基于ITS2序列的羌活及其混伪品的DNA条形码鉴定

目的对羌活及其混伪品进行分子鉴定,以确保该药材的质量以及临床疗效。方法利用PCR测序法,对样品进行核基因ITS2片段扩增并双向测序,所得序列经CodonCode Aligner拼接后,用软件MEGA4.0进行相关数据分析,并构建邻接(NJ)树。利用已建立的ITS2数据库及其网站预测ITS2二级结构。结果羌活与宽叶羌活ITS2序列长度均为228 bp,二者种内平均K2P(Kimura-2-parameter)遗传距离均远远小于其与混伪品的种间平均K2P遗传距离;由所构建的系统聚类树图可以看出,羌活与宽叶羌活均表现出了单系性,而同时又与其他混伪品明显分开;比较ITS2二级结构发现,羌活基原植物与其混伪品在4个螺旋区的茎环数目、大小、位置以及螺旋发出时的角度均有明显差异。结论 ITS2序列作为DNA条形码可以方便快捷地鉴别羌活及其混伪品,为其种质资源鉴定及临床安全用药提供了重要分子依据。

Identification of Notopterygii Rhizoma et Radix and its adulterants using DNA barcoding method based on ITS2 sequence

Objective To discriminate Notopterygii Rhizoma et Radix and its adulterants,and secure the quality and clinical curative effect of this medicinal material.Methods The second internal transcribed spacer(ITS2) of ribosomal DNA was amplified and sequenced by bidirectional sequencing of PCR products.Sequence assembly and consensus sequence generation were performed by using CodonCode Aligner.Phylogenetic study was performed using software MEGA 4.0 in accordance with Kimura-2-parameter(K2P) model,and the phylogenetic tree was constructed by using the neighbor-joining(NJ) method.The ITS2 secondary structure was predicted using ITS2 web server.Results The length of ITS2 sequence of Notopterygium incisum and N.franchetii was 228 bp.Their mean intraspecific genetic distance(K2P distance) was far lower than their mean interspecific genetic distance with the adulterants.In the cluster dendrogram,both N.incisum and N.franchetii showed monophyletic,and simultaneously distinguished from their adulterants.To compare the ITS2 secondary structure between the origin plant of Notopterygii Rhizoma et Radix and its adulterants,we noticed that it was obviously distinguished from the adulterants in terms of number,size,position of loop,and the angle of helix exsertion.Conclusion ITS2 barcode could be used to identify Notopterygii Rhizoma et Radix and its adulterants effectively,and then provide important molecular evidence for the authentication of germplasm rescouces and clinic safety of medicinal use.