基于ITS2序列的建泽泻及其混伪品鉴定研究

Identification of Alisma orientale(Sam.)Juzep.and its adulterants using ITS2 sequence

目的利用ITS2条形码鉴别建泽泻及其混伪品。方法提取28份建泽泻及其混伪品基因组DNA,PCR扩增ITS2序列并进行双向测序,测序结果提交至GenBank,同时从GenBank下载泽泻科植物及其混伪品10种17条ITS2序列,对提交与下载的45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NJ系统进化树直观反映鉴定结果。结果建泽泻、川泽泻ITS2序列长度均为311bp,存在1个变异位点;建泽泻与泽泻科泽泻属物种距离较近,与泽泻科其他属及其混伪品之间遗传距离较远;NJ树结果显示建泽泻及其混伪品均可明显区分,表现出良好的单系性。结论 ITS2序列作为DNA条形码可以准确快捷地鉴别建泽泻及其混伪品,为其种质资源鉴定及临床安全用药提供了新的技术手段。

OBJECTIVE The aim of this study was to identify Alisma orientale（Sam.）Juzep.and its adulterants using the ITS2 barcode.METHODS The total DNAs were extracted from twenty-eight samples of A.orientale as well as adulterants.The ITS2 sequences of these samples were amplified and sequenced.The acquired sequences were submitted to the GenBank and the other ITS2 sequences of 17 samples from ten species were downloaded from the GenBank.Total 45ITS2 sequences were aligned and the genetic distances among them were analyzed with MEGA 5.1.The sequences 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.orientale and A.plantago-aquatica were 311 bp.Only one mutation existed among them.There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 sequences.The NJ tree showed that A.orientale is obviously different from its adulterants,which showed high monophyly.CONCLUSION As a DNA barcode,ITS2 sequences can stably and accurately distinguish A.orientalefrom its adulterants and also provide a new clue to identify the medical germplasm resources and ensure the clinical safety in utilization of Chinese materia medica.