基于性状鉴别和DNA条形码联用的虎尾海马生药学研究

Pharmacognosical Identification of Hippocampus comes Based on the Combination of the Morphological Characteristics and DNA Barcodes

目的:采用性状-线粒体COI条形码联用的方法对市售海马药材伪品虎尾海马Hippocampuscomes进行生药学研究,提供主要海马药材伪品虎尾海马的性状和分子的鉴定特征,为海马药材的鉴定提供新方法。方法:利用外观性状鉴别方法对虎尾海马典型性状特征进行研究;扩增获得虎尾海马样品的COI条形码序列,比较各序列的碱基组成、GC含量、变异率等,分析虎尾海马与常见海马药材的正品和伪品序列,明确其种内和种间遗传距离;利用NJ(neighbor-joining)法构建虎尾海马与常见海马样品的系统进化发育树,分析其亲缘关系。结果:虎尾海马最典型性状鉴别特征为尾部有交替出现的花斑,头冠低小且有5个显著的圆形突起;虎尾海马样品的COI序列长度为649bp,碱基GC平均含量为39.73%,种内的平均遗传距离为0.0011,其种内遗传距离远小于与另外8种海马药材的种间遗传距离,存在显著的条形码间隔;NJ系统发育树显示虎尾海马与其他海马药材各自聚为一支,其分支支持率为100%,与刺海马的亲缘关系最近。结论:基于性状鉴别-线粒体COI条形码的生药学研究方法能够准确鉴定虎尾海马,与其他海马进行准确区分,为虎尾海马的快速鉴定提供了新的手段,对改善市售海马混乱的流通现状、控制和提高海马药材质量具有重要意义。

Objective: To investigate pharmacognosically the seahorse adulteration Hippocampus comes using the combination of morphological characteristics and DNA barcodes,and provide key features and new method for the quick identification of H . comes and other hippocampus species. Methods: The typical characteristics of H.comes were obtained based on morphological observation according to classification and identification methods in traditional pharmacognosical research.DNA barcoding technology was applied to provide specific molecular identification marker for H.comes .After the amplification of COI sequence from H.comes species,the base composition,GC content and proportion of variable sites were calculated using the MEGA 7.0.The intra-specific and inter-specific divergence and barcoding gap were analyzed and a phylogenetic analysis tree was constructed by the neighbor-joining (NJ )methods. Results: The most typical features of H.comes were striped tail and low coronet with 5 distinct points.The length and GC contents of COI sequence were 649 bp and 39.73%,respectively.The average genetic distance among 20 samples of H.comes was 0.001 1,and the maximum intra-specific distance was far less than the minimum inter-specific distance,with an obvious barcoding gap.The NJ phylogenetic tree showed that 20 samples of H.comes formed a clade with a 100% bootstrap value,and H.comes is closely related to H.histrix. Conclusion: The pharmacognosical identification based on the combination of the morphological characteristics and DNA barcodes can identify H.comes accurately and quickly.Our results provided a novel method for the identification of H.comes ,which has significant impacts for the clinical medication safety control and trading of hippocampus species in Chinese medicinal material market.