芡实及其近缘种药材ITS2条形码鉴定研究

目的:应用ITS2条形码鉴定芡实及其近缘种药材。方法:提取20份芡实及其近缘种药材样品基因组DNA,通过PCR扩增ITS2序列并双向测序,测序后用Sequin 12.3提交至GenBank;从GenBank上下载所有芡实及其近缘种30种102条ITS2序列;对提交与下载的122条序列,应用MEGA5.1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NJ系统进化树直观反映鉴定结果。结果:芡实药材ITS2序列长度均为214 bp,为1个单倍型,与其近缘种之间遗传距离较远,NJ树结果显示芡实及其近缘种药材可明显区分开,表现出良好的单系性。结论:ITS2序列作为DNA条形码能稳定、准确地鉴别芡实药材,为保障临床安全用药提供了新的技术手段,为拓宽DNA条形码技术在中药鉴定中的应用进行了新的探索。

Identification of Euryales Semen and Its Closely Related Species Using ITS2 Barcode

Objective: To identify Euryales Semen and its closely related species using the ITS2 barcode. Method:The total genomic DNAs were extracted from twenty samples of Euryales Semen and its closely related species. The ITS2 regions of the samples were amplified and bidirectional sequenced. Obtained sequences were submitted to the GenBank with Sequin 12.3. ITS2 sequences of 102 samples belonging to thirty species were downloaded from GenBank. The 122 ITS2 sequences were aligned and the genetic distances were analyzed with MEGA 5.1. Identification analyses were performed using BLAST1 and nearest distance methods, and were presented intuitively by constructing neighbor-joining (NJ) tree. Result: The length of ITS2 region of Euryales Semen was 214 bp,which was only one haplotype. There was significant divergence of the ITS2 regions among the samples. The NJtree showed that Euryales Semen could be obviously differed from its closely related species, which had good monophyly. Conclusion: ITS2 regions as a DNA barcode can stably and accurately distinguish Euryales Semen from its closely related species and also provide a new technique to ensure clinical safety in utilization of traditional Chinese medicines. The new exploration could broaden the application of DNA barcoding technology in identification of Traditional Chinese Medicine.