基于杆状病毒表达系统的烟粉虱CYP4C64体外表达条件优化及其对呋虫胺的代谢活性

Optimization of tobacco whitefly Bemisia tabaci CYP4C64 expression condition using a baculovirus expression system and its metabolic activity toward dinotefuran

中文关键词:昆虫P450  杆状病毒表达系统  体外表达  烟粉虱  新烟碱类杀虫剂

keywords:insect cytochrome P450  baculovirus expression system  in vitro expression  Bemisia tabaci  neonicotinoid insecticides

中文摘要:为解决烟粉虱Bemisia tabaci细胞色素P450（简称P450）体外异源表达不稳定、重复性差的问题，以CYP4C64为模型基因，基于杆状病毒表达系统对其体外表达条件进行优化，并进一步对其进行体外代谢检测及种群水平功能验证。结果表明：P450与细胞色素P450还原酶（cytochrome P450 reductase,CPR）共表达时存在最适比例50∶1,CPR表达过高或不足均会显著降低功能性P450的形成效率；同时，P450活性表达具有明显的时间依赖性，在转染粉纹夜蛾Trichoplusia ni H5细胞系后约44 h达到峰值，随后迅速下降。基于此，通过CPR比例优化和表达时间窗口构建P450体外表达体系。该表达体系可获得具有稳定催化活性的功能性CYP4C64蛋白，能够催化呋虫胺发生N-脱甲基反应生成低毒代谢物呋虫胺脲。同时，CYP4C64在烟粉虱抗性种群中显著高表达，RNA干扰降低其表达后显著提高了烟粉虱对呋虫胺的敏感性，而在转基因黑腹果蝇Drosophila melanogaster中过表达CYP4C64则显著增强了其对呋虫胺的耐受性。表明本研究所建烟粉虱CYP4C64体外表达体系具有较好的可重复性，可用于烟粉虱相关代谢机制研究。

Abstract:To address the instability and poor reproducibility of heterologous in vitro expression of cytochrome P450(P450) from tobacco whitefly Bemisia tabaci, CYP4C64 was selected as a model gene and its in vitro expression conditions was systematically optimized using a baculovirus expression system. Its metabolic activity was subsequently evaluated in vitro, and its function was further validated at the population level. The results showed that co-expression of P450 with cytochrome P450 reductase(CPR) required an optimal P450 ratio of 50∶1. Both excessive and insufficient CPR expression significantly reduced the efficiency of functional P450 formation. In addition, P450 activity exhibited a pronounced time-dependent pattern, reaching a peak approximately 44 h after transfection of Trichoplusia ni H5 cells, followed by a rapid decline. An in vitro P450 expression system was established by optimizing the CPR ratio and expression time window. This system produced functional CYP4C64 protein with stable catalytic activity and catalyzed the N-demethylation of dinotefuran to generate the less toxic metabolite dinotefuran-urea. Furthermore, CYP4C64 was significantly overexpressed in resistant populations of B. tabaci. RNA interference-mediated knockdown of CYP4C64 significantly increased the susceptibility of B. tabaci to dinotefuran, whereas overexpression of CYP4C64 in transgenic Drosophila melanogaster significantly enhanced tolerance to dinotefuran. These findings collectively validate the role of CYP4C64 at both the metabolic-function and resistance-phenotype levels. These results indicate that the B. tabaci CYP4C64 in vitro expression system established in this study has good reproducibility and can be used for studies of metabolic mechanisms in B. tabaci.