基于ITS序列对独活17个种的分子鉴定

Identification of 17 species of Angelicae Pubescentis Radix based on ITS analysis

目的通过对17个种共26个独活样本的ITS序列分析,为国内中药市场上的常见独活的分子鉴定提供依据,并探索其科学分类标准。方法对26个样本的ITS进行PCR扩增和测序。通过MEGA 5.0软件比较各序列间的差异性,计算K2P遗传距离;采用邻接法构建NJ系统树并应用Network 4.2.0.1软件进行中间连接网法分析构建单倍型网状图。结果 NJ系统树和单倍型网状图显示,26个样本聚集为5大类群,综合分析后将17种独活归为4大类;重齿毛当归Angelica pubescens的ITS序列具有特征碱基片段,可明显区别于其他样本;除牛尾独活类中3个样本不能通过ITS序列鉴定到种以外,其他样本均可以通过ITS序列鉴定到种。结论 ITS序列可为药用独活的鉴定和分类提供有力证据,四带芹类可以作为独活的首选替补品,牛尾独活类其次,九眼独活为最后之选。

Objective To provide the evidence for the molecular identification of Angelicae Pubescentis Radix（APR） and discuss the scientific classification standard through ITS analysis on 26 samples of APR from 17 species.Methods ITS of 26 populations was amplified and sequenced.The differences among the different samples were compared and K2P genetic distances of ITS sequence were calculated.NJ tree was constructed and haplotype network map was obtained by using the Network 4.2.0.1 software.Results NJ tree and haplotype network map suggested that 26 populations were clustered into five groups,and the 17 APR could be sorted into four types after comprehensive analysis.Angelica pubescens was different from other species due to the distinct base sequence in the ITS sequence.All species were identified by ITS analysis except for the three species in Heracleum L.Conclusion The ITS sequence is powerful for the identification and classification of medicinal APR.Tetrataenium-type plant could be the first choice as succedaneum and followed by Heracleum-type plant,then Aralia-type plant be the last candidate as well.