鳀科(Engraulidae)鱼类DNA条形码电子芯片研究

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(Homodimers)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.

Development of DNA Barcode-Microarray for Identification of Engraulidae Fishes

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.