基于ITS2序列及二级结构对连钱草与其地方习用品及易混品的鉴别研究  

Molecular Authentication of Glechomae Herba and Its Substitute and Adulterants by Integrating ITS2 and Its Secondary Structure

作　　者：郑梦迪 张彦[1] 张寒[1] 汪兴军 ZHENG Meng-di;ZHANG Yan;ZHANG Han;WANG Xing-jun(Department of Pharmacy,Xi’an Medical University,Xi’an 710021,China)

机构地区：[1]西安医学院药学院,陕西西安710021

出　　处：《现代中药研究与实践》2022年第1期1-6,共6页Research and Practice on Chinese Medicines

基　　金：陕西省科技厅自然科学基础研究计划项目(2021JQ-782);陕西省教育厅项目(21JK0900)。

摘　　要：目的利用ITS2(Internal Transcribed Spacer 2)序列及其二级结构对传统中药连钱草与其地方习用品及易混品进行分类鉴定。方法提取连钱草药材基原植物活血丹基因组DNA,扩增ITS序列并测序;同时从GenBank数据库中下载活血丹等共8个物种42条ITS序列,基于隐马尔可夫模型注释到ITS2序列。运用MEGA7软件进行种内种间序列差异分析,并构建Neighbor-jioning(NJ)进化树。同时利用ITS2 Database预测各样本ITS2二级结构,并利用4Sale软件比对一级结构和二级结构的联合矩阵,最终采用ProfDistS软件基于距离法构建Profile neighbor joining(PNJ)进化树。结果各物种种内最大遗传距离远小于种间最小遗传距离,Barcoding Gap存在于活血丹与各易混品之间;各物种的二级结构均存在差异;系统发育树显示各样本均独立分支。结论ITS2可以作为准确鉴别中药连钱草与其地方习用品及易混品的DNA条形码,为连钱草的基原鉴别及安全用药提供了科学依据。Objective To identify Chinese medicinal materials Glechomae Herba and its local varieties based on ITS2(Internal Transcribed Spacer 2)and its secondary structure.Methods DNA of Glechoma longituba is extracted,its sequences are amplified and sequenced.42 ITS sequences of 8 species including Glechoma longituba are downloaded from GenBank,and ITS2 sequences are obtained by using hidden Markov model annotation.MEGA7 software is used to align the sequences,analyze the differences between species and within species,and construct Neighbor-jioning(NJ)phylogenetic tree.ITS2 database is used to predict ITS2 secondary structure of each sample,and 4 Sale software is used to align the joint matrix of primary structure and secondary structure.Finally,ProfDists software is used to construct the Profile neighbor joining(PNJ)evolutionary tree based on distance method.Results The minimum interspecific genetic distance is far greater than the maximum intraspecific genetic distance,an obvious barcoding gap is noted between Glechoma longituba and its substitute and adulterants.The secondary structure of all species is significantly different.The phylogenetic trees show that each species clustered into seperate branches.Conclusion ITS2,as a DNA barcode,can accurately identify from its local and easily mixed species,which provides a scientific basis for the original identification and safe drug use of Glechomae Herba.

关 键 词：连钱草 ITS2 二级结构 分子鉴定 

分 类 号：R282.5[医药卫生—中药学]