熏蒸剂磷化氢对赤拟谷盗过氧化氢酶基因的表达调控机制研究

Phosphine is is by far the most widely used fumigant for the control of insect pests of stored grain. However, widespread high level resistance to phosphine in recent years now theatens the world food security seriously, and strengthen the research on the toxicity of phosphine has become a consensus due to a lack of economical alternative fumigant till now. Catalase is one of an important target enzymes of phosphine and the inactivation of catalase and subsequent oxidative stress is considered to be one of the permary mechanisms of phosphine.however, the regulation mechanism of phosphine towards catalase is still unclear. For the first time，we found that phosphine could inhibited the expression of genes encoding catalase in Drosophila melanogaster，indicating the regulation of catalase gene was performed at transcriptional level. Moreover,we found the nitric oxide(NO) synthesis level was also induced after phosphine fumigation. Combined with the reports that mutant in the metabolic pathway of NO could blockade the inhibition of catalase genes by phosphine and produce resistance, it could be assumed that regulation of catalase genes mediated by NO is one of the primary mechanisms of phsophine. A transcriptome analyses was further perfromed in the red flour beetle(Tribolium Castaneum) which is one of important pest in stored products in order to guide the prictical use of phosphine. The phosphine/NO/catalase pathway was confirmed by pathway analysis. Thus, in order to elucidate the molecular mechanisms of the toxicity of phosphine and establish the scientific resistance management strategy, we plan to employ the red flour beetle as test pests, establish the mode of action of the catalse regulated by phosphine, and determin the function and signal transduction of NO in the regulation of genes encoding catalase.

磷化氢是世界上应用最为广泛的熏蒸剂，然而磷化氢抗性昆虫普遍发生，严重威胁世界粮食安全，急需加强磷化氢毒理机制研究以应对上述挑战。过氧化氢酶是磷化氢的主要靶点，然而其调控机制还不清楚。申请者发现磷化氢可抑制黑腹果蝇过氧化氢酶基因的表达，表明磷化氢对过氧化氢酶的调控在转录水平进行。同时申请者发现磷化氢可激活黑腹果蝇一氧化氮合成，结合一氧化氮代谢途径关键酶的突变可以阻断磷化氢对过氧化氢酶的抑制作用并引发抗性的报道，可以推测一氧化氮可能介导了的磷化氢对过氧化氢酶的调控。为指导实际应用，申请者利用双末端RNA测序技术进行了磷化氢熏蒸重要仓储害虫赤拟谷盗的转录组研究，通路分析确认了磷化氢/一氧化氮/过氧化氢酶模型可信度较高。本项目计划在前期工作基础上，系统阐明磷化氢对赤拟谷盗过氧化氢酶的调控模式，明确一氧化氮在磷化氢调控过氧化氢酶中的功能和调控机制，从而为科学使用磷化氢和治理抗性提供理论指导。

磷化氢是世界上应用最为广泛的熏蒸剂，然而磷化氢抗性昆虫普遍发生，严重威胁世界粮食安全，急需加强磷化氢毒理机制研究以应对上述挑战。磷化氢主要通过诱导氧化胁迫杀虫，过氧化氢酶是其主要靶点，然而磷化氢对过氧化氢酶的调控机制还不清楚。项目组建立了磷化氢诱导模式昆虫氧化胁迫的实验室模型，在酶活性、基因表达和蛋白丰度三个水平研究了磷化氢对昆虫抗氧化酶系的调控模式，明确了磷化氢可在转录水平特异性抑制过氧化氢酶基因表达，且该调控模式具有明显的时间剂量依赖关系。为阐明磷化氢对过氧化氢酶基因的精细调控机制，项目组克隆了过氧化氢酶基因启动子，利用双荧光素酶表达系统在S2细胞系中，通过启动子缺失和突变分析，确定了DRE原件为磷化氢转录抑制过氧化氢基因的充分和必要条件。项目组进一步对DRE原件结合蛋白DREF开展研究，结果表明磷化氢可抑制DREF表达，且DREF过量表达和RNAi均会阻断磷化氢对过氧化氢酶的调控。这些结果表明，磷化氢可通过DRE/DREF系统调控过氧化氢酶酶基因的表达，从而首次在分子水平阐明了磷化氢对靶基因的调控机制。