果蝇CYP6G1真核表达及其对新烟碱类杀虫剂的体外代谢

Eukaryotic expression of Drosophila CYP6G1 and its metabolism on neonicotinoid insecticides in vitro

【目的】CYP6G1是黑腹果蝇Drosophila melanogaster体内重要的P450酶,其底物谱广泛,除了介导黑腹果蝇对DDT的抗性之外,还与其对新烟碱类和氨基甲酸酯类杀虫剂的抗性密切相关,但目前CYP6G1的代谢功能与黑腹果蝇抗药性之间的因果关系还缺乏一些直接证据。【方法】本研究通过建立Bac-to-Bac昆虫杆状病毒真核表达系统,功能性地表达了果蝇细胞色素P450酶CYP6G1,以吡虫啉为阳性对照,利用超高效液相色谱(ultra-performance liquid chromatography,UPLC)和超高效液相色谱串联质谱(ultra-performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)分析CYP6G1对噻虫啉和噻虫嗪的体外代谢情况。【结果】孵育2 h后,重组CYP6G1能够羟基化吡虫啉和噻虫啉,使它们的含量分别减少17.6%±7.1%和4.2%±2.3%;重组CYP6G1可以促进噻虫嗪转化为噻虫胺,使噻虫嗪含量减少5.8%±2.1%。【结论】本研究结果为果蝇CYP6G1的结构功能研究提供一定理论依据。

【Aim】CYP6G1 is one of the important P450 enzymes in Drosophila melanogaster,and has a wide substrate spectrum.In addition to mediating DDT resistance in D.melanogaster,CYP6G1 is also implicated in its resistance to neonicotinoid and carbamate insecticides.However,there are still some research gaps between the metabolic function of CYP6G1 and insecticide resistance in D.melanogaster.【Methods】In this study,the cytochrome P450 enzyme CYP6G1 was expressed functionally by the Bac-to-Bac insect baculovirus expression system,and the metabolism of thicloprid and thiamethoxam by the recombinant CYP6G1 in vitro was analyzed by ultra-performance liquid chromatography(UPLC)and ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)with imidacloprid as a positive control.【Results】After 2-h incubation,the recombinant CYP6G1 metabolized 17.6%±7.1%of imidacloprid and 4.2%±2.3%of thiacloprid,respectively,by participating in the hydroxylation reaction.In addition,the recombinant CYP6G1 could promote the conversion of thiamethoxam to clothianidin and reduced the thiamethoxam content by 5.8%±2.1%.【Conclusion】The results of this study provide a theoretical basis for the study on the structure and function of CYP6G1.