基于ITS2序列对藏药材植物的分子鉴定

Molecular identification of Tibetan medicinal plants based on ITS2 sequence

目的选用ITS2序列作为条形码来鉴定44种藏药材植物。方法用高盐低pH法提取藏药材植物的基因组DNA,通过PCR扩增藏药材植物的ITS2序列,共得到ITS2序列145条,分属于24科,39属,44种。在GenBank数据库中通过序列比对选取了部分藏药材的同源序列。将ITS2序列在Bioedit软件中进行序列比对,在MEGA软件中计算双参数(K2P)种内和种间遗传距离,并基于邻接法构建系统进化树来分析物种的系统发育关系。结果 ITS2区域有显著的种内、种间差异,基于ITS2区域的系统进化分析与形态学结果一致,并可以反映物种之间的亲缘关系。此外,藏药材的ITS2序列二级结构各有不同,为物种鉴定提供了另一种方法。结论 ITS2序列是藏药材植物鉴定和系统发育研究的非常有效的单条形码,为藏药材植物资源的利用和保护提供了科学依据。

Objective The barcoded ITS2 DNA sequence was used to identify 44 Tibetan medicinal plants. Methods Genomic DNA of Tibetan medicinal plants were extracted with high salt and low pH method. The PCR technique was performed to obtain the ITS2. A total of 145 ITS2 sequences was obtained belonging to 24 families, 39 genera, and 44 species. Some homologous sequences were also selected according to sequence alignment from Genbank database. The ITS2 sequences were aligned using Bioedit and the intraspecific and interspecific Kimura 2-parameter genetic distance was calculated using MEGA, and the neighbor-joining (NJ) phylogenetic trees were constructed to analyze phylogenetic relationship. Results ITS2 regions have significant intra-and inter-specific difference, phylogenetic analysis based on ITS2 regions concurred with the result of morphological classification, and it could also determine the phylogenetic relationship between species. In addition, the secondary structure of the ITS2 sequence of Tibetan medicinal plants is different, providing another method for species identification. Conclusion ITS2 can be used as a very effective single-locus barcode in the identification and phylogenetic study of Tibetan medicinal plants. The barcoding technique provides a scientific baseline for the utilization of resources and conservation of Tibetan medicinal plants.