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棉蚜细胞色素P450 CYP6J1的克隆与表达
Cloning and Expression of Cytochrome P450 CYP6J1 from Aphis gossypii
王亚美1, 黄丽娜1, 艾新宇1, 魏原杰1, 刘小宁1*, 高希武2*
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作者单位:1. 新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐 830046;
2. 中国农业大学农学与生物技术学院, 北京 100193
中文关键字:棉蚜;P450 CYP6J1;克隆;蛋白表达
英文关键字:Aphis gossypii; P450 CYP6J1; cloning; protein expression
中文摘要:为了深入了解细胞色素P450 CYP6J1蛋白的结构和功能,本实验克隆获得了棉蚜Aphis gossypii P450 CYP6J1基因,对该基因进行信息学及原核表达分析,通过SDS-PAGE检测目的蛋白的表达结果,并用Western-blot进行验证。结果表明, P450CYP6J1序列长1398 bp,编码氨基酸数为465,理论分子量为53.67 kD,理论等电点为8.80。氨基酸序列分析表明该序列具有完整的开放阅读框,且没有信号肽。同源性分析表明,棉蚜cDNA序列推导的氨基酸与豌豆蚜Acyrthosiphon pisum的保守性最为接近,一致性可达92%。在大肠杆菌Escherichia coli BL21中表达获得的His-CYP6J1蛋白,并用Western-blot检测目的蛋白大小正确。这些研究结果为棉蚜P450 CYP6J1多克隆抗体制备提供了基础。
英文摘要:In order to understand the structure and function of cytochrome P450 CYP6J1 protein, P450CYP6J1 gene from cotton aphid (Aphis gossypii) was cloned and analyzed using bioinformatic method. P450 CYP6J1 protein was first expressed in prokaryotic, followed by SDS-PAGE and Western-blot detection. The results showed that the length of open reading frame region was 1398 bp, encoding 465 amino acids residues with the predicted molecular weight of 53.67 kD and isoelectric point of 8.80. Amino acid sequence analysis showed that P450 CYP6J1 protein had no signal peptide. Homology analysis showed that the amino acids of P450 CYP6J1 of A. gossypii was similar with that of pea aphid (Acyrthosiphon pisum) (homologous similarity 92%). The His-CYP6J1 protein was obtained after being expressed in Escherichia coli BL21, and the target protein was proved by Western-blot. These results provided a foundation for preparation of P450 CYP6J1 polyclonal antibody from A. gossypii.
2016,(35): 378-383 收稿日期:2015-12-08
DOI:10.11984/j.issn.1000-7083.20150393
分类号:Q78;Q969.35
基金项目:国家自然科学基金项目(31330064,31471781)
作者简介:王亚美(1990-),硕士研究生,研究方向:生化与分子生物学,E-mail:wangyamei0114@sina.com
*通讯作者:刘小宁,博士,研究方向:农业昆虫与害虫防治,E-mail:liuxn0103@sina.com;高希武,gaoxiwu@263.net.cn
参考文献:
贺丽虹, 赵淑娟, 胡之璧. 2008. 植物细胞色素P450基因与功能研究进展[J]. 药物生物技术, 15(2):142-147.
邱星辉, 何凤琴, 李梅. 2003. 细胞色素P450介导的昆虫抗药性[J]. 生命的化学, 23(4):279-281.
张学涛, 柳建伟, 李芬, 等. 2012. 北疆地区棉蚜对不同杀虫剂敏感度水平测定[J]. 植物保护, 38(2):163-166.
Ahuja I, Rohloff J, Bones AM. 2010. Defence mechanisms of Brassicaceae:implications for plant-insect interactions and potential for integrated pest management. A review[J]. Agronomy for Sustainable Development, 30(2):311-348.
Amenya DA, Naguran R, Lo TC, et al. 2008. Over expression of a cytochrom P450 (CYP6P9) in major African malaria vector, Anopheles funestus, resistant to pyrethroids[J]. Insect Biochemistry and Molecular Biology, 17(1):19-25.
Carino FA, Koener JF, Plapp Jr. FW, et al. 1994. Constitutive overexpression of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides[J]. Insect Biochemistry and Molecular Biology, 24(4):411-418.
Crinnion WJ. 2009. Chlorinated pesticides:threats to health and importance of detection[J]. Alternative Medicine Review, 14:347-359.
Deeni YY, Paine MJ, Ayrton AD, et al. 2001. Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein[J]. Archives of Biochemistry and Biophysics, 396(1):16-24.
Denholm I, Rowland MW. 1992. Tactics for managing pesticide resistance in arthropods:theory and practice[J]. Annual Review of Entomology, 37(1):91-112.
Eldefrawi ME, Miskus R, Sutcher V. 1960. Methylenedioxyphenyl derivatives as synergists for carbamate insecticides on susceptible, DDT and parathion-resistant house flies[J]. Economic Entomology, (53):231-234.
Feyereisen R, Andersen JF, Carino FA, et al. 1995. Cytochrome-P450 in the house-fly-structure, catalyticactivity and regulation of expression of Cyp6a1 in an insecticide-resistant strain[J]. Pesticide Science, 43:233-239.
Graslund S, Nordlund P, Weigelt J, et al. 2008. Protein production and purification[J]. Nature Methods, 5(2):135-146.
Komarek M, Cadkova E, Chrastny V, et al. 2010. Contamination of vineyard soils with fungicides:a review of environmental and toxicological aspects[J]. Environment International, 36:138-151.
Kubota A, Kim EY, Jwata H. 2009. Alkoxyresorufin (methoxy-, ethoxy-, pentoxy- and bexzyloxyresorufin 0-dealkylase activities by in vitro-expressed cytochrome P450 1A4 and 1A5 from common cormorant (Phalacrocorax carbo)[J]. Comparative Biochemistry and Physiology, Part C, 149:544-551.
Liu XN, Zhang L, Zhang XT, et al. 2013. Molecular cloning and recombinant expression of cytochrome P450 CYP6B6 from Helicoverpa armigera in Escherichia coli[J]. Molecular Biology Reports, 40:1211-1217.
Scott JG, Lee SST. 1993. Purification and characterization of a cytochrome P450 from insecticide susceptible and resistant strains of housefly, Musca domestica L., before and after phenobarbital exposure[J]. Archives of Insect Biochemistry and Physiology, 24(1):1-19.
Scott JG, Wen Z. 2001. Cytochromes P450 of insects:the tip of the iceberg[J]. Pest Management Science, 57(10):958-967.
Snyder MJ, Van Antwerpen. 1998. Evidence for a diazepam-binding inhibitor (DBI) benzodiazepine receptor-like mechanism in ecdysteroidogenesis by the insect prothoracic gland[J]. Cell Tissue Research, 294:161-168.
Tan W,Guo Y. 1996. Effects of host plant on susceptibility to deltamethrin and detoxification enzyme of Heliothis armigera (Lepidoptera:Noctuidea)[J]. Economic Entomology, 86:11-14.
Uchinomiya S, Nonaka H, Wakayama S, et al. 2013. In-cell covalent labeling of reactive His-tag fused proteins[J]. Chemical Communications, 49:5022-5024.
Wang XP, Hobbs AA. 1995. Isolation and sequence analysis of a cDNA clone for a pyrethroidinducible cytochrome P450 from Helicoverpa armigera[J]. Insect Biochemisry and Molecular Biology, 25(9):1001-1009.
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