线粒体COⅠ、Cyt b和16S rRNA基因在6种金枪鱼鉴定中的适用性分析

Applicability Analysis of Mitochondrial COⅠ, Cyt b and 16S rRNA Genes in Identification of Six Tuna Species

中文关键词: 金枪鱼  分子鉴定  COⅠ  Cyt b  16S rRNA

英文关键词: Tuna  Molecular identification  COⅠ  Cyt b  16S rRNA

中文摘要:

      探讨细胞色素氧化酶亚基Ⅰ基因(COⅠ)、细胞色素b基因(Cyt b)及16S rRNA基因对主要渔获区的蓝鳍金枪鱼(Thunnus thynnus)、马苏金枪鱼(Thunnus maccoyii)、黄鳍金枪鱼(Thunnus albacares)、大目金枪鱼(Thunnus obesus)、长鳍金枪鱼(Thunnus alalunga)和正鲣(Katsuwonus pelamis) 6种重要的生食金枪鱼及其易混品种的物种鉴定和进化分析的适用性。采用3对通用引物对6种金枪鱼共63个样品的COⅠ、Cyt b和16S rRNA 3种序列片段进行PCR扩增、测序，并运用DnaSP 5.10、Mega 7.0等软件进行了DNA序列分析、遗传差异分析和进化树分析。结果显示，16S rRNA较为保守，不能很好区分6种金枪鱼，且不能对同一物种不同地理群体进行聚类分析；Cyt b和COⅠ配合使用能很好区分6种金枪鱼，且存在一定同一物种地理群体聚类的趋势。建议COⅠ与Cyt b基因联合用于上述6种金枪鱼的分子鉴别研究。本研究为生食金枪鱼及其制品的物种鉴定及金枪鱼产业的健康发展提供了技术支撑。

英文摘要:

      Tuna is rich in nutrients and one of three major fish recommended by the International Nutrition Society. Large tuna such as Thunnus albacares, T. maccoyii, T. obesus, and T. thynnus are used as top-grade aquatic products, mainly in the form of sushi or other fresh fish products. Small tuna such as T. alalunga and Katsuwonus pelamis are mainly used in canned tuna and processed katsuobushi. The price of different species of tuna varies greatly, especially because selling tuna without the head or skin can lead to mislabeling and substandard products, which harms the interests of consumers and undermines market functions. This study was aimed at the identification T. albacares, T. maccoyii, T. obesus, T. thynnus, T. alalonga, and K. pelamis. Using tuna DNA as a template, three pairs of universal primers were used to amplify and sequence the DNA fragments of three genes (16S rRNA, Cyt b, and COⅠ) from six species of tuna. The sequencing results were manually corrected to remove the primer regions, which were compared with reference sequences in GenBank by BLAST analysis on the National Center for Biotechnology Information website. The COⅠ gene was analyzed by the Barcode of Life Data System (www.boldsystems.org). As a result, DNA sequences of 16S rRNA (576 bp), Cyt b (652 bp) and COⅠ (307 bp) were obtained from 63 samples of six species of tuna. Genetic diversity parameters such as Number of Haplotypes (h), Haplotype diversity (Hd), Average number of nucleoside difference (k), and Nucleotide diversity (π) were calculated by DnaSP 5.10 software. C-conserved sites, PI-parsimony-informative sites, and V-variable sites were calculated using Mega 7.0 software, which performed 1000 bootstrap tests with neighbor joining. The Kimura 2-parameter model was used to construct a phylogenetic evolutionary tree. The experimental results showed that 16S rRNA was relatively conservative and could not distinguish among the six tuna species. Cyt b and COⅠ could be used to identify the species. However, it is possible that COⅠ and Cyt b sequences can be used together as a DNA barcode for tuna research. A multi-gene DNA-barcode species-identification method that provides technical support for the identification of tuna and its fish products would be of great significance in promoting the accurate identification and healthy development of aquatic products.