别鉴定出西南鬼灯檠9个化合物, 羽叶鬼灯檠14个化合物和七叶鬼灯檠17个化合物，并阐明了七叶鬼灯檠中6个化合物可能的质谱裂解规律。课题分别从DNA条形码和化学成分两方面揭示羽叶鬼灯檠和七叶鬼灯檠亲缘关系较西南鬼灯檠近。. 本研究首次将DNA条形码技术应用于多基源岩陀药材的鉴定研究中，GeneBank数据库收录了课题研究获得的岩陀共291条序列，建立了该药材DNA条形码鉴定识别体系。研究结果将为岩陀药材资源和品质评价提供科学依据，为修订完善其质量标准提供新的手段，对开发和培育岩陀优良品种提供分子生物学方面的指导，也对该药材资源保护和充分利用具有重要的参考意义；化学标志物的揭示可为其药代动力学、体内代谢产物结构鉴定、药效物质基础及作用机制等深入研究提供科学的参考依据。另外，我们的研究揭示不同地理环境不同品种的岩陀药材在DNA和化学成分方面有所区别，那么进入体内发挥药理药效的物质基础和作用机制是否也有差异，值得后续研究。