摘要
目的:通过DNA条形码技术对金钱草样品、地方习用品及其常见的混伪品进行研究,比较分析ITS2和rbcL基因的鉴定效率,为金钱草的分子鉴别提供参考依据。方法:采用CTAB法提取12个金钱草样品、浙江地区习用品种浙金钱草和其他常见的4种混伪品基因组DNA,PCR扩增各样品的ITS2和rbcL基因序列并进行测序,通过对序列的变异位点、种内种间遗传距离和NJ树聚类进行分析,比较两个序列在金钱草种内及其混伪品鉴定中的优劣。结果:金钱草ITS2和rbcL序列的金钱草种内变异位点分别为3和1个,且ITS2在金钱草及其混伪品样品中较rbcL有更丰富的变异位点。基于ITS2和rbcL序列的金钱草与其混伪品间最小种间遗传距离均大于最大种内遗传距离,不存在条形码重叠部分;NJ树聚类分析中金钱草样品聚为一支,与浙金钱草的亲缘关系最近,并能够与其他几种混伪品明显区分。结论:ITS2和rbcL均可用于鉴别金钱草正品与地方习用品及其常见的混伪品,也为解释浙金钱草长期作为浙江地区金钱草药材的代替习用品寻找到遗传学方面的理由。该研究为金钱草的分子鉴定提供了技术支持,有利于金钱草药材市场规范化,为浙金钱草后续的开发研究提供了一定的参考依据。
Objective: To study the Lysimachia christinae,substitute and its adulterants by DNA barcoding, to compare the identification efficiency of ITS2 and rbcL genes, so as to provide the reference for molecular identification of Lysimachia christinae.Methods: CTAB method was used to extract genomic DNA of twelve Lysimachia christinae samples, substitute in Zhejiang and four common adulterants.The ITS2 and rbcL gene sequences of each samples were amplified and sequenced by PCR.Through the analysis of viriation sites, intraspecific and interspecific genetic distances and NJ tree clustering, the advantages and disadvantages of the two sequences in the identification of Lysimachia christinae intraspecific and its adulterants were compared.Results: The intraspecific variation sites in Lysimachia christinae based on ITS2 and rbcL sequences were 3 and 1,respectively.In addition, ITS2 had more mutation sites than rbcL in Lysimachia christinae and its adulterants.The minimum interspecific genetic distance of Lysimachia christinae and its adulterants based on the ITS2 and rbcL sequences was greater than the maximum interspecific genetic distance, and there was no bar code overlap;In the cluster analysis of NJ tree, the samples of Lysimachia christinae were clustered into one branch, which had the closest relationship with Lysimachia hemsleyana,and could distinguish clearly with other several kinds of adulterants.Conclusion: The ITS2 and rbcL can be used to identify Lysimachia christinae,substitute and its common adulterants, in addition, genetic reasons are also found to explain that Lysimachia hemsleyana has long been used as a substitute for Lysimachia christinae in Zhejiang.This study provides technical support for the molecular identification of Lysimachia christinae,which is conducive to the standardization of Lysimachia christinae market and provides a certain reference for the subsequent development and research of Lysimachia hemsleyana.
作者
孙思雅
陈云
王琪瑞
葛宇清
程汝滨
SUN Si-ya;CHEN Yun;WANG Qi-rui;GE Yu-qing;CHENG Ru-bin(College of Pharmacy,Zhejiang Chinese Medical University,Hangzhou 310053,China;School of Traditional Chinese Medicine,Zhejiang Pharmaceutical College,Ningbo 315100,China;The First School of Clinical Medicine,Zhejiang Chinese Medical Universi-ty,Hangzhou 310006,China)
出处
《中药材》
CAS
北大核心
2020年第6期1336-1342,共7页
Journal of Chinese Medicinal Materials
基金
浙江中医药大学校级科研基金项目(2018ZG29)。
关键词
金钱草
浙江习用品
市场混伪品
DNA条形码
分子鉴定
Lysimachia christinae Hance
Substitute in Zhejiang
Market adulterants
DNA barcoding
Molecular identification
