DNA条形码鉴定洋金花及其伪品

Identification of Daturae Flos and its adulterants based on DNA barcoding technique

为建立有毒中药洋金花及其伪品DNA条形码鉴定方法,采用国际通用的条形码序列ITS2、psbA-trnH、matK和rbcL对洋金花原植物白花曼陀罗及其伪品毛曼陀罗、曼陀罗和木本曼陀罗4个种共计20份材料进行了比较研究。PCR及测序成功率分别为ITS2(100%)、matK(100%)、psbA-trnH(90%)、rbcL(85%)。采用CodonCode Aligner进行序列拼接,采用MEGA 4.1计算白花曼陀罗及其伪品的种内、种间的K2P距离,并基于K2P模型构建NJ树。结果显示ITS2序列共有30个单核苷酸多态性(SNPs)位点、psbA-trnH序列有33个单碱基的插入和缺失I。TS2和psbA-trnH序列种间遗传距离大于种内,matK和rbcL种内和种间没有明显"Barcoding Gap"。4个条形码序列及其组合获得的分子系统树(ITS2、psbA-trnH、matK、rbcL、matK+rbcL)均分成了两大支,木本曼陀罗单聚为一支,该分子证据支持将Brugmansia提升为属水平。实验结果表明ITS2及psbA-trnH序列可以作为洋金花及其伪品鉴定用的条形码序列。

To identify the original plant of Daturae Flos from its adulterants by DNA barcoding,the sequences of ITS2,psbA-trnH,matK,rbcL of four species including Datura metel,Darura innoxia,Darura stramonium and Brugmansia arborea were compared and analyzed.The PCR and sequencing success rate of the four regions（ITS2,psbA-trnH,matK,rbcL） was 100%,90%,100% and 85%,respectively.Sequences were assembled with CodonCode Aligner.K2P distances were calculated and NJ tree was performed by MEGA 4.1.Thirty SNPs were found among ITS2 sequences,and 33 insert/deletes were found among psbA-trnH intergenic regions.The interspecific K2P distance of ITS2 and psbA-trnH was obviously higher than that of the intraspecific one.As to matK and rbcL,there was no ＂Barcoding Gap＂ existing between inter-and intra-specific distances.The NJ trees of the four regions/combinations were built separately.Samples of Brugmansia arborea were clustered into one clade,and the other species of Datura L.formed another clade.The results showed that either ITS2 or psbA-trnH was useful to identify Daturae Flos from its adulterants.