基于ITS2和psbA-trnH序列鉴别金线兰及其近缘种  

Identification of Anoectochilus roxburghii and its closely related species based on ITS2 and psbA-trnH sequences

作　　者：吴岩斌[1] 张超 吴建国[1] 吴锦忠[1] 郑承剑 WU Yan-bin;ZHANG Chao;WU Jian-guo;WU Jin-zhong;ZHENG Cheng-jian(College of Pharmacy,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;Department of Chinese Medicine Authentication,Faculty of Pharmacy,Naval Medical University,Shanghai 200433,China)

机构地区：[1]福建中医药大学药学院,福建福州350122 [2]海军军医大学药学系中药鉴定学教研室,上海200433

出　　处：《中草药》2022年第18期5807-5812,共6页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(82174081,81773846);福建省科技厅高校产学合作项目(2020Y4015);上海市浦江人才计划(21PJD082)。

摘　　要：目的 应用ITS2和psbA-trnH条形码鉴定金线兰Anoectochilus roxburghii及其近缘种。方法 提取金线兰及其近缘种的DNA,对ITS2和psbA-trnH序列进行扩增和测序,计算物种种内、种间遗传距离,构建邻接法(neighbor-joining,NJ)系统聚类树直观反映鉴定结果。结果 经PCR扩增后测序,金线兰及其近缘种ITS2序列长度为250~254bp,GC含量为48.2%~51.6%;psbA-trnH序列长度为636~704 bp,GC含量为31.8%~32.0%。根据K2P遗传距离计算,金线兰的种内遗传距离明显小于种间遗传距离。基于ITS2序列构建的NJ树结果显示,除长片金线兰A. longilobus外,金线兰与其余混伪品可以明显区分。基于psb A-trnH序列构建的NJ树结果显示,除长片金线兰和丽蕾金线兰A. lylei外,金线兰与其余金线兰属近缘种可以明显区分。结论 ITS2和psb A-trnH序列可以鉴别金线兰与其遗传距离较远的近缘种。Objective To identify Anoectochilus roxburghii and its closely related species using ITS2 and psbA-trnH barcode.Methods Total genomic DNA was isolated from A. roxburghii and its related species. Nuclear DNA ITS2 and chloroplast gene psbA-trnH sequences were amplified and sequenced. The Kimura 2-parameter(K2P) distances were calculated. Identification analyses was performed using Neighbor-joining(NJ) methods. Results After PCR amplification, the ITS2 regions of tested samples were sequenced and their lengths were from 250—254 bp, and the GC content was 48.2%—51.6%. The lengths of psbA-trnH regions ranged in size from 636 to 704 bp, with GC content ranging from 31.8%—32.0%. Based on K2P model, the intra-specific genetic distances of A. roxburghii in ITS2 and psbA-trnH regions were smaller than the inter-specific ones of A.roxburghii with its closely related species. The NJ tree based on ITS2 sequence showed that A. roxburghii can be distinguished clearly from its adulterants except for A. longilobus, while the NJ tree based on psbA-trnH sequence showed that A. roxburghii can be distinguished clearly from other Anoectochilus species except for A. longilobus and A. lylei. Conclusion ITS2 and psbA-trnH sequences can be used as potential DNA barcode to distinguish A. roxburghii from its related species with genetic distances.

关 键 词：金线兰属 金线兰 DNA条形码 ITS2序列 psbA-trnH序列 

分 类 号：R286.2[医药卫生—中药学]