基于trnL-trnF序列分析的何首乌PCR-RFLP分子鉴别

目的:建立何首乌(Fallopia multiflora)DNA分子鉴别技术。方法:对何首乌与其近缘种及其混淆品的trnL-trnF(trnL基因和trnF基因间隔区)序列进行比较分析。结果:何首乌与其近缘种及其混淆品的trnL-trnF差异率为2.1%～22%,何首乌种内各居群间trnL-trnF差异率为0%～1.5%。基于何首乌与其近缘种及其混淆品的trnL-trnF序列的差异,找出一个位于trnL5'-trnL3'间区的何首乌特征性Xba I酶切位点(T↓CTAGA),用Xba I酶对不同采集地的何首乌样品trnL-trnF序列扩增产物酶切后均得到含约804～819 bp和256 bp两个片段的PCR-RFLP图谱,而其混淆品trnL-trnF序列扩增产物因不能被Xba I酶切,图谱呈单一条带。结论:利用建立的PCR-RFLP方法可以很好地区分何首乌及其混淆品植物。

Molecular Authentication of Fallopia multiflora by PCR-RFLP Based on the trnL-trnF Analysis

Objective:To establish a method for the molecular authentication of Fallopia multiflora.Methods: The trnL-trnF regions of Fallopia multiflora and its closely related species and/or adulterants were sequenced and analyzed.Results: It was found that the trnL-trnF sequence divergences between Fallopia multiflora and its closely related species and/or adulterants were 2.1%~22%.While the intra-species trnL-trnF divergences of Fallopia multiflora were 0%~1.5%.Based on the trnL-trnF regional variations,an endonuclease Xba I(T↓CTAGA) restriction site specific to Fallopia multiflora was detected.The Fallopia multiflora trnL-F polymerase chain reaction product could be cleaved by Xba I into two pieces,804~819 bp and 256 bp each,whereas the restriction endonuclease could not digest the trnL-trnF polymerase chain reaction product of its closely related species or adulterants.The restriction patterns analyzed for restriction enzyme Xba I were found to be identical in all Fallopia multiflora individuals from different geographical regions in China.Conclusion: The assay based on polymerase chain reaction amplification of the trnL-trnF fragment of chloroplast DNA and subsequent restriction fragment length polymorphism can be used as a general test to identify Fallopia multiflora.