Abstract: Objective　To establish a DNA barcoding technique for the molecular identification of flies based on COⅠ gene and to make up for the deficiency of morphological identification of flies. Methods　Sixteen flies commonly found at Yantai Port were used as subjects in this study. After morphological identification, total DNA was extracted from the single hind foot of each fly. According to reference, the international COⅠ primer LCO1490: 5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′ and HCO2198: 5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′ were used to set up a PCR system. Sequence of COⅠ was successfully amplified, followed by sequencing and aligning of purified PCR product. Phylogenetic study of COⅠ gene was performed using Mega 5.05 by neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) methods. Results　 The amplified COⅠ fragments of sixteen flies were 690 bp in size, with an average A＋T content of 68.53%. The sequence alignment and phylogenetic study showed that the results of COⅠ gene-based identification were consistent with morphological identification. Conclusion　The DNA barcoding technique established in this study is able to amplify COⅠ gene from flies and thereby identify flies with the same result as morphological identification; this technique is particularly suitable for the identification of a trace amount of samples.

Key words: DNA barcoding, Mitochondrial cytochrome c oxidase subunitⅠ gene, Fly, Classification and identification

摘要： 目的　为解决蝇类形态学鉴定的不足,建立基于细胞色素C氧化酶亚基Ⅰ(COⅠ)基因的DNA条形码技术对蝇类进行分子鉴定。方法　选取烟台口岸常见的16只蝇类作为研究对象,形态学鉴定后,取蝇的单后足代表微量组织,提取总DNA。依据资料,选取COⅠ国际通用引物LCO1490:5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′,HCO2198:5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′,建立PCR体系,完善PCR程序,有效扩增蝇类的COⅠ基因。纯化PCR产物后进行序列测定和比对。此外,应用Mega 5.05软件分别用最大似然法、邻接法、最大简约法分析系统发育,构建系统发育树比较进化关系。结果　16只蝇类COⅠ基因扩增片段长度为690 bp,其中A＋T平均含量高达68.53%。序列比对结果及系统进化关系显示,16只蝇类COⅠ基因的分子鉴定与形态学鉴定结果一致。结论　应用DNA条形码技术可成功扩增蝇类的COⅠ基因,其分子鉴定与形态学鉴定结果完全一致,尤其适用于微量组织样本的鉴定。

关键词: DNA条形码, 细胞色素C氧化酶亚基Ⅰ基因, 蝇类, 分类鉴定