基于ITS2序列的翻白草与委陵菜分子鉴定

目的 应用DNA条形码技术对翻白草及易混品委陵菜进行分子鉴定，保障药材质量和临床用药安全。方法 提取翻白草及委陵菜的基因组DNA，扩增内转录间隔区2（ITS2）序列并双向测序，所得序列用SeqMan软件校对、拼接；在线双序列比对翻白草对照药材（F_R）及委陵菜对照药材（W_R）的ITS2序列，并预测其二级结构；从GenBank下载部分相关序列，运用MEGA X软件进行多序列比对，分析序列变异位点，计算种内、种间遗传距离，采用邻接法（NJ）构建系统进化树。结果 所有样品均成功扩增出ITS2序列，翻白草和委陵菜的ITS2序列长度均为210 bp。双序列比对结果表明，F_R与W_R的ITS2序列相似性为92.9%，二级结构存在明显差异。多序列分析结果表明，翻白草ITS2序列平均鸟嘌呤（G）+胞嘧啶（C）占比为63.0%，存在2个变异位点，种内遗传距离为0~0.009 6；委陵菜ITS2序列平均G+C占比为64.4%，存在5个变异位点，种内遗传距离为0~0.019 3；种间遗传距离为0.054 8~0.075 8。NJ树结果显示，翻白草、委陵菜聚为不同分支，可明显区分。结论 利用ITS2序列可以准确鉴别翻白草与委陵菜，为保障其安全用药提供了新的技术手段。

Molecular Identification of Potentilla discolor Bge. and P. chinensis Ser. Based on ITS2 Sequences

Objective To carry out molecular identification for Potentilla discolor Bge.and its adulterants P. chinensis Ser. by DNA barcoding technology, further ensure the quality and medication safety in clinic.Methods The total genomic DNAs were extracted from all samples. The internal transcribed spacers 2 (ITS2) sequence was amplified by PCR and sequenced bi-directionally, and the obtained sequences were validated and assembled by SeqMan software. The pairwise sequence alignment and the prediction of ITS2 secondary structure were performed on-line control drugs of P. discolor Bge (F_R) and P. chinensis Ser. (W_R). Moreover, some related sequences were downloaded from GenBank. MEGA X software was used for multiple sequence alignment and variation site analysis. The genetic distance within and between species were calculated. Additionally, the phylogenetic tree was constructed by neighbor-joining (NJ) method.Results The ITS2 sequences were successfully amplified from all samples, while the ITS2 sequences of P. discolor Bge and P. chinensis Ser. were both 210 bp. The pairwise sequence alignment showed that the sequence similarity of ITS2 between F_R and T_R was 92.9%, and obvious differences were detected in the secondary structures. The multiple sequence analysis showed that the average guanine and cytosine (G+C) content of the ITS2 in P. discolor Bge.was 63.0%, and two variation sites were found, while the intraspecific genetic distance was 0-0.009 6. However, the average GC content of the ITS2 in P. chinensis Ser.was 64.4%, and five variation sites were found, while the intraspecific genetic distance was 0-0.019 3. The interspecific genetic distance was 0.054 8-0.075 8. The NJ tree showed that P. discolor Bge.and P. chinensis Ser.could be distinguished obviously as they were clustered in different clades.Conclusion ITS2 sequence could be used to identify P. discolor Bge. and P. chinensis Ser. effectively, which may provide a new technical method for ensuring medication safety in clinic.