| 摘要: |
| 在自然或人为因素下容易出现鳗苗种质混杂的现象，由于鳗鲡苗种在形态上十分相似，难以区分。为了保障鳗农的合法利益和提高养殖效益，急需建立一种能够在现场快速、高效使用的鳗鲡种质鉴定方法。本研究通过比对找出5种常见鳗鲡养殖种类：日本鳗鲡（Anguilla japonica）、美洲鳗鲡（A. rostrata）、花鳗鲡（A. marmorata）、太平洋双色鳗鲡（A. bicolor pacifica）、欧洲鳗鲡（A. anguilla）线粒体细胞色素B（cytochrome b，cyt b）基因的差异序列，基于5个cyt b基因序列差异较大的片段，设计多对引物，分别经过PCR验证和条件优化，挑选出4对引物：aj S1/A1、r-a S1/A2、bp-m S5/A3和m-a S4/A4。将这4对引物组合在同一个反应体系中进行PCR扩增，进行条件优化，筛选出组合PCR的最优条件：退火温度为58℃；退火时间为45s；循环数为27。结果表明，通过扩增产物凝胶电泳中条带的有无及大小可快速准确的鉴定出五个鳗鲡种类。本研究建立的组合PCR方法在3小时内可完成整个种类鉴定过程，同时可使用便携式小型仪器完成操作，可满足现场快速、高效鉴定的要求。此外，通过MEGA5.0软件构建5种鳗鲡线粒体cyt b基因序列NJ进化树，发现花鳗鲡和太平洋双色鳗鲡先聚为一支，再与日本鳗鲡聚在一起，而欧洲鳗鲡和美洲鳗鲡聚在一起，进化树图显示的遗传距离和它们的地理分布的远近相似。 |
| 关键词: 组合PCR 种质鉴定 鳗鲡 细胞色素B |
| DOI：10.11693/hyhz20180400097 |
| 分类号:S931 |
| 基金项目:福建省科技厅高校产学合作项目，2016N5009号；福建省科技厅区域发展项目，2016N3002号；鳗鲡现代产业技术教育部工程研究中心开放基金，RE201804号。 |
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| IDENTIFICATION OF FIVE CULTURAL ANGUILLA SPECIES BY COMBINED PCR TECHNIQUE BASED ON DIFFERENT SEQUENCES OF MITOCHONDRIA CYTOCHROME b GENE |
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LAI Xiao-Jian1,2, LIN Xiang-Ri3, LUO Bi-Lian1,2, JIANG Xing-Long1,2
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1.Fisheries College, Jimei University, Xiamen 361021, China;2.Engineering Research Center of the Modern Industrial Technology for Eel, Ministry of Education, Xiamen 361021, China;3.Xiamen Ocean Vocational College, Xiamen 361100, China
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| Abstract: |
| Different Anguilla spp.species were easily mixed naturally or artificially. Due to Anguilla spp. species are very similar in shape, it is very difficult to separate Anguilla spp. by naked eye. In order to protect the legitimate interests of eel farmers and improve aquaculture efficiency, a rapid and efficient identification method of Anguilla spp. was needed urgently. In the study, the combined PCR technique was used. Firstly, the cytochrome b gene of 5 common cultural species (Anguilla japonica, A. rostrata, A. marmorata, A. bicolor Pacifica and A. anguilla) were aligned, and then over ten pairs of primers were designed from the differential sequences. Secondly, annealing temperature and cycles of PCR were optimized and 4 pairs of primers:aj S1/A1, r-a S1/A2, bp-m S5/A3 and m-a S4/A4 were selected. Finally, the 4 pairs of primers were combined into one PCR, and the conditions of the combined PCR were optimized and validated. The results showed that the 5 Anguilla spp. species were identified through the combined PCR amplification and the result of gel electrophoresis. A rapid, efficient identification method of Anguilla spp., which met the demands of eel culture industry, was established in the study. For phylogenetic analysis, mitochondrial cyt b gene sequences of the 5 Anguilla spp. were used to construct neighbor-joining phylogenetic tree by MEGA5.0 software. The results found that A. mamorata and A. bicolor pacifica were gathered into one clade and then gathered with A. japonica, while A. anguilla and A. rastrata were clustered into another clade. The phylogenetic tree showed that the genetic distance was similar to their geographical distribution. |
| Key words: combined PCR idioplasm identification Anguilla spp. cytochrome b |
