褐飞虱共生菌调控宿主解毒代谢基因细胞色素P450表达的机制研究

Symbiont plays an important role in physiological functions of host insects, including nutrition, metabolism, immunity, and reproduction. It has been confirmed that symbionts can directly participate in the degradation of insecticides, which can reinforce the host metabolism of the pesticides. Nevertheless, the mechanism of symbiotic bacteria modulate the gene expression of the host in detoxification rarely reported. Previously, we found that the sensitivity of tetracycline-treated Nilaparvata lugens population was significantly increased, and the activity of host detoxification enzyme P450 was significantly decreased, indicating that N. lugens symbiont is involved in the regulation of host detoxification metabolism, but its mechanism is not yet clear. The objective of the project is to investigate the following main tasks. Identify the symbiotic bacteria that can regulate the detoxification enzyme P450 in N. lugens. Screen the P450 gene that is regulated by the symbiotic bacteria. And screen the factors that modulate P450 expression and its functions, and determine the signal pathways of the symbiotic bacteria that regulate the detoxification gene P450 expression in N. lugens. The aim of the project is to elucidate the expression mechanisms of symbiont modulate detoxification genes cytochrome P450 in N. lugens. The results will provide a theoretical understanding of the insect toxicology and further elucidate the interaction mechanism between insects and symbionts.

共生菌在宿主昆虫的营养、代谢、免疫和生殖等诸多生理功能上发挥着重要作用。目前已证实昆虫共生菌可直接参与杀虫剂的降解而增强宿主对药剂的代谢，而昆虫共生菌通过调控宿主基因表达间接介导宿主解毒代谢鲜有报道。申请人前期研究发现四环素处理褐飞虱种群的药剂敏感性显著上升，宿主解毒酶细胞色素P450的活力显著下降，表明褐飞虱共生菌参与调控宿主解毒代谢，但对其调控机制尚不明确。本项目拟在前期研究基础上，鉴定调控褐飞虱P450介导宿主解毒代谢的共生菌；筛选褐飞虱共生菌调控宿主差异表达的解毒代谢基因P450；研究褐飞虱共生菌诱导宿主解毒代谢基因P450表达的相关因子及功能，明确共生菌调控褐飞虱解毒代谢基因P450表达的信号途径。通过以上研究，揭示褐飞虱共生菌调控宿主解毒代谢基因P450表达的分子机制。研究结果有助于完善昆虫毒理学理论及深入研究昆虫与共生菌的互作机制提供理论依据。

褐飞虱作为抗药性最严重的粮食作物害虫，近年来抗药性事件频发。解毒代谢增强是褐飞虱对化学杀虫剂产生抗性的主要原因。本研究发现，抗生素破坏共生细菌结构影响褐飞虱核心解毒酶基因的表达进而抑制宿主的解毒代谢，暗示共生细菌与褐飞虱解毒酶基因表达存在潜在的互作机制。基于不同抗生素塑造不同共生细菌结构的褐飞虱种群，且显著影响褐飞虱解毒代谢基因的表达模式，通过Procrustes分析揭示了四环素处理褐飞虱种群塑造的共生细菌结构与宿主解毒酶基因表达谱显著相关，共生细菌差异分析筛选四环素特异性抑制的共生细菌Wolbachia。为进一步验证共生细菌Wolbachia与宿主解毒酶基因表达之间的关系，基于四环素和高温胁迫成功破坏褐飞虱共生细菌结构并成功抑制共生细菌Wolbachia，发现抑制共生细菌Wolbachia的褐飞虱种群对杀虫剂的敏感性显著升高，解毒酶P450和GST活力显著下降，分别有30（4条GST基因和26条P450基因）和20（7条GST基因和13条P450基因）条解毒酶基因在四环素和高温处理后显著下调表达，其中有11条（1条GST基因和10条P450基因）解毒酶基因在两种处理中均显著下调，包括核心解毒代谢P450基因NlCYP4CE1和NlCYP6AY1，表明这些解毒酶基因的下调表达是高温和四环素影响宿主杀虫剂敏感性的潜在机制，并使用不同种群的褐飞虱与果蝇进行了验证。为明确共生细菌Wolbachia调控褐飞虱解毒代谢机制，基于褐飞虱异源物质感受器家族转录因子筛选，发现共生细菌Wolbachia调控褐飞虱解毒代谢基因的核心效应因子NlCncC在Wol+的褐飞虱种群中表达量显著高于Wol-褐飞虱种群。沉默NlCncC可显著抑制褐飞虱解毒酶基因特别是核心P450基因NlCYP4CE1和NlCYP6AY1的表达，从而降低解毒酶活力，增加褐飞虱对杀虫剂的敏感性。综上，本研究初步阐明共生细菌Wolbachia调控褐飞虱解毒代谢的机制，研究结果有助于深化对昆虫共生细菌与宿主互作理论的理解。