小菜蛾性信息素降解酶功能表达及与性信息素结合蛋白的互作研究

Most moths rely on the species-specific sex pheromone communication for mate finding and reproductive isolation between species. Its biochemical and molecular mechanism is the research hotspot in animal olfactory molecular mechanism, and has great scientific significance and application potential. Sex pheromone components must be rapid degradation by specific pheromone degrading enzymes after activating the antennal neurons, which is in order to keep the sensitivity of antennal neurons to receive new signals. In this project, we select the important agricultural pest Plutella xylostella as the object. First, we molecular characterize its pheromone degrading enzyme (esterase and aldehyde oxidase) genes, and investigate their tissue localization to obtain the antennae-enriched expression genes, then further using protein expression in vitro, affinity purification and enzyme activity measurement to obtain enzyme kinetic parameters, and finally assay the interaction between pheromone degrading enzymes and “pheromone binding protein /pheromone complex”. This findings will further clarify the degrading enzymes in physiological function and molecular mechanism. Moreover, we analysis enzyme substrate spectrum, the substrate (including inhibitor) affinity, and structure-activity relationship between enzyme and substrate, which will provide a scientific guidance and a screening model for new insect behavior blockers or interference agents.

大多数蛾类昆虫依靠高度特异的性信息素通讯寻找配偶以实现种间生殖隔离，其生化和分子机制是当今动物嗅觉分子机制的前沿热点，具有重要科学意义和应用潜力。当性信息素激活触角神经元后，性信息素分子将快速的被特异的性信息素降解酶清除，以保持触角神经元的敏感性从而接受下一个新的信号。本项目拟以重要农业害虫小菜蛾为对象，首先对性信息素降解酶（酯酶和醛氧化酶）基因进行分子克隆、组织定位，以获得触角高表达的酶基因，进一步通过体外表达纯化、酶学特性的研究，并结合酶与“性信息素结合蛋白/性信息素复合物”的互作关系研究，进一步阐明降解酶在性信息素感受中的生理作用及分子机制；而后对酶的底物谱及底物（包括抑制剂）亲和力等测定，分析酶和底物间的构效关系，为建立以性信息素降解酶为靶标的新型害虫行为阻断剂或干扰剂的筛选模型，提供理论基础和科学指导。

大多数蛾类昆虫依靠高度种特异性的性信息素通讯寻找配偶以及实现种间生殖隔离，其生化和分子机制是当今动物嗅觉分子机制的前沿热点，具有重大科学意义和应用潜力。由于释放的性信息素微量且不连续，当进入触角感器淋巴液，随后被性信息素结合蛋白运输给性信息素受体，并被触角神经元感受后，性信息素分子必须快速的被特异性的性信息素降解酶所灭活，以此保持触角神经元的敏感性从而接受下一个新的信号。本项目以重要农业害虫小菜蛾为对象，克隆到对性信息素降解酶（酯酶和醛氧化酶）基因，3个醛氧化酶基因（AOX）和49个羧酸酯酶（CCE）基因。进一步的qPCR显示其中2个醛氧化酶基因和5个羧酸酯酶基因为触角高表达。选取表达量较高的1个AOX1和两个CCE016a和016c基因进行真核昆虫细胞表达，并通过亲和纯化获得纯的酶蛋白。最后通过酶活性实验证明，三个酶蛋白可以降解对应的醛类性信息素和酯类性信息素。为建立以性信息素降解酶为靶标的新型害虫行为阻断剂或干扰剂的筛选模型，提供科学指导。

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