基于ITS2序列的黔产金线莲及其常见易混品分子鉴定研究  

Molecular Identification of Guizhou Anoectochilus roxburghii and its Common Mixed Products Based on ITS2 Sequence

作　　者：黄哲 杨烨 王世清 党友超 王锦 HUANG Zhe;YANG Ye;WANG Shiqing;DANG Youchao;WANG Jin(School of Pharmacy,Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China;Zhejiang Sundoc Pharmaceutical Science and Technology Co.,Ltd.,Hangzhou 310051,China)

机构地区：[1]贵州中医药大学药学院,贵州贵阳550025 [2]浙江圣兆药物科技股份有限公司分析中心,浙江杭州310051

出　　处：《现代中药研究与实践》2023年第4期11-15,共5页Research and Practice on Chinese Medicines

基　　金：贵州省科技计划项目(黔科合基础[2020]1Y373);2018年度学术新苗培养及创新探索专项(黔科合平台人才[2017]5735号-24);贵州中医药大学博士启动基金资助项目(2018,3043-043180015)。

摘　　要：目的 对黔产金线莲及其常见易混品进行分子鉴定。方法 以ITS2序列为模版,对所收集的西南齿唇兰Anoectochilus elwesii、兴仁金线兰Anoectochilus xingrenensis、植物斑叶兰Goodyera schlechtendaliana等5种植物样品进行ITS2序列扩增及测序,同时在GenBank下载台湾金线莲Anoectochilus formosanus、小斑叶兰Goodyera repens等共6种相关植物ITS2序列,采用MEGAX6.4软件确定ITS2序列的GC含量,基于K2P模型计算种内和种间遗传距离,构建NJ系统发育树,ITS2 database在线分析二级结构,研究ITS2条形码序列对金线莲的鉴别效果。结果 金线莲的ITS2序列长度在329 bp,种内距离为0.001 8。而金线莲与兴仁金线兰、西南齿唇兰、银线莲、斑叶兰、小斑叶兰的最小种间距离为0.038 8,最大种间距离为0.109 9,表明金线莲的种内平均K2P遗传距离远小于种间遗传距离,并已达到序列差异的标准阈值。构建的邻接树显示金线莲单独聚成一支,与其他植物种类区分明显。结论 建立的鉴定方法简便,结果准确,能有效区分金线莲及其常见易混品植物。Objective To identify Anoectochilus roxburghii and its plants of different genera in the same family.Methods 5 samples of Anoectochilus elwesii,Anoectochilus xingrenensis and Goodyera schlechtendaliana were collected using ITS2 sequence as template.ITS2 sequence amplification and sequencing.At the same time,six related ITS2 sequences of Anoectochilus formosanus and Goodyera repens were downloaded from GenBank.The GC content of ITS2 sequence was determined by MEGAX6.4 software.The intraspecific and interspecific genetic distances were calculated based on K2P model,and the NJ phylogenetic tree was constructed.The secondary structure of ITS2 database was analyzed online to study the identification effect of ITS2 barcode sequence on Anoectochilus roxburghii.Results The ITS2 sequence length of Anoectochilus roxburghii was 329 bp,and the intraspecific distance was 0.001 8.The minimum interspecific distance between Anoectochilus roxburghii and Anoectochilus xingrenensis,Anoectochilus elwesii,Goodyera hachijoensis,Goodyera procera,Goodyera repens was 0.038 8,and the maximum interspecific distance was 0.109 9.This indicated that the intraspecific average K2P genetic distance was far smaller than the interspecific genetic distance and had reached the standard threshold of sequence difference.Adjacent trees constructed showed that Anoectochilus roxburghii clustered together and distinguished itself from other plant species.Conclusion The method is simple and accurate,and it can effectively distinguish Anoectochilus roxburghii and its common mixed products.

关 键 词：金线莲 易混品 ITS2 分子鉴定 

分 类 号：R282.5[医药卫生—中药学]