大青叶及其混淆品的ITS2序列鉴定研究

目的:对中药材大青叶及其混淆品进行分子鉴定,以确保该药材的质量以及临床疗效。方法:采用PCR直接测序法,测定核基因ITS2区,所得序列经CodonCode Aligner拼接后,用软件MEGA4.0进行相关数据分析,构建NJ(邻接)树,并利用Koetschan等建立的ITS2数据库及其网站预测ITS2二级结构。结果:大青叶基源植物菘蓝ITS2序列长度为191bp,其种内平均K2P遗传距离(0.002)远小于其与混淆品的种间平均K2P遗传距离(0.882);由所构建的系统聚类树图可以看出,各物种均表现出了单系性,而同时又与其它物种明显区分开;比较ITS2二级结构发现,各物种在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作为条形码可以方便快捷的鉴别大青叶正品及其混淆品,对药典中其它药材的相关研究也具有重要的参考价值。

Molecular Identification of Isatidis Folium and Its Adulterants by ITS2 Sequences

This study aimed to discriminate between Isatidis Folium and its adulterants in order to guarantee the quality and clinical curative effect of this medical material.In this study,the second internal transcribed spacer(ITS2) of ribosomal DNA were amplified and sequenced.Sequence assembly and consensus sequence generation were performed by using the CodonCode Aligner.Phylogenetic study was performed by using software MEGA 4.0 software in accordance with the Kimura 2-parameter(K2P) model.And the phylogenetic tree was constructed using the neighbor-joining methods.The ITS2 secondary structure was predicted by using the ITS2 database and website which was built by Koetschan et al.The results showed that the length of ITS2 sequence of the origin plant of Isatidis Folium was 191 bp.The mean intraspecific genetic distance(K2P distance,0.002) was far lower than its mean interspecific genetic distance(0.882).In the cluster dendrogram,all species showed monophyletic,and distinguished from others.To compare the ITS2 secondary structure of the origin plant of Isatidis Folium and its adulterants,we noticed that it was obviously distinguished from other species in the amount,size,position of loop and the angle of helix exertion.It is concluded that ITS2 sequence can be used as DNA barcode to identify Isatidis Folium and its adulterants effectively.This study may provide an important example for the authentication of other medicinal herbs listed in the Chinese Pharmacopoeia.