中药白头翁及混伪品的DNA条形码分子鉴定

Molecular identification of Pulsatilla chinensis(Bunge)Regel and adulterants using different DNA barcodes

目的:应用ITS2、psbA-trnH、matK、rbcL 4条DNA条形码区分白头翁药材及混伪品(委陵菜、翻白草、白鼓钉)。方法:提取样品总DNA,对目的条带进行PCR扩增和双向测序获得序列。通过软件MEGA6.06对序列进行比对、遗传距离分析和进化树构建。采用相似性搜索法(BLAST)评估各序列鉴定成功率。结果:4条DNA条形码最大种内遗传距离均远小于其与混伪品的最小种间遗传距离,PCR扩增效率和测序成功率均为100%,ITS2序列种、属水平鉴定成功率均为100%;进化树显示ITS2、psbA-trnH与matK序列能有效将白头翁药材及混伪品明显区分开,而rbcL序列仅可鉴别出白鼓钉,且ITS2、psbA-trnH从种水平上对白头翁的鉴定能力大于matK序列。结论:ITS2与psbA-trnH序列可对白头翁及混伪品进行鉴别,确认了条形码在白头翁及混伪品中的普适性,为质量标准建立提供了依据。

Objective:To identify Pulsatilla chinensis(Bunge)Regel and its adulterants using DNA barcodes of ITS2,psbA-trnH,matK and rbcL sequences,providing a scientific basis for the application of DNA barcode technology in the identification of Chinese herbal medicines,and ensure the safety and accuracy of Pulsatilla chinensis(Bunge)Regel of its clinical application.Methods:The total DNA of Pulsatilla chinensis(Bunge)Regel and its adulterants were extracted,and then amplified by polymerase chain reaction.All of sequences were obtained by bi-directional DNA sequencing after detection of products.Finally,sequence alignment,genetic distance analysis and phylogenetic tree construction were performed by software MEGA 6.06.Using BLAST to evaluate the identification ability of the sequences.Results:The maximal intraspecific genetic distance of four sequences of Pulsatilla chinensis(Bunge)Regel is much smaller than the minimum interspecies genetic distance.The success rate of PCR amplification and sequencing of the four barcodes are 100%,and the species and genus level identification success rate of ITS2 sequence are 100%.NJ trees showed that ITS2,psbA-trnH and matK sequences could effectively distinguish Pulsatilla chinensis(Bunge)Regel and its adulterants,while rbcL sequences could only identify Polycarpaea corymbosa(L.)Lam,and the ability of ITS2 and psbA-trnH sequences to identify Pulsatilla chinensis(Bunge)Regel at the species level is greater than the matK sequence.Conclusion:ITS2 and psbA-trnH sequences can identify Pulsatilla chinensis(Bunge)Regel and its adulterants.This study confirmed the applicability of DNA barcodes in Pulsatilla chinensis(Bunge)Regel and its counterfeit products,and provided basis for the establishment of quality standards.