孙思雅;方昀;来梦茹;葛宇清;张光霁;程汝滨;
SUN Si-ya;FANG Yun;LAI Meng-ru;GE Yu-qing;ZHANG Guang-ji;CHENG Ru-bin;College of Pharmaceutical Science,Zhejiang Chinese Medical University;the First Affiliated Hospital,Zhejiang Chinese Medical University;

• 1:浙江中医药大学药学院
• 2:浙江中医药大学第一临床医学院

摘要(Abstract)：

利用性状鉴别与DNA条形码联用的方法对药用海马棘海马进行系统研究,为棘海马快速准确的鉴定奠定基础。结合文献与样品观察获得的棘海马典型鉴别特征包括棘刺发达、短吻,具有单或双颊刺,育儿袋接壤的棘刺更加尖锐。提取棘海马和其他海马样品的基因组总DNA,对COⅠ和ATP6基因序列进行PCR扩增和双向测序,通过Blast对序列的准确性进行检验,利用MEGA 7计算序列的GC量,种内种间距离以及采用邻接法构建NJ系统发育树。结果显示,COⅠ序列和ATP6序列长度分别为649 bp和602～603 bp,GC碱基平均量分别为39. 96%,35. 37%,COⅠ序列和ATP6序列的最小种间遗传距离均大于最大种内遗传距离,存在明显的条形码间隔。基于COⅠ基因序列和ATP6基因序列构建的NJ系统发育树结果显示,棘海马样品单独聚为一个大分支,明显区分于其他海马样品,且与线纹海马亲缘最近。此外棘海马的种内存在3个稳定的亚群结构,提示COⅠ和ATP6序列可用于后续棘海马的地理生态学研究。该研究表明,通过性状鉴别和DNA条形码序列联用的方法确定了棘海马的典型性状和分子鉴别特征,为棘海马的准确鉴定和后续应用提供了技术保证,并为扩大海马药用资源和保证海马药材临床用药安全提供了新的依据。
The combination of morphological characteristics and DNA barcodes was used to a systematic study of Hippocampus spinosissimus,laying the foundation for rapid and accurate identification for the medical seahorse species. According to the reported literature and observation on seahorse samples,the typical characteristics of the H. spinosissimus include highly developed spiny,much short nose,single or double cheeks and strongly developed spines bordering pouch. Genomic DNAs of H. spinosissimus and other related seahorse species were extracted using the TIANamp Marine Animals DNA Kit. The COⅠ and ATP6 genes were amplified and sequenced in both directions. After the verification by Blast,the GC content,intraspecific and interspecific genetic distance,and the Neighbor joining( NJ) phylogenetic trees were analyzed by MEGA 7. The lengths of the COⅠ and ATP6 genes were 649 bp and 602-603 bp,respectively,with the average GC content of 39. 96% and 35. 37%. The maximum intraspecific genetic distances in H. spinosissimus based on COⅠ and ATP were both far less than the minimum interspecific genetic distance between H. spinosissimus and other seahorses,suggesting a significant barcoding gap. NJ analysis results of COⅠ and ATP6 exhibited that all H. spinosissimus species clustered together,indicating that the two DNA barcode could identify H. spinosissimus from other seahorses accurately and quickly. In addition,H. spinosissimus shared a close genetic relationship between H. kelloggi according to the NJ tree. Furthermore,there exits three stable subgroup structure of H. spinosissimus,indicating that COⅠ and ATP6 barcodes could be applied the indicator for the geographical ecology research of H. spinosissimus. The results obtained the typical morphological and molecular identification characteristics of H. spinosissimus,which played central roles for the development of species identification. This study provides an important basis data for expanding the medical seahorse resources and ensuring the safety of clinical medicine.

关键词(KeyWords)： 棘海马;DNA条形码;COⅠ;ATP6;性状鉴别
Hippocampus spinosissimus;DNA barcode;COⅠ;ATP6;morphological identification

Abstract:

Keywords:

基金项目(Foundation): 国家中医药管理局公益性行业科研专项经费项目(201507002-4-4);; 浙江省教育厅新苗人才计划项目(752214F00803)

作者(Author): 孙思雅;方昀;来梦茹;葛宇清;张光霁;程汝滨;
SUN Si-ya;FANG Yun;LAI Meng-ru;GE Yu-qing;ZHANG Guang-ji;CHENG Ru-bin;College of Pharmaceutical Science,Zhejiang Chinese Medical University;the First Affiliated Hospital,Zhejiang Chinese Medical University;

Email:

DOI: 10.19540/j.cnki.cjcmm.20190829.109

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