基于性状鉴别和DNA条形码联用的小海马生药学研究

Pharmacognosy Study of Hippocampus japonicus Based on the Combination of Morphological Characteristics and DNA Barcodes

目的:采用性状鉴别和DNA条形码联用的方法对小海马药材进行生药学研究,为小海马的鉴别技术开发和遗传多样性研究提供数据支撑。方法:利用外观性状鉴别法对小海马典型性状进行研究;扩增测序小海马样品的COⅠ和16S rRNA序列,比较两个序列的碱基组成、GC含量、变异位点、信息位点等;基于MEGA 7.0中的K2P模型计算小海马的种内和种间遗传距离,NJ法构建小海马与市场常见海马品种的系统聚类树。结果:小海马最典型的性状鉴别特征为体型小,身体横向扁平,龙骨突出。14条小海马样品COⅠ、16S rRNA序列长度分别为649和573 bp,其中16S rRNA序列中的变异位点和信息位点均少于COⅠ序列。COⅠ和16S rRNA序列的最大种内遗传距离均远小于最小种间遗传距离,存在显著的条形码间隔区。NJ树结果显示COⅠ和16S rRNA序列均能将小海马与其他海马明显区分;小海马与三斑海马的亲缘关系最近。结论:COⅠ和16S rRNA序列均能将小海马与其他常见海马药材进行区分。COⅠ序列更适合作为鉴定小海马的标准条形码,16S rRNA可作为补充序列。基于性状和条形码联用的生药学研究方法能够准确提供小海马的性状和分子鉴别特征,为小海马的快速鉴定提供了新的方法,对控制和提高海马药材质量具有重要意义。

Objective:To investigate the pharmacognosy of Hippocampus japonicus using the combination of morphological characteristics and DNA barcodes,in order to provide basic data for the identification of Hippocampus japonicus and the research of genetic diversity.Methods:The typical characteristics of Hippocampus japonicus were studied by the method of appearance identification.After the amplification and sequencing of COⅠand 16S rRNA sequences from samples of Hippocampus japonicus,the base composition,GC content,variable sites and phylogenetical informative sites were calculated and compared,respectively.The intraspecific and interspecific genetic distances of Hippocampus japonicus were determined based on K2P model using MEGA 7.0.The systematic clustering tree of Hippocampus japonicus and common varieties in the market was constructed by NJ method.Results:The most typical feature of Hippocampus japonicus included the small size with a laterally flattened body and prominent keel.The lengths of the measured COⅠand 16S rRNA sequences of 14 samples of Hippocampus japonicus were 649 and 573 bp,respectively,the variable sites and informative sites of 16S were less than COⅠ,and the maximum intraspecific genetic distances of 16S rRNA and COⅠwere all far lower than the minimum interspecific genetic distances,with an obvious bar code spacing.The NJ tree showed that Hippocampus japonicus could differed obviously from others by COⅠand 16S rRNA sequences,and Hippocampus japonicus was closely related to Hippocampus trimaculatus.Conclusion:The COⅠand 16S rRNA sequences can distinguish Hippocampus japonicus from its adulterants,the COⅠcan be used as the standard DNA barcode for Hippocampus japonicas identification,while the 16S rRNA is the alternative supplementary barcode.The pharmacognosy method based on the combination of traits and barcodes can accurately provide the traits and molecular identification characteristics of Hippocampus japonicus,which provides a new method for its rapid identification and it is of great significance for controlling and improving the quality of Hippocampus.