龙胆科藏药蒂达类DNA条形码鉴定研究.docVIP

龙胆科藏药蒂达类DNA条形码鉴定研究 　　[摘要]应用ITS2序列对多基原龙胆科藏药“蒂达”进行DNA条形码鉴定，为保障该类药材的准确鉴定及其临床用药安全提供科学依据。以青藏高原地区龙胆科中獐牙菜属Swertia、花锚属Halenia、扁蕾属Gentianopsis、喉毛花属Comastoma、肋柱花属Lomatogonium 5个属，共计13个种，151份实验样本为研究对象，提取“蒂达”药材的基因组DNA、扩增ITS2序列并双向测序。采用CodonCode Aligner V3.7.1对测序峰图进行校对拼接。ITS2序列经比对后长度为231 bp，13个种共31种单倍型，G+C平均含量达61.40%。最小距离法和NJ树结果显示，龙胆科藏药“蒂达”类11个种能够各自聚为一支相互区分，并表现出良好的单系性，二叶獐牙菜和华北獐牙菜不能准确鉴定。ITS2序列可准确、稳定的鉴别龙胆科藏药“蒂达”类药材，从而为进一步构建复杂多基原藏药品种真伪鉴别方法提供了科学参考。 　　[关键词]藏药；“蒂达”；龙胆科；DNA条形码；ITS2序列；物种鉴定 　　[Abstract]The ITS2 barcode was used toidentify Tibetan medicine Dida， and tosecure its quality and safety in medication. A total of 13 species， 151 experimental samples for the study from the Tibetan Plateau， including Gentianaceae Swertia， Halenia， Gentianopsis， Comastoma， Lomatogonium ITS2 sequences were amplified， and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner V3.7.1. The Kimura 2-Parameter （K2P） distances were calculated using MEGA 6.0. The neighbor-joining （NJ） phylogenetic trees were constructed. There are 31 haplotypes among 231 bp after alignment of all ITS2 sequence haplotypes， and the average G+C content of 61.40%. The NJ tree strongly supported that every species clustered into their own clade and high identification success rate， except that Swertia bifolia and Swertia wolfangiana could not be distinguished from each other based on the sequence divergences. DNA barcoding could be used as a fast and accurate identification method to distinguish Tibetan medicine Dida to ensure its safe use. 　　[Key words]Tibetan medicina； Dida； Gentianaceae； DNA barcoding； ITS2； species identification 　　“蒂达”系藏药中最具有代表性的多基原药材品种之一，据《晶珠本草》[1-2]等文献记载其具有清肝利胆、利尿、续筋骨、止血等多种功效，临床上被广泛用于急性黄疸性肝炎、病毒性肝炎、胆囊炎、尿路感染、血病、赤巴病、跌打损伤、痢疾、流行性感冒等多种疾病的治疗。根据藏药“蒂达”来源，可分为印度“蒂达”，尼泊尔“蒂达”和西藏“蒂达”[3]，从药用形状，花色，药性和功效来看，藏医将“西藏蒂达”分为“金”、“银”、“铜”、“铁” 4类。文献记载[4]“蒂达”实质上系“味苦，治疗肝胆疾病”的一类功效相近的药物的“总称”[5]，基原极为复杂，其基原主要涉及到龙胆科Gentianaceae，虎耳草科Saxifragaceae，十字花科Crucife