目的 采用性状鉴别与DNA条形码联用的方法对市售海马药材鲍氏海马(Hippocampus barbouri)进行生药学研究,为其后续鉴定技术开发与应用奠定基础。方法 通过文献结合样品观察总结鲍氏海马的典型性状鉴别特征;提取鲍氏海马样品基因组DNA, 对COI、16S和ATP6基因序列进行扩增和测序,通过MEGA 7分析各序列,计算不同条形码在种内和种间遗传距离的差异,并构建系统进化树,分析不同条形码在鲍氏海马鉴定中的优劣。结果 鲍氏海马的典型鉴别特征包括鼻子有条纹,眼眶内有5条辐射状条纹和尾刺的长短间隔;COI、16S、ATP6序列长度分别为649~650、574和603 bp,不同序列的最大种内遗传距离分别为0.012、0.003和0.003,远小于鲍氏海马与其他海马品种间的最小遗传距离,存在显著的条形码间隔;基于COI、16S和ATP6条形码构建的NJ树结果均显示,鲍氏海马样品单独聚为一个分支,与其他正伪品海马能明显区分,与刺海马的亲缘关系最近,且COI与ATP6构建的NJ树结构更加稳定。结论 基于性状鉴别与DNA条形码联用的生药学研究可获得鲍氏海马的性状和分子鉴定特征,COI、16S和ATP6序列可用于鲍氏海马的后续分子鉴定技术,本实验为鲍氏海马的后续开发和保障海马的药材质量提供了新的技术手段。
Abstract
OBJECTIVE To obtain the key identification information of Hippocampus barbouri (H. barbouri), investigate the pharmacognosy of the seahorse adulteration H. barbouri, using the combination of morphological and DNA barcoding technique. METHODS Typical morphological characteristics of H. barbouri were studied based on the observations according to the previous reports and traditional pharmacognosy methods. Genomic DNAs of H. barbouri and other related seahorse species were extracted and the DNA barcoding genes of COI, 16S and ATP6 sequences were amplified and sequenced, respectively. In order to determine advantages of three barcodes for the identification of hippocampus, MEGA 7 was performed to analyze the intraspecific and interspecific distances and construct the NJ phylogenetic trees based on three different barcoding genes. RESULTS The most typical distinguishing features of H. barbouri included the streak of the nose, five radial lines in the eyelid, and the long and short intervals of the tail thorns. The lengths of COI, 16S and ATP6 sequences were 649-650, 574 and 603 bp, respectively, with the maximum K2P intraspecific distances were 0.012, 0.003 and 0.003, respectively. The intraspecific distances of the three barcodes were far smaller than the corresponding minimum K2P interspecific distances of H. barbouri and other seven related seahorse species, existing an obvious barcoding gap. The NJ phylogenetic trees based on COI, 16S and ATP6 sequences showed that H. barbouri samples formed of a group indicating that H. barbouri could be distinguished from other seahorse species through the three DNA barcoding markers. In addition, H. barbouri showed close genetic relationship with H. histrix according to the NJ trees. Furthermore, the NJ tree structures of COI and ATP6 were more stable than that of 16S. CONCLUSION The pharmacognostical investigation bases on the morphological identification and DNA barcodes obtained the key identification information of H. barbouri, providing the possibility for quick and accurate identification of this species. The COI, 16S and ATP6 barcoding genes can be used for further molecular identification markers for seahorse species. This study provides a new technical method for the development and quality control of hippocampus.
关键词
鲍氏海马 /
性状鉴别 /
DNA条形码 /
分子鉴定
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Key words
Hippocampus barbouri /
morphological identification /
DNA barcode /
molecular identification
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中图分类号：
R969
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脚注
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基金
国家中医药管理局公益性行业科研专项经费项目资助(201507002-4-4);浙江省教育厅新苗人才计划项目资助(752214F00803)
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