棉蚜细胞色素P450 CYP6J1的克隆与表达
Cloning and Expression of Cytochrome P450 CYP6J1 from Aphis gossypii

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, P450 CYP6J1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, P450 CYP6J1 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