三斑海马及其混伪品的DNA条形码分子鉴定研究

Molecular identification of Hippocampus trimaculatus and its adulterants based on DNA barcoding

目的建立三斑海马Hippocampus trimaculatus的COI、16 S rRNA和ATP6的条形码序列数据库,应用DNA条形码技术从分子水平快速准确鉴定三斑海马和其他正伪品海马,探讨海马属药材鉴定的新方法。方法提取三斑海马药材的基因组DNA,PCR扩增COI、16 S rRNA和ATP6的序列并进行双向测序,所得序列采用软件Codon Code Aligner V4.2对测序峰图进行校对拼接,应用Clustal X软件进行序列比对,MAGA 5.0软件计算三斑海马的种内种间遗传距离(Kimura 2-Parameter,K2P),构建邻接树(Neighbor-joing tree,NJ Tree)聚类分析不同品种海马药材的鉴定结果。结果获得的三斑海马线粒体COI、16 S rRNA、ATP6序列长度分别649、572、603~605 bp,其中3个条形码序列的种内变异位点碱基数分别为8、4和15,种内的变异率较小,COI、16 S rRNA、ATP6序列的平均种内K2P遗传距离0.002、0.001和0.006,均远小于三斑海马的种间K2P距离;NJ树结果显示三斑海马与其他海马均可明显区分,具有良好的单系性。结论COI、16 S rRNA、ATP6序列作为条形码均可以鉴定三斑海马及其他混伪品海马药材,为动物类药材及其混伪品和近源物种的分子鉴定提供依据,为对保障海马临床用药安全提供了新的技术手段。

Objective The aim of present study was to establish a DNA barcoding datebase of COI,16 S rRNA and ATP6 sequences from Hippocampus trimaculatus and identify H.trimaculatus and other adulterants quickly and accurately.Methods Total genomic DNA was isolated using the muscle tissue from tail of H.trimaculatus.The DNA barcoding genes of COI,16 S,and ATP6 sequences were amplified by universal primers and PCR products were sequenced from both directions.Sequences assembly and consensus sequence generation were performed using the Codon Code Aligner V4.2.Sequence alignment was performed by using Clustal X software.The Kimura 2-Parameter(K2P)distances were calculated using the MEGA5.0.Identification analyses were performed using neighbor-joining(NJ)methods.Results The length of the measured sequences of mitochondrial COI,16 S,and ATP6 were 649572 and 603—605 bp,respectively.The number of intraspecific variation sites of three genes was 8 bp,4 bp,and 15 bp,respectively.The average intraspecific K2P genetic distance of COI,16 S,and ATP6 were 0.002,0.001,and 0.006.The genetic variation among H.trimaculatus was obviously less than the interspecific genetic variation.The NJ tree showed that H.trimaculatus could be distinguished obviously from other Hippocampus species,which showed high monopholy.Conclusion Our results indicated that COI,16 S,and ATP6 sequences are effective DNA barcodes for the identification of H.trimaculatus species and its adulterants,with view to providing the basis data for molecular identification of animal medicinal materials,adulterants and near-source species,and contributing to the clinical medication safety of H.trimaculatus.