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• 生物工程 •    下一篇

多基因DNA条形码鉴定6个鳗鱼物种

陈文炳1,邵碧英2,缪婷玉彭娟2,陈彬2,张志灯1,江树勋2   

  • 收稿日期:2017-06-02 修回日期:2017-11-03 出版日期:2018-01-25 发布日期:2018-01-05
  • 通讯作者: 陈文炳 E-mail:621213wbc@163.com
  • 基金资助:
    福建省科技计划农业引导性项目

Polygenic DNA Barcoding Six Eel Species

  • Received:2017-06-02 Revised:2017-11-03 Online:2018-01-25 Published:2018-01-05
  • Contact: CHEN Wen-Bing E-mail:621213wbc@163.com

摘要: 根据鳗鱼的养殖、加工、贸易企业及执法部门的需求,建立6种鳗鱼的物种多基因DNA条形码精准鉴定方法。以鳗鱼DNA为模板,采用3对通用引物对6种鳗鱼的3个基因(16S rRNA、Cyt b、CoⅠ)部分DNA片段进行PCR扩增、测序,结果6种鳗鱼各获得3条16S rDNA(638-640 bp)、Cyt b(464-466 bp)、CoⅠ(690 bp)基因部分DNA序列,从中选取各物种序列同源的片段设计3对新引物对6种鳗鱼的16S rRNA、Cyt b、COⅠ基因部分DNA片段进行PCR扩增,其产物大小分别为(504-507)bp、400bp、609b,再从各片段中筛选出具有6种鳗鱼物种特异性强的、碱基数分别为262bp、280bp、300bp的3条DNA片段序列,作为 6种鳗鱼物种的3个基因的标准DNA条形码,应用DNAMAN6.0软件进行同源性分析,建立了鳗鱼物种的多基因条形码检测方法。应用该方法对30个待检鳗鱼样品进行检测,结果表明,各样品基于3个基因DNA条形码的比对,物种判别结果互相吻合,从而精准判别样品的所属物种。该方法稳定、精准、易于操作,可应用于6种鳗鱼的物种鉴定,值得推广。

关键词: 16S rRNA基因, Cyt b基因, COⅠ基因, 多基因DNA条形码, 鳗鱼物种鉴定

Abstract: In order to meet the requirements of enterprises and law enforcement, the accurate methods that DNA barcoding six eel species, including Anguilla rostrata, Anguilla Anguilla, Anguilla japonica,Anguilla mossambica,Anguilla marmorata,Anguilla bicolor pacifica were developed, based on 3 genes encoding 16S rRNA, cytochrome b (Cyt b) and Cytochrome C Oxidase Ⅰ ( COⅠ). General primers were used to amplify DNA fragments of partial gene of 16S rRNA , Cyt b and COⅠ in above six eel species and their PCR products were sequenced to get the DNA fragments with the length of 638-640 bp from 16S rRNA, 464-466 bp from Cyt b and 690 bp from COⅠin every eel species respectively. Three pairs of primers were designed to use for establishing PCR-DNA Barcode method according to the sequences of above three fragments, and their PCR products were sequenced to get the DNA fragments with the length of 504-507 bp, 400 bp and 609 bp, corresponding to the DNA fragments of 16S rRNA, Cyt b and COⅠrespectively. The highly specific DNA fragments with the length of 262 bp, 280 bp and 300 bp also corresponding to 16S rRNA, Cyt b and COⅠwere selected as standard DNA barcodes among eel species specificity to identify eel species conveniently. Above method was applied to identify 30 samples in six eel species. The results show that DNA barcoding methods for A. rostrata, A. Anguilla, A. japonica A. mossambica, A. marmorata, A. bicolor pacifica are correspond to three kinds of standard DNA barcodes from 16S rRNA, Cyt b and COⅠ. It shows that the method is stable, precise and easy to operate, and can be applied to the identification of six species of eels, which is worth popularization.

Key words: 16S rRNA , Cyt b, COⅠ, Polygenic DNA-Barcode, Identification of eel species

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