基于ITS2序列的东北透骨草及其混伪品DNA分子鉴定

目的应用ITS2条形码鉴定东北透骨草及其混伪品,为东北透骨草药材鉴定提供新方法。方法提取35份东北透骨草及其混伪品的基因组DNA,通过PCR扩增ITS2序列并进行双向测序,测序结果提交至GenBank;从GenBank下载13种东北透骨草及其混伪品ITS2序列42条;对提交与下载的77条序列,应用MEGA7.0软件进行序列比对,计算种内和种间遗传距离,构建Neighber-jioning(NJ)系统进化树,并预测ITS2二级结构。结果东北透骨草3种基原的种内最大K2P遗传距离均远远小于其与混伪品的种间最小K2P遗传距离;NJ树结果显示东北透骨草及其混伪品药用植物均可明显区分,表现出良好的单系性;比较ITS2二级结构发现,东北透骨草与其混伪品在4个螺旋区的茎环数目、大小、位置以及螺旋发出时的角度均有明显差异。结论 ITS2序列作为DNA条形码能稳定、准确鉴别东北透骨草,为保障安全用药提供了新的技术手段。

Molecular identification of Tougucao and its adulterants based on ITS2 sequence

Objective ITS2 barcoding was used to discriminate Tougucao and its adulterants in order to provide a new method for the identification of Tougucao. Methods The total genomic DNAs were extracted from 35 samples of Tougucao and its adulterants. The ITS2 sequences of these samples were amplified and bidirectional sequenced by PCR. The obtained sequences were submitted to the GenBank and the ITS2 sequences of 42 samples belonging to 13 species were downloaded from the GenBank. Genetic distance, neighbor joining (NJ) phylogenetic tree and secondary structures of ITS2 sequences were analyzed by using MEGA 7.0. Results The maximum intraspecific genetic distance (K2P distance) was much smaller than the minimum interspecific genetic distance between Tougucao and its adulterants. The NJ tree showed that Tougucao and its adulterants could be distinguished obviously, which showed high monophyly. Comparing the secondary structure of ITS2 among Tougucao and its adulterants, it was found that there were significant differences in the number, size, position of loop, and the angle of helix exsertion. Conclusion As a DNA barcode, ITS2 sequences can stably and accurately distinguish Tougucao from its adulterants and it can also provide a new technique to ensure the clinical safety in utilization of Chinese materia medica.