基于matK和ITS2及二级结构对药材香薷及其混伪品的鉴别研究

Identification of Moslae Herba and Its Adulterants Based on matK, ITS2 and Its Secondary Structure

关键词:  香薷  matK  ITS2  分子鉴定

Key words:  Moslae Herba  matK  ITS2  molecular identification

摘要:

目的 快速、准确鉴别药材香薷及其混伪品，保障香薷的药材质量和用药安全。方法 收集石香薷、江香薷和香薷植物材料分别进行matK和ITS2序列的扩增与测序，测序结果经Codon Code Aligner软件校对，同时从GenBank下载石香薷、江香斋及其易混品种海州香薷、香薷、密花香薷、牛至等物种的matK和ITS序列。其中，ITS序列经隐马尔可夫模型去除两端的5.8S和28S序列，共得到16个物种的ITS2序列50条；经Clustal软件校对共获得9个物种的matK序列28条。通过Mega7.0软件分析matK和ITS2序列，计算所有物种种内和种间遗传距离，构建邻接法(neighbor joining，NJ)聚类树，通过ITS2 Database预测ITS2二级结构，采用4Sale软件比对二级结构，通过ProfDistS软件构建基于联合ITS2一级序列及其二级结构的剖面邻接(profile neighbor-joining，PNJ)系统发育树。结果 基于matK和ITS2序列的遗传距离均表明香薷正品与其各种混伪品之间存在明显barcoding gap。NJ和PNJ进化树的拓扑关系一致，可以区分药材香薷及其混伪品。香薷的ITS2二级结构与其各混伪品具有显著差异。结论 建议matK和ITS2序列均可以作为鉴别香薷与其混伪品的DNA条形码，ITS2二级结构信息的加入可丰富鉴定结果，为香薷药材的准确鉴别、香薷属与石荠苎属植物的科学分类提供参考。

Abstract:

OBJECTIVE To quickly and accurately identify Moslae Herba and its adulterants, so as to ensure the quality of Moslae Herba and the safety of medication. METHODS The matK and ITS2 sequences of Mosla chinensis, Mosla chinensis 'Jiangxiangru' and Elsholtzia ciliate were amplified and sequenced. The sequencing results were checked by Codon Code Aligner software. The matK and ITS sequences of Moslae Herba, Mosla chinensis 'Jiangxiangru' and its adulterants Elsholtzua splendens, Elsholtzia ciliata, Elsholtzia densa, Origanum vulgare were downloaded from GenBank. The hidden Markov model was used to remove 5.8S and 28S from ITS sequences. A total of 50 ITS2 sequences of 16 species were obtained. A total of 28 matK sequences of 9 species were obtained by proofreading with Glustal software. Mega7.0 software was used for matK and ITS2 sequences analysis, intraspecific and interspecific genetic distances caculation, and neighbor joining(NJ) phylogenetic tree construction. ITS2 secondary structure was predicted by ITS2 database and aligned by 4Sale software. Profile neighbor-joining(PNJ) phylogenetic tree was constructed based on combined ITS2 sequence and its secondary structure by ProfDistS software. RESULTS The genetic distance based on matK and ITS2 sequences showed that there was an obvious barcoding gap between the authentic Moslae Herba and its adulterants. The NJ and PNJ evolutionary trees had the same topological relationship, which could effectively identify Moslae Herba and its adulterants. The ITS2 secondary structures of Moslae Herba were significantly different from its adulterants. CONCLUSION It is suggested that matK and ITS2 can be used as DNA barcodes to identify Moslae Herba and its adulterants. The addition of ITS2 secondary structure information can enrich the identification results, and provide reference for the accurate identification of Moslae Herba and the species classification of gnus Elsholtzia and genus Mosla.