Department of Entomology, College of Plant Protection/Key Laboratory of Monitoring and Management of Plant Diseases and Insects, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu, China.
College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.
Pest Manag Sci. 2018 Jun;74(6):1265-1271. doi: 10.1002/ps.4808. Epub 2018 Feb 21.
Cytochrome P450s (CYPs) are known to play a major role in metabolizing a wide range compounds. CYP6FU1 has been found to be over-expressed in a deltamethrin-resistant strain of Laodelphax striatellus. This study was conducted to express CYP6FU1 in Sf9 cells as a recombinant protein, to confirm its ability to degrade deltamethrin, chlorpyrifos, imidacloprid and traditional P450 probing substrates.
Carbon monoxide difference spectrum analysis indicated that the intact CYP6FU1 protein was expressed in insect Sf9 cells. Catalytic activity tests with four traditional P450 probing substrates revealed that the expressed CYP6FU1 preferentially metabolized p-nitroanisole and ethoxyresorufin, but not ethoxycoumarin and luciferin-HEGE. The enzyme kinetic parameters were tested using p-nitroanisole. The michaelis constant (K ) and catalytic constant (K ) values were 17.51 ± 4.29 µm and 0.218 ± 0.001 pmol min mg protein, respectively. Furthermore, CYP6FU1 activity for degradation of insecticides was tested by measuring substrate depletion and metabolite formation. The chromatogram analysis showed obvious nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of deltamethrin, and formation of the unknown metabolite. Mass spectra and the molecular docking model showed that the metabolite was 4-hydroxy-deltamethrin. However, the recombinant CYP6FU1 could not metabolize imidacloprid and chlorpyrifos.
These results confirmed that the over-expressed CYP6FU1 contributes to deltamethrin resistance in L. striatellus, and p-nitroanisole might be a potential diagnostic probe for deltamethrin metabolic resistance detection and monitoring. © 2017 Society of Chemical Industry.
细胞色素 P450 （CYPs）被认为在代谢广泛的化合物中起着重要作用。已经发现 CYP6FU1 在拟除虫菊酯抗性品系的褐飞虱中过表达。本研究旨在 Sf9 细胞中表达 CYP6FU1 作为重组蛋白，以确认其降解拟除虫菊酯、毒死蜱、氯吡虫啉和传统 P450 探针底物的能力。
一氧化碳差示光谱分析表明，完整的 CYP6FU1 蛋白在昆虫 Sf9 细胞中表达。用四种传统 P450 探针底物进行的催化活性测试表明，表达的 CYP6FU1 优先代谢对硝基苯甲醚和乙氧基荧光素，但不代谢乙氧基香豆素和荧光素-HEGE。用对硝基苯甲醚测试酶动力学参数。米氏常数（K ）和催化常数（K ）值分别为 17.51 ± 4.29 μM 和 0.218 ± 0.001 pmol min mg 蛋白。此外，通过测量底物消耗和代谢物形成来测试 CYP6FU1 对杀虫剂的降解活性。色谱分析显示，拟除虫菊酯在烟酰胺腺嘌呤二核苷酸磷酸（NADPH）依赖性消耗和未知代谢物形成方面明显减少。质谱和分子对接模型表明，代谢物为 4-羟基-拟除虫菊酯。然而，重组 CYP6FU1 不能代谢氯吡虫啉和毒死蜱。
这些结果证实，过表达的 CYP6FU1 导致褐飞虱对拟除虫菊酯的抗性，对硝基苯甲醚可能是检测和监测拟除虫菊酯代谢抗性的潜在诊断探针。© 2017 化学工业协会。
