藏药臭蒿及其近缘种药材的ITS2 DNA条形码鉴别

臭蒿Artemisia hedinii有清热凉血,退黄,消炎的功能,在藏药中占有重要地位。蒿属植物种类繁多,在青藏高原分布广泛,且其外形较为相似,传统鉴定方法难以准确快速鉴定。文章以ITS2序列为DNA条形码,建立了一种快速准确鉴定臭蒿及其易混蒿属藏药植物的方法。从青藏高原采集蒿属藏药21个样品,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;并从Gen Bank上下载同属序列11条;运用MEGA 6.0对所有32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。结果显示:臭蒿、牛尾蒿、黄花蒿、艾蒿的遗传距离较近,但四者种间最小距离均远大于种内最大距离,NJ树显示4种蒿属植物种内之间各单独形成一个分支,ITS2序列二级结构也显示出差异。以ITS2序列作为DNA条形码,可达到对臭蒿及其近缘蒿属藏药植物进行准确、快速的识别和鉴定的目的,准确地弄清物种之间的系统进化关系的目的,为该属药用植物的质量控制、安全用药及合理开发利用提供理论依据。

ITS2 sequence analysis and identification of medicinal Artemisia plants

Artemisia hedinii occupies an important position in the Tibetan medicine. Plants in Artemisia vary a lot and are widely distributed in the Qinghai-Tibet Plateau, many plants in Artemisia look similar, making traditional identification methods laborious. In this article, ITS2 sequences were used as DNA barcoding to identify four kinds of confusable Tibetan medicine plants in Artemisia, aiming to establish a rapid and accurate identification methods. Twenty-one samples in Artemisia were collected from the Qinghai-Tibet Plateau, ITS2 sequence PCR amplification and sequencing were conducted after the extraction of DNA. Another 11 sequence downloaded from Genbank were added to the analysis. Genetic distance calculation and analysis, building Neighbor Joining (NJ) phylogenetic tree were conducted by MEGA 6.0, also comparison of secondary structures of ITS2 sequences among samples. A. hedinii, A. annua, A. dubia and A. argyi shared close genetic distance, but the maximum distance between the four species was much greater than the minimum distance within each species, NJ tree showed that the four species went to four separate branches, differences among secondary structures of ITS2 sequences also made it clear to identify these medical plants. It could be an accurate and rapid method for identification and recognition, as well as the evolutionary relationships between the species by using ITS2 sequence as DNA barcode for plants of Tibetan Artemisia. The study provides theoretical basis for quality control, medication safety and rational exploitation.