基于ITS2序列及其二级结构对防己及其混伪品的鉴定

目的:利用核糖体DNA第2内部转录间隔区(ITS2)及其二级结构对传统中药材防己及其混伪品进行分子鉴定及聚类分析。方法:从Gen Bank上下载36条防己及其混伪品的ITS序列,利用Geneious R11.0.3进行序列比对,运用MEGA7.0计算种内、种间K2P遗传距离,构建邻接(NJ)系统进化树,同时利用ITS2数据库在线软件预测各样本的二级结构,并利用4Sale软件进行二级结构的比对,最终运用ProfDistS软件基于距离法构建了PNJ进化树。结果:各种间平均遗传距离均远大于种内平均遗传距离,NJ树显示防己及其混伪品聚为6个分支;混伪品与正品的二级结构均有一定的差异;PNJ进化树比NJ树显示出更多的分支和更高的分辨率。结论:虽然ITS2可以作为防己及其混伪品的DNA条形码,但是若包含ITS2二级结构所蕴含的系统发育信息,鉴定结果更加精准。

Identification of Stephaniae Tetrandrae Radix and Its Adulterants Based on ITS2 and Its Secondary Structure

Objective: To identify Stephaniae Tetrandrae Radix and its adulterants based on internal transcribed spacer 2 (ITS2) and its secondary structure. Method: Total 36 ITS sequences were downloaded from GenBank and aligned by Geneious R11.0.3. The intraspecific and interspecific K2P genetic distance was calculated by MEGA 7.0, and the neighbor joining (NJ) phylogenetic tree was built by the same software.Simultaneously, the secondary structure of each sample was predicted by the ITS2 Database online softwareand aligned by 4sale software. Finally, ProfDistS software was used to construct the profile neighbor-joining (PNJ) phylogenetic tree based on the distance method. Result: The average interspecific genetic distance was far greater than the average intraspecific genetic distance. NJ tree showed that Stephaniae Tetrandrae Radix and its adulterants were clustered into 6 branches;both Stephaniae Tetrandrae Radix and its adulterants had different secondary structures. The PNJ tree showed more branches and higher resolution than NJ tree. Conclusion: ITS2 can be used as a DNA barcode for distinguishing Stephaniae Tetrandrae Radix from its adulterants, but the identification results would be more accurate when considering the phylogenetic information contained in ITS2 secondary structure.