ITS2序列作为DNA条形码鉴定紫堇属植物的有效性研究

摘要

[目的]利用DNA条形码技术对6种紫堇属植物进行分子鉴定,评价内转录间隔区2(internal transcribed spacer 2,ITS2)序列对紫堇属植物的鉴别能力.[方法]从江苏南京、安徽合肥、浙江磐安、浙江缙云等地采集紫堇(Corydalis edulis)、黄堇(C.pallida)、小花黄堇(C.racemosa)、延胡索(C.yanhusuo)、东北延胡索(C.ambigua)和刻叶紫堇(C.incisa)6种紫堇属植物共29份样品.提取其DNA后用ITS2序列的通用引物进行PCR扩增并测序,运用CodonCode Aligner软件拼接和校对序列,MEGA5.1软件分析序列并构建K2P模型的邻接树.[结果]ITS2序列对所研究的29个样品的扩增和测序成功率均为100%.ITS2序列长度在225bp~248bp间,保守位点190个,变异位点71个,变异比例为27.2%.首次扩增获得小花黄堇和紫堇的ITS2序列并上传至GenBank.6个物种的种内遗传距离为0~0.0206,种间遗传距离为0.0403~0.1678,小花黄堇和黄堇的遗传距离最小;黄堇与刻叶紫堇的遗传距离最大.不同物种在邻接树中各自进化为单系.6种紫堇属植物的ITS2二级结构均有不同程度的差异.[结论]ITS2序列能够准确鉴定紫堇、黄堇、小花黄堇、延胡索、东北延胡索和刻叶紫堇这6种紫堇属植物,对中药鉴定具有潜在的应用价值.%[Objective] Using DNA barcoding technology to identify 6 species of the genus Corydalis, and evaluate the identification ability of the ITS2 sequence. [Methods] Twenty-nine samples of Corydalis edulis, C. pallida, C. racemosa, C. yanhusuo, C. ambigua and C. incise were collected from Nanjing Jiangsu, Hefei Anhui, Pan'an Zhejiang, and Jinyun Zhejiang, respectively. The genomic DNA extracted from the plants was PCR amplified with the universal primers of ITS2. Sequences were assembled and proofread using the CodonCode Aligner and analyzed by MEGA 5.1. The neighbor-joining (NJ) phylogenetic tree was constructed based on the Kimura-2-parameter model. [Results] ITS2 regions of all samples were successfully PCR amplified and sequenced. The lengths of them were 225bp~248bp. The conserved sites of them were 190. The variable sites were 71. The variation ratio was 27.2%. ITS2 sequences of C. edulis and C. racemosa were amplified for the first time and uploaded to GenBank. The intra-specific genetic distance of all samples was 0~0.0206. The inter-specific genetic distance of them was 0.0403~0.1678. The inter-specific genetic distance between C. racemosa and C. pallida was the smallest. The inter-specific genetic distance between C. pallida and C. incisa was the largest. Each species showed monophyletic on NJ-tree. The ITS2 secondary structure of each species was different. [Conclusion] ITS2 region as a barcode can accurately identify the genus Corydalis, and has a potential value of application for identification of traditional Chinese medicine.