桔小实蝇电压门控钠离子通道基因cDNA克隆及其生物信息学分析

doi:10.3864/j.issn.0578-1752.2012.19.011

摘要/Abstract

摘要： 【目的】克隆获得桔小实蝇（Bactrocera dorsalis）电压门控钠离子通道基因cDNA序列，明确其典型特征，为研究桔小实蝇抗性分子机理奠定基础。【方法】采用RT-PCR和PCR技术，克隆桔小实蝇钠离子通道基因cDNA序列，利用相关软件对其序列进行生物信息学分析。【结果】克隆得到1条长为6 446 bp的cDNA序列，包含1个6 405 bp的完整开放阅读框，共编码2 134个氨基酸。同源比对发现，桔小实蝇与果蝇（Drosophila melanogaster，NP_001188635）和家蝇（Musca domestica，AAB47604）钠离子通道基因相似度分别高达91.7%和86.9%，而与人的钠离子通道Nav1.2基因（Homo sapiens，NP_066287）相似度为42.3%。所克隆序列包含昆虫钠离子通道所有典型特征。【结论】成功地从桔小实蝇中克隆得到钠离子通道基因完整开放阅读框。该钠离子通道基因存在丰富的选择性剪接，发现了3个可能与抗性相关的碱基取代。钠离子通道基因有可能发展成为一种昆虫系统发育研究的分子标记。

关键词: 桔小实蝇, 钠离子通道, 基因克隆, 生物信息学分析, 系统发育分析

Abstract: 【Objective】 The objective of this study is to clone voltage-gated sodium channel gene (VGSC) from Bactrocera dorsalis (Hendel), and to identify its typical hallmarks. It will provide basic molecular information for clarifying insecticide resistance mechanism of B. dorsalis.【Method】 The cDNA sequence was isolated using RT-PCR and PCR methods. Based on the sequencing results, the bioinformatics analysis of nucleic acid and putative amino acid was conducted.【Result】An almost full-length cDNA sequence (6 446 bp) of VGSC was obtained, including a complete open reading frame (ORF) of 6 405 bp, which encoded 2 134 amino acids and included all the typical hallmarks of VGSC. The amino acid shared 91.7%, 86.9% and 42.3% identity with sodium channel genes of Drosophila melanogaster (NP_001188635), Musca domestica (AAB47604), and Homo sapiens Nav1.2 (NP_066287), respectively. 【Conclusion】A complete ORF of VGSC was sequenced with clone strategy from oriental fruit fly for the first time. Abundant alternative splicing phenomena existed in the VGSC of B. dorsalis. Three potential mutation points, which may confer resistance to pyrethroids and DDT, were observed. It is suggested that VGSC would be used as a molecular marker in phylogenetic study of insect.

Key words: Bactrocera dorsalis, sodium channel, gene cloning, bioinformatics analysis, phylogenetic analysis

蒋玄赵, 魏丹丹, 申光茂, 豆威, 王进军. 桔小实蝇电压门控钠离子通道基因cDNA克隆及其生物信息学分析[J]. 中国农业科学, 2012, 45(19): 3996-4003.

JIANG Xuan-Zhao, WEI Dan-Dan, SHEN Guang-Mao, DOU Wei, WANG Jin-Jun. Cloning and Bioinformatics Analysis of Voltage-Gated Sodium Channel Gene cDNA in Bactrocera dorsalis (Hendel)[J]. Scientia Agricultura Sinica, 2012, 45(19): 3996-4003.

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参考文献

[1]林进添, 曾玲, 梁广文, 陆永跃, 王琳. 桔小实蝇雄成虫的空间分布格局. 华南农业大学学报, 2005, 26(2): 43-46.

Lin J T, Zeng L, Liang G W, Lu Y Y, Wang L. Distribution spatial pattern of Bactrocera dorsalis males. Journal of South China Agricultural University, 2005, 26(2): 43-46. (in Chinese)

[2]Jin T, Zeng L, Lin Y Y, Lu Y Y, Liang G W. Insecticide resistance of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), in mainland China. Pest Management Science, 2011, 67(3): 370-376.

[3]章与苹, 曾玲, 陆永跃, 梁广文. 华南地区桔小实蝇抗药性动态监测. 华南农业大学学报, 2007, 28(3): 20-23.

Zhang Y P, Zeng L, Lu Y Y, Liang G W. Monitoring of insecticide resistance of Bactrocera dorsalis adults in South China. Journal of South China Agricultural University, 2007, 28(3): 20-23. (in Chinese)

[4]Williamson M S, Denholm L, Bell C A, Devonshire A L. Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica). Molecular and General Genetics, 1993, 240(1): 17-22.

[5]Hartshorne R P, Catterall W A. Purification of the saxitoxin receptor of the sodium channel from rat brain. Proceedings of the National Academy of Sciences of the United States of America, 1981, 78(7): 4620-4624.

[6]Hartshorne R P, Messner D J, Coppersmith J C, Catterall W A. The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical β subunits. The Journal of Biological Chemistry, 1982, 257(23): 13888-13891.

[7]Soderlund D M, Knipple D C. The molecular biology of knockdown resistance to pyrethroid insecticides. Insect Biochemistry and Molecular Biology, 2003, 33(6): 563-577.

[8]Loughney K, Kreber R, Ganetzky B. Molecular analysis of the para locus, a sodium channel gene in Drosophila. Cell, 1989, 58(6): 1143-1154.

[9]Ingles P J, Adams P M, Knipple D C, Soderlund D M. Characterization of voltage-sensitive sodium channel gene coding sequences from insecticide-susceptible and knockdown-resistant house fly strains. Insect Biochemistry and Molecular Biology, 1996, 26(4): 319-326.

[10]Dong K. A single amino acid change in the para sodium channel protein is associated with knockdown-resistance (kdr) to pyrethroid insecticides in German cockroach. Insect Biochemistry and Molecular Biology, 1997, 27(2): 93-100.

[11]Shao Y M, Dong K, Tang Z H, Zhang C X. Molecular characterization of a sodium channel gene from the silkworm Bombyx mori. Insect Biochemistry and Molecular Biology, 2009, 39(2): 145-151.

[12]Dong K. Insect sodium channels and insecticide resistance. Invertebrate Neuroscience, 2007, 7(1): 17-30.

[13]Larkin M A, Blackshields G, Brown N P, Chenna R, McGettigan P A, McWilliam H, Valentin F, Wallace I M, Wilm A, Lopez R, Thompson J D, Gibson T J, Higgins D G. Clustal W and clustal X version 2.0. Bioinformatics, 2007, 23(21): 2947-2948.

[14]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maxium parsimony methods. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.

[15]Guindon S, Dufayard J F, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum- likelihood phylogenies: assessing the performance of phyML 3.0. Systematic Biology, 2010, 59(3): 307-321.

[16]Catterall W A. From ionic currents to molecular review mechanisms: The structure and function of voltage-gated sodium channels. Neuron, 2000, 26(1): 13-25.

[17]Thackeray J R, Ganetzky B. Developmentally regulated alternative splicing generates a complex array of Drosophila para sodium channel isoforms. The Journal of Neuroscience, 1994, 14(5): 2569-2578.

[18]Song W, Liu Z, Tan J, Nomura Y, Dong K. RNA editing generates tissue-specific sodium channels with distinct gating properties. The Journal of Biological Chemistry, 2004, 279(31): 32554-32561.

[19]Thackeray J R, Ganetzky B. Conserved alternative splicing patterns and splicing signals in the Drosophila sodium channel gene para. Genetics, 1995, 141(1): 203-214.

[20]Lee S H, Ingles P J, Knipple D C, Soderlund D M. Developmental regulation of alternative exon usage in the house fly Vssc1 sodium channel gene. Invertebrate Neuroscience, 2002, 4(3): 125-133.

[21]Plummer N W, Meisler M H. Evolution and diversity of mammalian sodium channel genes. Genomics, 1999, 57(2): 323-331.

[22]Soderlund D M. Pyrethroids, knockdown resistance and sodium channels. Pest Management Science, 2008, 64(6): 610-616.

[23]Vais H, Williamson M S, Goodson S J, Devonshire A L, Warmk J W, Usherwood P N, Cohen C J. Activation of Drosophila sodium channels promotes modification by deltamethrin. Reductions in affinity caused by knock-down resistance mutations. The Journal of General Physiology, 2000, 115(3): 305-318.

[24]Miyazaki M, Ohyama K, Dunlap D Y, Matsumura F. Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica). Molecular and General Genetics, 1996, 252(1/2): 61-68.

[25]Williamson M S, Martinez-Torres D, Hick C A, Devonshire A L. Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides. Molecular and General Genetics, 1996, 252(1/2): 51-60.

[26]O’Reilly A O, Khambay B P S, Williamson M S, Field L M, Wallace B A, Davies T G E. Modelling insecticide-binding sites in the voltage-gated sodium channel. Biochemical Journal, 2006, 396(2): 255-263.