摘要
目的:基于内转录间隔区(ITS)序列,运用DNA条形码技术对滇鸡血藤药材及其混伪品进行序列比对分析,根据单核苷酸多态性位点构建滇鸡血藤单倍型数据库,建立快速、准确鉴定滇鸡血藤的分子鉴定方法。方法:以滇鸡血藤及其混伪品为材料,利用DNA提取试剂盒,分别提取滇鸡血藤及其混伪品的总DNA,对ITS序列进行聚合酶链式反应扩增及测序分析,使用DNAMAN软件统计其片段长度及变异位点个数,使用MEGA软件对数据进行分析比对,计算Kimura2-parameter遗传距离,并利用邻接法建立系统发育树进行分析。结果:滇鸡血藤ITS序列片段长度为675 bp,共包括19种单倍型,与其主要混伪品的差异位点为212、223、224 bp,系统发育树显示,ITS序列能够区分滇鸡血藤及其混伪品。结论:ITS条形码可以作为区分鉴定滇鸡血藤及其混伪品的分子条形码,包含19个单倍型的单倍型数据库覆盖了滇鸡血藤全部的单倍型。
Objective:Based on internal transcribed spacer(ITS)sequence,this study used DNA barcode technology forsequence alignment analysis of Kadsurae Caulis and its adulterants.A haplotype database was constructed based on single nucleotide polymorphism(SNP)loci to establish a rapid and accurate molecular identification method for Kadsurae Caulis.Methods:Kadsurae Caulis and its adulterants were used as materials to extract their total DNA using a DNA extraction kit.The ITS sequences were amplified by polymerase chain reaction(PCR)and subjected to sequencing analysis.The length of the fragment and the number of variation sites were calculated by DNAMAN software.The data were compared by using MEGA software to calculate the Kimura2-parameter(K2P)genetic distances and to build a phylogenetic tree by using the neighbor-joining(NJ)method for analysis.Results:The length of Kadsurae Caulis ITS sequence was 675 bp,including 19 haplotypes,and the difference sites with its main adulterants were identified at positions 212,223,224 bp.Phylogenetic tree showed that ITS could separate Kadsurae Caulisand its adulterants.Conclusion:ITS barcode can be used as a molecular barcode for the identification of Kadsurae Caulis and its adulterants.The haplotype database containing 19 haplotypes covers all haplotypes of Kadsurae Caulis.
作者
刘心雨
姜丹
杨楚楚
徐庆一
徐裕彬
陈绪军
任广喜
刘春生
LIU Xin-yu;JIANG Dan;YANG Chu-chu;XU Qing-yi;XU Yu-bin;CHEN Xu-jun;REN Guang-xi;LIU Chun-sheng(College of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 102488;Beijing Jujing Health Technology Group Co.,Ltd.,Beijing 100068;Beijing Weiren Chinese Medicine Slices Factory Co.,Ltd.,Beijing 101300)
出处
《中国现代中药》
CAS
2024年第1期10-17,共8页
Modern Chinese Medicine
基金
中央高校横向课题项目(2021110031010987)。
