王业胜+李奇威+周林+管润锋+曾常青+朱爽

[摘要] 目的 以核糖体转录间隔区（rDNA ITS）序列作为分子条形码，对一种常用中药材钩藤进行分子鉴定和遗传分析，并阐明其在钩藤属内种间的分子系统发育关系。 方法 通过改良十六烷基三甲基溴化铵（CTAB）法对2015年7月采集的钩藤进行总DNA提取，利用通用引物对rDNA ITS序列进行PCR扩增，经克隆、测序后，运用MEGA软件进行系统发育分析和构建系统发育树。 结果 测序得到该钩藤的rDNA ITS区序列长度为718 bp，序列分析结果显示其与GenBank数据库中已有的钩藤（Uncaria rhynchophylla）rDNA ITS区序列之间相似度达99.8%，并且在系统发育树中并排聚类成一支。 结论 基于ITS区序列测定分析的分子生物学方法和构建系统发育树的分子遗传学方法，可以对钩藤属药用植物进行分子鉴定和遗传分析，为钩藤属药用植物的种间分类地位以及种类鉴定提供准确的分子证据。

[关键词] 钩藤；ITS序列；分子鉴定；系统发育树

[中图分类号] S567.239 [文献标识码] A [文章编号] 1674-4721（2016）03（a）-0004-04

[Abstract] Objective To clarify the phylogenetic relationship of one species of Uncaria，molecular identification and genetic analysis were carried out by using the rDNA ITS sequence as molecular barcoding. Methods Molecular identification and genetic analysis were carried out with the rDNA ITS sequence as molecular marker.Total DNA was extracted with modified CTAB method and thereby rDNA ITS regions were amplified with universal primer，sequenced and phylogenetic analysed with MEGA 6.0 at July 2015. Results The entire rDNA ITS sequence of Uncaria was 718 bp.Sequence analysis showed that the rDNA ITS sequence was closely related to Uncaria rhynchophylla available in GenBank and the similarity reached 99.8%. Conclusion Based on molecular biology methods of rDNA ITS region analysis，molecular identification is available in accurate classification on traditional Chinese medicinal material plants in Genus Uncaria and provide molecular evidence for taxonomy and identification of different species in Genus Uncaria.

[Key words] Uncaria；ITS sequence；Molecular identification；Phylogenetic tree

钩藤是茜草科钩藤属植物，主要分布于我国广东、广西、四川、云南、贵州等地区。《中国药典》2015年版规定的钩藤为茜草科植物钩藤[Uncaria rhynchophylla （Miq.）Miq.ex Havil.]、大叶钩藤[Uncaria macrophylla Wall.]、毛钩藤[Uncaria hirsuta Havil.]、华钩藤[Uncaria sinensis （Oliv.） Havil.]或无柄果钩藤[Uncaria sessilifructus Roxb.]的干燥带钩茎枝[1]。

钩藤为常用中药，药用历史悠久，具有息风定惊、清热平肝的功效。但是钩藤药材在市场上的分类鉴定比较混乱，影响了中药材用药的准确性和安全性。因此，有必要运用DNA分子标记技术进一步对钩藤属植物不同种间进行鉴定。核糖体转录间隔区（rDNA ITS）序列作为分子条形码的鉴定能力已经在药用植物很多个科属基原植物及药材的鉴定中得到验证[2-12]。……