基于ITS2和psbA-trnH序列鉴别五味子和华中五味子  

Identification of Schisandra chinensis and S.sphenanthera Based on ITS2 and psbA-trnH Sequences

作　　者：杨楚楚 任广喜[1] 齐玉鑫 徐庆一 田俊强 程豪杰 陈晓洲 姜丹[1] 刘春生[1] YANG Chuchu;REN Guangxi;QI Yuxin;XU Qingyi;TIAN Junqiang;CHENG Haojie;CHEN Xiaozhou;JIANG Dan;LIU Chunsheng(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China;BabyCare Co.,Ltd.,Beijing 100020,China)

机构地区：[1]北京中医药大学中药学院,北京102488 [2]葆婴有限公司,北京100020

出　　处：《中国现代中药》2025年第1期43-51,共9页Modern Chinese Medicine

基　　金：北京中医药大学BURC联合重点项目(2023110031007136)。

摘　　要：目的:基于核内转录间隔区2(ITS2)和psbA-trnH序列,运用DNA条形码技术对五味子和华中五味子进行序列比对分析,建立快速、准确的分子鉴定方法,筛选最佳DNA条形码序列用于鉴别五味子和华中五味子真伪。方法:以五味子和华中五味子为材料,利用DNA提取试剂盒,分别提取五味子和华中五味子的总DNA,对ITS2序列和psbA-trnH序列进行聚合酶链式反应扩增及测序分析,使用DNAMAN 6.0软件统计其片段长度及变异位点个数,使用MEGA 5.0软件对数据进行分析比对,计算Kimura 2-parameter遗传距离,通过数据库预测二级结构,并利用邻接法建立系统发育树进行分析。结果:五味子和华中五味子ITS2序列片段长度为231 bp,鉴别位点为腺嘌呤(A)-胸腺嘧啶(T)、鸟嘌呤(G)-T、T-A;在psbA-trnH序列中,存在7个稳定的鉴别位点,分别为GA、T-G、胞嘧啶(C)-A、T-A、缺失-T、A-T、G-T,五味子和华中五味子ITS2序列二级结构存在明显差异,能将两者进行区分。结论:ITS2和psbA-trnH条形码均可以用于区分五味子和华中五味子,但psbA-trnH条形码种内遗传更稳定,种间鉴别位点更多,研究为五味子和华中五味子的快速、准确鉴定和用药安全提供科学依据。Objective:To establish a fast and accurate molecular identification method(DNA barcoding)for Schisandra chinensis and S.sphenanthera based on the internal transcribed spacer 2(ITS2)and chloroplast psbA-trnH intergenic region sequences and screen the optimal DNA barcode sequence for distinguishing between S.chinensis and S.sphenanthera.Methods:DNA extraction kits were used to extract total DNA from S.chinensis and S.sphenanthera.The ITS2 sequence and psbA-trnH sequence were amplified and sequenced by polymerase chain reaction.DNAMAN 6.0 was used to measure lengths of the fragments and the number of variation sites.MEGA 5.0 was used for sequence alignment.The Kimura 2-parameter model was adopted to calculate the genetic distance,and a database was used to predict the secondary structure.A phylogenetic tree was established with the neighbor-joining method.Results:The ITS2 sequences of S.chinensis and S.sphenanthera had a length of 231 bp,with adenine(A)-thymine(T),guanine(G)-T,and T-A as the sites for identification.The psbA-trnH sequence contained 7 stable sites for identification,which were G-A,T-G,C(cytosine)-A,T-A,deletion-T,A-T,and G-T.The secondary structure of the ITS2 sequence showing significant differences can be used for distinguishing between S.chinensis and S.sphenanthera.Conclusion:Both ITS2 and psbA-trnH sequences can be used as DNA barcodes to distinguish between S.chinensis and S.sphenanthera.The psbA-trnH barcode is more genetically stable within species and has more variation sites for distinguishing between species.This study provides a scientific basis for the rapid and accurate identification of S.chinensis and S.sphenanthera as well as for the evaluation of drug safety.

关 键 词：五味子 华中五味子 DNA条形码 内转录间隔区2序列 叶绿体基因间隔区序列 

分 类 号：R282[医药卫生—中药学]