基于ITS2序列的市售木通药材及其混伪品的分子鉴定12

Molecular identification of commercially available Akebiae Caulis and its adulterants based on ITS2 sequence

目的建立基于核糖体内部转录间隔区2(internal transcribed spacer 2,ITS2)序列的木通药材DNA条形码鉴定方法,对市售木通药材进行物种分析。方法收集河北、安徽、贵州等地的样品总计45份,其中木通药材及其混伪品的原植物样品18份,市售木通药材样品27份。通过提取DNA、聚合酶链式反应(polymerase chain reaction,PCR)扩增、双向测序获得ITS2序列,基于邻接(neighbor joining,NJ)系统发育树和单核苷酸多态性(single nucleotide polymorphisms,SNP)位点进行物种鉴定分析。结果基于木通药材及其混伪品原植物ITS2序列构建的NJ树分析结果表明,木通药材正品及其混伪品在NJ树上聚为独立的分支,木通药材正品及其混伪品在NJ树上可明确区分;基于NJ树对27份市售木通药材的物种分析表明,市售样品中仅有4份为正品木通,2份为小木通,21份为粗齿铁线莲,正品率为14.8%。结论基于ITS2序列的DNA条形码技术可以准确区分中药材木通及其混伪品,市售木通药材物种较混乱。

Objective A DNA barcoding identification method based on internal transcribed spacer 2(ITS2)sequences was established to analyze the species of commercially available Mutong(Akebiae Caulis).Methods A total of 45 samples from Hebei,Anhui,Guizhou provinces and other places were collected,including 18 samples of original plants samples and 27 samples of commercially available medicinal materials.Their ITS2 sequences have been obtained after DNA extraction,polymerase chain reaction(PCR)amplification and bi-directional sequencing.Species identification analysis were based on neighbor-joining(NJ)phylogenetic tree and single nucleotide polymorphisms(SNPs).Results The results of NJ phylogenetic tree constructed by the original plants of Akebiae Caulis and its adulterants showed that the authentic Akebiae Caulis and its main adulterants gathered into independent branches.The authentic Akebiae Caulis and its adulterants could be clearly distinguished by the NJ phylogenetic tree.Based on the NJ tree,the analysis of the species of 27 commercial samples showed that only four samples were Akebiae Caulis,two samples were Clematis armandii,and 21 samples were Clematis argenfilucida.The authenticity rate of commercially available medicinal materials was 14.8%.Conclusion The DNA barcoding based on the ITS2 sequence can accurately distinguish Akebiae Caulis and its adulterants.The varieties of commercially available Akebiae Caulis are more chaotic.