瓜蚜对黄瓜中葫芦素代谢适应的分子机制

Aphis gossypii Glover as important pest insects to cucurbitaceae family plant such as cucumber (Cucumis sativus L.) caused serious damage to the production of cucumber vegetable. Cucurbitacins are important defensive plant secondary metabolites of cucurbitaceae plant to resist against insects herbivory. The adaption of Aphis gossypii Glover to host plant cucumber is correlated with the metabolic adaption of Aphis gossypii Glover to cucurbitacin of cucumber. The metabolism of plant secondary metabolites by pest insects can induce the metabolic ability of insects, and then affect the insecticide sensitivity of insects. Therefore, it is worth to study the molecular mechanism for metabolic adaption of Aphis gossypii Glover to cucurbitacin of cucumber (Cucumis sativus L.) and its relation with the insecticides sensitivity of Aphis gossypii..The project intends to conduct research from the following four aspects: (1) the influence of Aphis gossypii feeding on the cucurbitacin level in cucumber; (2) the effects of cucurbitacin feeding on P450s and other detoxify enzyme activities and transcript levels of detoxify enzyme genes in Aphis gossypii; (3) Heterologous expression of the P450s genes and other detoxify enzyme genes of Aphis gossypii which respond significantly to cucurbitacin stress, and study the metabolic ability of these genes to cucurbitacin and insecticides. (4) Verify the function of the cucurbitacin metabolism related P450s genes and other detoxify enzyme genes of Aphis gossypii, by conducting RNAi. Through conducting the project, the detoxify enzyme genes of Aphis gossypii which be involved in the metabolism of cucurbitacin will be clarified, and the molecular mechanism for metabolic adaption of Aphis gossypii Glover to cucurbitacin and the induction of cucurbitacin to insecticides sensitivity of Aphis gossypii will be understand..The project exhibit important theoretical and practical value in uncovering the coevolution relationship between insect-host plant, and sustainable control of Aphis gossypii based on cooperating the insect resistance of host plants and chemical control.

瓜蚜(棉蚜)是黄瓜等葫芦科植物的重要害虫。葫芦素是葫芦科植物防御植食性害虫的重要次生物质。瓜蚜对黄瓜寄主的适应性与其对葫芦素的代谢适应相关。昆虫对植物次生物质的代谢会诱导其体内解毒代谢增强，进而影响其杀虫剂敏感度。因此，瓜蚜对葫芦素的代谢适应机制及其与瓜蚜杀虫剂敏感度的关系值得研究。.本项目拟通过(1)瓜蚜取食对黄瓜叶片中葫芦素水平的影响;(2)取食葫芦素对瓜蚜P450s等解毒酶活性及相关基因转录水平的影响;(3)异源表达对葫芦素胁迫有显著响应的瓜蚜P450s等解毒酶，研究其对葫芦素及杀虫剂的代谢能力;(4)RNAi验证与瓜蚜代谢葫芦素密切相关的P450s等解毒酶基因等4个方面研究，弄清参与代谢葫芦素的瓜蚜解毒酶基因，阐明瓜蚜代谢适应葫芦素的分子机制及其影响瓜蚜对杀虫剂敏感度的机制。研究对揭示昆虫-寄主植物的协同进化关系、协调植物抗虫性和化学防治、实现瓜蚜的可持续治理，具有理论和实践价值。

针对重要的蔬菜害虫瓜蚜以及葫芦科植物的防御物质葫芦素，本项目研究了瓜蚜对葫芦素B的代谢适应机制及其与瓜蚜对杀虫剂敏感度的关系。已按照计划，研究了葫芦素B对瓜蚜的毒力，瓜蚜取食对甜瓜叶片中葫芦素B含量的诱导作用，两种不同寄主黄瓜、甜瓜，以及葫芦素B分别处理，对瓜蚜的生长发育及种群的影响。通过研究明确葫芦素B对瓜蚜的毒力、对瓜蚜取食的响应以及对瓜蚜种群的不利影响等，阐明了葫芦素B对瓜蚜的防御作用。为阐明瓜蚜对葫芦素B代谢适应的生化机制，研究了取食不同寄主、取食葫芦素B对瓜蚜解毒酶活性的影响，发现葫芦素B对瓜蚜CarE和GSTs酶活性的诱导具有剂量效应及时间效应，100 mg/L葫芦素B处理24h显著诱导了CYP450酶活性的增加，表明瓜蚜解毒酶相关的机制参与了对葫芦素B的解毒代谢。通过转录组测序筛选了葫芦素B处理后上调表达的瓜蚜解毒酶基因，并验证了葫芦素B处理对瓜蚜P450基因表达的诱导作用，最后通过RNAi干扰研究证实了瓜蚜P450基因CYP6CY13和CYP6CY14对葫芦素B的解毒功能，研究明确了P450介导的瓜蚜代谢适应葫芦素B的分子机制。为阐明瓜蚜对葫芦素B的适应性与其化学杀虫剂敏感度的关系，筛选了瓜蚜抗噻虫嗪品系、抗呋虫胺品系，研究明确了瓜蚜对噻虫嗪、呋虫胺的抗性机制，并比较研究了呋虫胺敏感和抗性瓜蚜对葫芦素B的适应性差异。发现，对呋虫胺具有抗性的瓜蚜，对葫芦素B的耐受性显著增加，同时葫芦素B处理对抗性瓜蚜的种群参数影响较小，表明，对呋虫胺具有代谢抗性的瓜蚜种群，其对寄主植物防御物质葫芦素B的适应性也增强了。因而针对瓜蚜P450基因进行RNAi的策略将有利于治理瓜蚜的抗药性，并发挥寄主植物的抗虫性。为开发基于瓜蚜的抗药性治理和增强寄主抗虫性的瓜蚜可持续治理策略，提供了新的思路。本项目的研究结果揭示了瓜蚜-寄主植物防御的协同进化关系，为协调植物抗虫性和化学防治、实现瓜蚜的可持续治理，提供了研究基础。

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