基于ITS2序列的杜仲及其主要混伪品的鉴定  

Identification of Eucommia ulmoides and its adulterants based on ITS2 sequences

作　　者：彭梓[1,2] 朱金国[2] 谭建锡[2] 王利兵[1,2] 

机构地区：[1]湖南农业大学,湖南长沙410128 [2]湖南出入境检验检疫局检验检疫技术中心,湖南长沙410004

出　　处：《中草药》2013年第21期3042-3047,共6页Chinese Traditional and Herbal Drugs

基　　金："十二五"国家科技支撑计划课题(2012BAK01B07)

摘　　要：目的分析杜仲及其混伪品的ITS2条形码序列,探讨杜仲及其混伪品鉴定的新方法,为杜绝市售商品药材混乱现象提供技术支撑。方法提取样品DNA,利用PCR方法对样品进行核基因ITS2片段扩增,采用DNA克隆测序技术对ITS2基因进行双向测序,所得序列经Seqman程序拼接后,用软件MEGA4.1进行相关数据分析,并构建邻接(Nighbor-joining,NJ)树。利用网站已建立的ITS2数据库预测ITS2二级结构。结果杜仲种内最大K2P遗传距离远远小于其与混伪品的种间最小K2P遗传距离;由构建的系统聚类树图可以看出,不同来源的杜仲样品聚成一支,表现为单性系,并与其他混伪品明显分开;比较ITS2二级结构发现,杜仲与其混伪品在4个螺旋区的茎环数目、大小、位置以及螺旋发出时的角度均有明显差异。结论 ITS2序列作为DNA条形码可以有效地鉴别杜仲及其混伪品,为其种质资源鉴定及临床安全用药提供了重要分子依据。Objective To identify Eucommia ulmoides and its adulterants using ITS2 barcode, to discuss the feasibility of this new method, and to provide the technical support for the prevention against the confusion of commercial medicinal materials. Methods DNA was extracted from each sample as template, the ITS2 region was amplified by PCR technology and sequenced bidirectionally using DNA cloning sequencing method. The sequence assembly and consensus sequence generation were performed using the Seqman program. Phylogenetic study was performed by MEGA 4.1, and the Nighbor-joining （NJ） dendrogram was constructed. The ITS2 secondary structure was predicted using the ITS2 website. Results The maximum Kimura-2-parameter （K2P） genetic distance orE. ulmoides was far lower than the minimum K2P genetic distance of the adulterants. In the cluster dendrogram, E. ulmoides samples from various sources showed the monophyletic and simultaneously distinguished from the adulterants. To compare the ITS2 secondary structure, it could be noticed that ITS2 secondary structure could distinguish E. ulmoides from its adulterants in terms of numbers, sizes, positions of loop, and the angles of helix exsertion in tbur helix regions. Conclusion ITS2 barcode could be used to distinguish E. ulmoides from its adulterants effectively, and provide the important molecular evidence for the identification of germplasm rescouces and clinic safety in medicinal use.

关 键 词：杜仲 混伪品 ITS2 DNA克隆 二级结构 

分 类 号：R282.12[医药卫生—中药学]