白纹伊蚊紫外光敏感视蛋白基因的克隆及序列特征

doi:10.11853/j.issn.1003.8280.2021.05.009

中国媒介生物学及控制杂志 ›› 2021, Vol. 32 ›› Issue (5): 553-559.DOI: 10.11853/j.issn.1003.8280.2021.05.009

白纹伊蚊紫外光敏感视蛋白基因的克隆及序列特征

郭秀霞, 程鹏, 刘丽娟, 王海防, 张崇星, 王怀位, 公茂庆

山东省寄生虫病防治研究所, 山东第一医科大学(山东省医学科学院), 山东省医药卫生媒介生物学重点实验室, 山东济宁 272033

收稿日期:2021-01-20 出版日期:2021-10-20 发布日期:2021-10-20
通讯作者: 公茂庆,E-mail:maoqingg@yahoo.com
作者简介:郭秀霞,女,硕士,助理研究员,主要从事病原生物学研究,E-mail:guoguoxiua@163.com
基金资助:
国家自然科学基金（81871685）；山东省自然科学基金（ZR2017YL004，ZR2020MC048）；山东省医学科学院医药卫生科技创新工程

Cloning and sequence features of ultraviolet sensitive opsin gene in Aedes albopictus

GUO Xiu-xia, CHENG Peng, LIU Li-juan, WANG Hai-fang, ZHANG Chong-xing, WANG Huai-wei, GONG Mao-qing

Shandong Institute of Parasitic Diseases, Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong Province Key Laboratory of Health and Vector Biology, Jining, Shandong 272033, China

Received:2021-01-20 Online:2021-10-20 Published:2021-10-20
Supported by:
Supported by the National Natural Science Foundation of China (No. 81871685), Natural Science Foundation of Shandong Province (No. ZR2017YL004, ZR2020MC048) and the Innovation Project of Shandong Academy of Medical Sciences

摘要/Abstract

摘要： 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。

关键词: 白纹伊蚊, 紫外光敏感视蛋白, 基因克隆, 序列分析

Abstract: 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.

Key words: Aedes albopictus, Ultraviolet sensitive opsin, Gene cloning, Sequence analysis

中图分类号:

R384.1
Q781

郭秀霞, 程鹏, 刘丽娟, 王海防, 张崇星, 王怀位, 公茂庆. 白纹伊蚊紫外光敏感视蛋白基因的克隆及序列特征[J]. 中国媒介生物学及控制杂志, 2021, 32(5): 553-559.

GUO Xiu-xia, CHENG Peng, LIU Li-juan, WANG Hai-fang, ZHANG Chong-xing, WANG Huai-wei, GONG Mao-qing. Cloning and sequence features of ultraviolet sensitive opsin gene in Aedes albopictus[J]. Chines Journal of Vector Biology and Control, 2021, 32(5): 553-559.

参考文献

[1] van Breugel F,Riffell J,Fairhall A,et al. Mosquitoes use vision to associate odor plumes with thermal targets[J]. Curr Biol,2015,25(16):2123-2129. DOI:10.1016/j.cub.2015.06.046.
[2] Leung NY,Montell C. Unconventional roles of opsins[J]. Annu Rev Cell Dev Biol,2017,33:241-264. DOI:10.1146/annurevcellbio-100616-060432.
[3] Cardé RT. Multi-cue integration:how female mosquitoes locate a human host[J]. Curr Biol,2015,25(18):R793-R795. DOI:10.1016/j.cub.2015.07.057.
[4] Feuda R,Marlétaz F,Bentley MA,et al. Conservation,duplication,and divergence of five opsin genes in insect evolution[J]. Genome Biol Evol,2016,8(3):579-587. DOI:10.1093/gbe/evw015.
[5] Henze MJ,Oakley TH. The dynamic evolutionary history of pancrustacean eyes and opsins[J]. Integr Comp Biol,2015,55(5):830-842. DOI:10.1093/icb/icv100.
[6] Giraldo-Calderón GI,Zanis MJ,Hill CA. Retention of duplicated long-wavelength opsins in mosquito lineages by positive selection and differential expression[J]. BMC Evol Biol,2017,17(1):84. DOI:10.1186/s12862-017-0910-6.
[7] Baik LS,Recinos Y,Chevez JA,et al. Multiple phototransduction inputs integrate to mediate UV light-evoked avoidance/attraction behavior in Drosophila[J]. J Biol Rhythms,2019,34(4):391-400. DOI:10.1177/0748730419847339.
[8] Li LF,Chi H,Liu HN,et al. Retention and losses of ultraviolet-sensitive visual pigments in bats[J]. Sci Rep,2018,8:11933. DOI:10.1038/s41598-018-29646-6.
[9] Arikawa K,Wakakuwa M,Qiu XD,et al. Sexual dimorphism of short-wavelength photoreceptors in the small white butterfly,Pieris rapae crucivora[J]. J Neurosci,2005,25(25):5935-5942. DOI:10.1523/JNEUROSCI.1364-05.2005.
[10] Gao J, Zhang HD,Guo XX, et al. Dispersal patterns and population genetic structure of Aedes albopictus (Diptera:Culicidae) in three different climatic regions of China[J]. Parasit Vectors,2021,14(1):12. DOI:10.1186/s13071-020-04521-4.
[11] Senthilan PR,Helfrich-Förster C. Rhodopsin 7-the unusual rhodopsin in Drosophila[J]. Peer J,2016,4:e2427. DOI:10.7717/peerj.2427.
[12] Baldwin JM,Schertler GFX,Unger VM. An alpha-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors[J]. J Mol Biol,1997,272(1):144-164. DOI:10.1006/jmbi.1997.1240.
[13] Katana R,Guan CL,Zanini D,et al. Chromophore-independent roles of opsin apoproteins in Drosophila mechanoreceptors[J]. Curr Biol,2019,29(17):2961-2969.e4. DOI:10.1016/j.cub. 2019.07.036.
[14] Yourick MR,Sandkam BA,Gammerdinger WJ,et al. Diurnal variation in opsin expression and common housekeeping genes necessitates comprehensive normalization methods for quantitative real-time PCR analyses[J]. Mol Ecol Resour,2019,19(6):1447-1460. DOI:10.1111/1755-0998.13062.
[15] Pohl N,Sison-Mangus MP,Yee EN,et al. Impact of duplicate gene copies on phylogenetic analysis and divergence time estimates in butterflies[J]. BMC Evol Biol,2009,9(1):99. DOI:10.1186/1471-2148-9-99.
[16] Zanini D,Giraldo D,Warren B,et al. Proprioceptive opsin functions in Drosophila larval locomotion[J]. Neuron,2018,98(1):67-74.e4. DOI:10.1016/j.neuron.2018.02.028.
[17] Salcedo E,Zheng LJ,Phistry M,et al. Molecular basis for ultraviolet vision in invertebrates[J]. J Neurosci,2003,23(34):10873-10878. DOI:10.1523/JNEUROSCI.23-34-10873.2003.
[18] Wang T,Montell C. Phototransduction and retinal degeneration in Drosophila[J]. Pflügers Arch-Eur J Physiol,2007,454(5):821-847. DOI:10.1007/s00424-007-0251-1.
[19] Owens GL,Rennison DJ. Evolutionary ecology of opsin gene sequence,expression and repertoire[J]. Mol Ecol,2017,26(5):1207-1210. DOI:10.1111/mec.14032.
[20] Jenkins AM,Muskavitch MAT. Crepuscular behavioral variation and profiling of opsin genes in Anopheles gambiae and An. stephensi (Diptera:Culicidae)[J]. J Med Entomol,2015,52(3):296-307. DOI:10.1093/jme/tjv024.
[21] 孙艳. 视蛋白基因参与蚊抗药性机制的研究[D]. 南京:南京医科大学,2012.Sun Y. Opsin,involved in mosquito resistance to deltamethrin[D]. Nanjing:Nanjing Medical University,2012.

白纹伊蚊紫外光敏感视蛋白基因的克隆及序列特征

Cloning and sequence features of ultraviolet sensitive opsin gene in Aedes albopictus

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