不同种源银柴胡及其伪品的ITS2序列分析与鉴别  

Identification of Stellariae Radix and Its Counterfeits from Different Provenances Based on ITS2 Sequences

作　　者：李振凯 冯璐 杨燕 宋乐 王红 李彦青 高跳 彭励[1] LI Zhen-kai;FENG Lu;YANG Yan;SONG Le;WANG Hong;LI Yan-qing;GAO Tiao;PENG Li(School of Life Sciences,Ningxia University,Yinchuan 750021,China)

机构地区：[1]宁夏大学生命科学学院,宁夏银川750021

出　　处：《中国现代中药》2022年第12期2335-2341,共7页Modern Chinese Medicine

基　　金：宁夏回族自治区自然科学基金项目(2021AAC03103);吴忠市科技创新驱动计划项目;吴忠市同心县科技发展计划项目。

摘　　要：目的:采用基于内转录间隔区2(ITS2)序列的分子鉴定法分别对17份不同种源银柴胡和6种伪品基原植物进行分析和鉴别。方法:对植物样品进行DNA提取、扩增、测序,并检索GenBank数据库,获取17份不同种源银柴胡及6种伪品的ITS2序列。采用ITS2数据库进行注释,去除5.8S和28S片段,获得完整的ITS2序列,利用MEGA 8.0软件进行序列比对、计算K2P遗传距离、构建邻接(NJ)系统发育树,采用美国国家生物技术信息中心(NCBI)数据库和“中药材DNA条形码鉴定系统”进行BLAST分析和物种鉴定,并利用ITS2数据库预测二级结构。结果:17份银柴胡样品共得到3个单倍型ITS2序列(YCH1、YCH2和YCH3),经与银柴胡标准ITS2序列比对,发现YCH1和YCH2与标准序列一致,但YCH3有4处碱基缺失,表现出0.936的遗传距离,在系统发育树中被单独聚为一个分支,并具有不同的二级结构;6种伪品的ITS2序列在长度、鸟嘌呤+胞嘧啶占比、变异位点、遗传距离、NJ系统发育树和二级结构上均与银柴胡ITS2序列表现出明显的差异。结论:基于ITS2序列的分子鉴定方法可以有效鉴别银柴胡真伪,不同种源银柴胡的遗传物质可能存在一定差异。Objective:To ensure the correct provenance and construct the seed breeding base of Stellariae Radix from the source,we identified 17 samples of Stellariae Radix from different sources and the original plants of 4 counterfeits based on internal transcribed spacer 2(ITS2)sequence.Methods:The DNA samples were extracted,amplified,sequenced,and searched against GenBank to obtain the ITS sequences of 17 samples of Stellariae Radix and the original plants of 6 counterfeits.MEGA 8.0 was used for sequence alignment,calculation of Kimura′s two-parameter(K2P)genetic distance,and construction of neighbor-joining(NJ)phylogenetic tree.Blast analysis and species identification were conducted via the NCBI database and the DNA barcoding system for Chinese herbal medicines.The secondary structure was predicted using the ITS2 Database online.Results:A total of 3 haplotypes(YCH1,YCH2,and YCH3)of ITS2 sequences were obtained from 17 Stellariae Radix samples.YCH1 and YCH2 were consistent with the standard ITS2 sequence of Stellariae Radix.YCH3 had 4 base deletions,showing a genetic distance of 0.936,which was clustered into a branch alone in the phylogenetic tree and had a unique secondary structure.The ITS2 sequences of the original plants of 6 counterfeits showcased significant differences in length,guanine+cytosine content,mutation site,genetic distance,position on the phylogenetic tree,and secondary structure compared with the standard ITS2 sequence of Stellariae Radix.Conclusion:The ITS2 sequence-based molecular method can effectively identify the authenticity of Stellariae Radix,and there may be genetic differences among different provenances.

关 键 词：银柴胡 内转录间隔区2 DNA条形码 种质资源 真伪鉴别 

分 类 号：R282.5[医药卫生—中药学]