基于ITS2条形码的乌头属藏药植物鉴别

目的乌头属藏药植物变异极为复杂,品种繁多,商品来源复杂,极易造成药材混乱,且大多乌头属藏药具有较大的毒性,使安全用药面临巨大挑战。采用ITS2序列为DNA条形码,建立一种快速、准确鉴定乌头属藏药药材的方法。方法从青藏高原地区采集展花乌头、凉山乌头、工布乌头、露蕊乌头、铁棒锤、甘青乌头、木里乌头、弯喙乌头、多裂乌头、缩梗乌头等乌头属藏药样品50份,分别提取样品DNA,对ITS2目标序列进行PCR扩增、测序,获得其ITS2序列信息;运用MEGA 6.0对50条ITS2序列进行对比,计算种间、种内遗传距离,构建neighbor joining(NJ)系统进化树,并比较样本ITS2序列间的二级结构差异。结果乌头属藏药植物样本种间最小距离大于种内最大距离,NJ树显示各种乌头属植物种内之间各单独形成一个分支,ITS2序列二级结构也有明显差异,能够将乌头属各种植物区分开。结论 ITS2序列作为DNA条形码可以将乌头属藏药植物进行准确、快速的识别和鉴定,为乌头属藏药的安全使用提供可靠的技术手段。

Identification of Tibetan medicinal plants of Aconitum genus by ITS2 sequences

Objective The complex morphological variation and sources of Tibetan medicinal plants from Aconitum genus make it difficult to identify the medicinal materials correctly. In addition, the toxicity of these materials could be a huge challenge for the safety of drug use. A rapid and accurate method for the identification and classification of Tibetan medicinal herbs of Aconitum genus was built by using ITS2 sequences as DNA barcodes. Methods A total of 50 samples of Aconitum chasmanthum, A. liangshanicum, A. kongboense, A. gymnandrum, A. pendulum, A. tanguticum, A. phyllostegium, A. campylorrhynchum, A. Polyschistum, and A. sessiliflorum were collected from the Qinghai-Tibet Plateau. PCR amplification and sequencing of ITS2 sequences were conducted after the extraction of DNA. Genetic distance, neighbor joining (NJ) phylogenetic tree and secondary structures of ITS2 sequences were analyzed using MEGA 6.0. Results The maximum distance between species was greater than the minimum distance within each species, NJ tree showed that the samples went to 12 separate branches, differences among the secondary structures of ITS2 sequences also made it clear to identify these species. Conclusion Using ITS2 sequence as DNA barcode can be an accurate and rapid method for identification and recognition for Tibetan medicinal plants of Aconitum genus, which provides a reliable technical means to ensure safety use of these Tibetan medicines.