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2016, 37(6): 19-25. doi: 10.11758/yykxjz.20151203001

鳀科(Engraulidae)鱼类DNA条形码电子芯片研究

1. 农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;

2. 青岛海洋科学与技术国家实验室 海洋渔业科学与食物产出过程功能实验室 青岛 266071;

3. 青岛海洋科学与技术国家实验室 海洋生物学与生物技术功能实验室 青岛 266071;

4. 大连海洋大学水产与生命学院 大连 116023;

5. 上海海洋大学水产与生命学院 上海 201306

收稿日期:2015-12-03
修回日期:2015-12-30

基金项目:   山东省科技发展计划项目(2012GHY11531)、科技基础性工作专项(2013FY110700)和泰山学者建设工程专项经费共同资助。 

关键词: 鳀科 , DNA条形码 , 探针 , DNA芯片 , 电子杂交

Development of DNA Barcode-Microarray for Identification of Engraulidae Fishes

1. Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;

2. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071;

3. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071;

4. College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023;

5. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306

Received Date:2015-12-03
Accepted Date:2015-12-30

Keywords: Engraulidae , DNA barcode , Probe , DNA Microarray , Electric hybridization

本研究将基于线粒体COI部分序列的DNA条形码和DNA芯片技术相结合,以鳀科11属30种鱼类为研究对象,在比对分析其DNA条形码序列的基础上,利用软件Oligo Array 2.1筛选探针,经OligoCalc优化探针,去除易形成发夹(Hairpin)、茎环(Stem-loop)及自身二聚体结构(Homo-dimers)的探针,再利用Oligo heat map对探针与靶标序列进行虚拟杂交,共有14个物种的24条探针能与靶标序列特异性结合。利用DNA条形码芯片技术能将14个物种鉴定到种,虽然物种识别能力仅占总物种数的46.7%,但鉴定准确率可达100%。因此,基于COI基因的DNA芯片技术对鳀科鱼类物种鉴定有一定的实用价值,但该技术对物种的识别能力尚有较大提升空间,通过筛选和优化得到高质量的分子探针则是突破该项技术的关键。

Species identification plays a key role in the protection of biodiversity. However, there have been great challenges in traditional morphological identification because of its innate limitations and the lack of experienced techniques. Recently thanks to the development of molecular biology, DNA-based identification is becoming a more accurate alternative method. The cytochrome oxidase I (COI) barcode is a short piece of DNA from a standard region of the mitochondrial genome, which can be used to identify and classify different species and has been proved to be highly effective in many species including fish. The advantage of COI is that it is long enough to identify variations among species yet short enough to be sequenced quickly at low cost. In this study, we analyzed DNA barcoding of Engraulidae species in order to explore the feasibility of DNA microarray technology in species identification. Oligonucleotide probes were designed based on the COI sequences of mitochondrial obtained from 101 individuals of 30 fish species in 11 genera. Forty-six specific probes obtained from 24 species were selected with online software Oligo Array 2.1, among which the probes potentially forming hairpin, stem-loop, or homo-dimers were identified with Oligonucleotide Properties Calculator and excluded. The remaining probes were virtually hybridized with the target sequences in Oligo Heat Map (OHM), and subsequently a total of 26 probes from 16 species bound to the target sequences specifically. The DNA microarray technology identified 14 species, accounting for 46.7% of the total with 100% accuracy. Therefore, DNA microarray based on COI sequences should have practical values in the identification of Engraulidae fish species. Nevertheless, this technique needs further improvement and the key step would be screening and optimization of high quality molecular probes.

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鳀科(Engraulidae)鱼类DNA条形码电子芯片研究