鸡血藤及其混伪品的DNA条形码分子鉴定研究

目的采用分子生物学鉴定技术,筛选合适的DNA条形码序列,建立快速、准确鉴定鸡血藤的方法。方法采集72份鸡血藤及其混伪品的样本,分别提取样品DNA,对ITS2、matK、psbA-trnH、ITS和rbcL序列扩增、测序;计算各序列扩增成功率和测序成功率,利用MEGA7.0分析比对序列特征;基于Kimura-2-Parameter(K2P)双参数模型计算种内、种间的遗传距离评估Barcoding gap;利用Phylosuite软件构建ITS2、matK和psbA-trnH和多基因(I-M-P)联合系统发育树。结果 ITS2的扩增成功率和测序成功率最高,均为100%,matK和psbA-trnH的测序成功率亦有94.4%、91.7%,而rbcL和ITS仅为69.4%和61.1%;ITS2较其他条形码序列具有明显的Barcoding gap,且种内、种间重叠少;系统发育树显示,ITS2和psbA-trnH可将鸡血藤及其混伪品明显聚为不同分支,matK不能把滇鸡血藤和南五味子分开;I-M-P系统发育树同ITS2和psbA-trnH结果相同。结论以ITS2为主、psbA-trnH序列为辅的鉴定方法能够对鸡血藤及其混伪品进行快速、准确的鉴定,为鸡血藤临床用药的安全性和合理使用的准确性提供依据。

Molecular identification of Spatholobi Caulis and its adulterants based on DNA barcoding

Objective Molecular biology identification technology was used to screen the appropriate DNA barcoding to establish a fast and accurate method for identifying Spatholobi Caulis. Methods A total of 72 samples of Spatholobi Caulis and its adulterants were collected, the sample DNA was extracted, the ITS2, matK, psbA-trnH, ITS, and rbcL sequences were amplified and sequenced, and the amplification success rate and sequencing success rate of each sequence were calculated. The alignment of all sequences was determined by MEGA 7.0 software, and the interspecies and intraspecific genetic distance of them were analyzed to evaluate the Barcoding gap, based on the Kimura-2-Parameter (K2P) two-parameter model. Phylosuite software was used to construct the ITS2, matK and psbA-trnH and multi-gene (I-M-P) phylogenetic tree. Results The amplification success rate and sequencing success rate of ITS2 were the highest (100%), and the sequencing success rates of matK and psbA-trnH were 94.4% and 91.7% respectively, while rbcL and ITS were only 69.4% and 61.1%. Compared with other barcoding, ITS2 has obvious Barcoding gap, and there was less overlap tree showed that ITS2 and psbA-trnH can obviously cluster Spatholobi Caulis and its adulterants into different branches, while matK cannot separate Kadsurae Caulis and Schisandrae Sphenantherae Fructus. I-M-P phylogenetic tree had the same result as ITS2 and psbA-trnH. between species. Conclusion The identification method based on ITS2 and supplemented by the psbA-trnH sequence can quickly and accurately identify S. Caulis and its adulterants, which can provide the basis for the safety and the accuracy of the clinical application.