基于ITS2序列的茅苍术及其近缘种DNA分子鉴定

Molecular identification of Atractylodes lancea and its closely related species based on ITS2 sequence

目的应用ITS2条形码鉴定茅苍术Atractylodes lancea及其近缘种药材。方法提取不同产地29份茅苍术、北苍术A.chinesis及白术A.macrocephala基因组DNA,通过PCR扩增ITS2序列并进行双向测序,测序结果提交至Gen Bank;从Gen Bank下载茅苍术及其菊科近缘种10种45条ITS2序列;对提交与下载的73条序列,应用MEGA 5.1软件进行序列比对,计算种内和种间距离,采用相似性搜索法、最近距离法进行鉴定分析,并构建Neighber-jioning(NJ)系统进化树直观反映鉴定结果。结果茅苍术药材ITS2序列长度均为229 bp,是1个单倍型;与菊科近缘种苍术属药材距离较近,与菊科其他属近缘种之间遗传距离较远,NJ树结果显示茅苍术及其近缘种药材均可明显区分,表现出良好的单系性,依据ITS2二级结构,也可以直观地将茅苍术与菊科近缘种药材区分。结论 ITS2序列作为DNA条形码能稳定、准确鉴别茅苍术药材,为保障临床安全用药提供了新的技术手段。

Objective The aim of present study was to identify Atractylodes lancea and its closely related species（Atractylodes chinesis and Atractylodes macrocephala） using the ITS2 barcode. Methods The total genomic DNAs were extracted from twenty-nine samples of A. lancea and its closely related species from different habitats. The ITS2 sequences of these samples were amplified and bidirectional sequenced by PCR. The obtained sequences were submitted to the Gen Bank and the ITS2 sequences of 45 samples belonging to ten species were downloaded from the Gen Bank. Total 73 ITS2 sequences were aligned and the genetic distances were analyzed using the MEGA 5.1. Identification analyses were performed using the BLAST1 and the nearest distance methods, and were presented intuitively by constructing Neighbor-joining（NJ） tree. Results The lengths of all ITS2 sequences of A. lancea were 229 bp presented as one haplotype pattern. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 sequences. The NJ tree showed that A. lancea could differed obviously from its closely related species, which showed high monophyly. The secondary structures of ITS2 in the helical regions displayed clear differences in stem loop number, size, position, and screw angle among the medicinal plants of Compositae. Conclusion As a DNA barcode, ITS2 sequences can stably and accurately distinguish A. lancea from its closely related species and also provide a new technique to ensure the clinical safety in utilization of Chinese materia medica.