目的 克隆白纹伊蚊紫外光敏感视蛋白（UV-opsin）基因，分析该基因编码蛋白的序列特征，为研究视蛋白基因的生物学功能提供分子基础。方法 根据已报道蚊虫的UV-opsin的保守序列设计简并引物，通过反转录-聚合酶链式反应（RT-PCR）扩增白纹伊蚊的UV-opsin基因，纯化PCR产物，经EcoRⅠ酶和XhoⅠ酶酶切，酶切产物连接至pET28a（+）载体构建原核表达质粒，阳性重组质粒经PCR鉴定后进行基因测序，利用Expasy、Protparam、Tmpred及ScanProsite等软件进行编码蛋白的结构特征分析及功能预测，利用Clustal X 1.81和MEGA 6.0软件进行序列同源性分析，构建系统进化树。结果 成功获得白纹伊蚊UV-opsin的cDNA序列。序列分析表明，该序列全长为1 146 bp（含终止密码子），编码381个氨基酸，理论蛋白相对分子质量为42 500，等电点为7.06。与白纹伊蚊（XP_029730343）序列同源性为99.74%，与埃及伊蚊（ABF18478）序列同源性为96.85%。构建系统进化树发现不同属、不同目的昆虫为独立分支，伊蚊属与库蚊属聚成一簇，之后与按蚊属合并。该视蛋白具有7个跨膜拓扑结构，含有1个G蛋白偶联受体位点，4个N-豆蔻酰化位点、3个N-糖基化位点、1个酪氨酸激酶磷酸化位点、3个蛋白激酶C磷酸化位点以及2个酪蛋白激酶Ⅱ磷酸化位点。该视蛋白含有保守的LRTPSN、DRY和QAKK基序，为视蛋白发挥作用的重要基序。结论 成功克隆获得了白纹伊蚊的UV-opsin序列，该序列具有典型的视蛋白结构特征，为进一步研究白纹伊蚊视蛋白的功能奠定了基础。
Objective To clone the ultraviolet sensitive opsin (UV-opsin) gene of Aedes albopictus, to characterize the sequences of UV-opsin gene and its encoded protein, and to provide a molecular basis for the study of its biological function. Methods The degenerate primers were designed according to the conserved sequences of the UV-opsin gene from mosquitoes, and the UV-opsin gene of Ae. albopictus was amplified by RT-PCR. The PCR product was purified and ligated into the pET28a(+) vectors with EcoRⅠ digestion and XhoⅠ to construct a prokaryotic expression plasmid. The positive recombinant plasmid was identified by PCR and sequenced. Structural features and function were analyzed and predicted using Expasy, Protparam, Tmpred, and ScanProsite. Sequence homology was analyzed by Clustal X 1.81 and a phylogenetic tree was constructed using MEGA 6.0 software. Results The cDNA sequence of Ae. albopictus UV-opsin was obtained, which was 1 146 bp in full length (including termination codon) and encoded 381 amino acid residues. The predicted molecular weight was 42 500 and the isoelectric point was 7.06. The homology was 99.74% with Ae. albopictus (XP_029730343) and 96.85% with Ae. aegypti (ABF18478). The phylogenetic tree showed that insects from the same genera and orders clustered together. Aedes was clustered with the genus Culex, and then with Anopheles. This UV-opsin had 7 transmembrane topology domains, 1 G-protein-coupled receptor, 4 N-myristoylation sites, 3 N-glycosylation sites, 1 tyrosine kinase phosphorylation site, 3 protein kinase C phosphorylation sites, and 2 casein kinase Ⅱ phosphorylation sites. The UV-opsin contained conserved motifs LRTPSN, DRY and QAKK, which were critical for its function. Conclusion The UV-opsin gene sequence of Ae. albopictus is cloned, which has the structural characteristics of opsins. The results provide a foundation for further research on the function of Ae. albopictus UV-opsin gene.
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