细胞色素P450参与褐飞虱对菖蒲活性物质β-细辛醚的应答机制研究

With long-term and large scale use of chemical pesticides, the resistance to most chemical pesticides and the resurgence of the rice brown planthopper (BPH) become a big problem in rice pest control. To explore new substitution of chemical pesticides, the environmentally friendly and sustainable substance from plants turned into focus of study recently. Here an active substance was extracted from Acorus plants in our preliminary work, which was finally identified as beta-asarone with double bioactivities of both insecticidal and chemical pesticides-synergistic effect on BPH. Furthermore, three differential expressed genes of cytochrome P450 (CYP) enzymes were screened out from the beta-asarone-treated BPH by analysis of genes differential expression. The fact of down-regulated expression of two of the three CYP enzymes verified by RT-PCR and decreased enzymes activity verified by enzyme assay suggests that the CYP enzymes are involved in response to beta-asarone. In this study, to clarify the response mechanism of the CYP enzymes in BPH, the following scientific experiments will be conducted: (1) the gene cloning, protein expression and enzyme activity assay of the three CYP enzymes, (2) the effect of beta-asarone on the activity of the three CYP enzymes, (3) the differential expression in vivo of the three CYP enzymes temporally and spatially induced by beta-asarone, (4) the growth and development change of the planthopper caused by the genes silence of the three CYP enzymes through RNA interference, and (5) the resistance monitoring of the resistant BPH to chemical pesticide buprofezin after beta-asarone treatment. These results will lay the foundation for the future use of beta-asarone as plant-derived pesticide, and provide new target of BPH for its effective control.

随着化学农药的长期大量使用，褐飞虱的抗药性和再猖獗问题日益严重，急需寻求新的化学农药替代物。植物源杀虫活性物质因其不易产生抗药性且对环境安全等特点，成为目前新型杀虫剂研究的热点。本研究团队从菖蒲的根茎提取物中分离到的活性物质β-细辛醚对褐飞虱具有杀虫和杀虫增效双重作用，极具研究价值。通过β-细辛醚处理前后差异基因表达分析，筛选到3个解毒相关的细胞色素P450酶基因。通过RT-PCR进一步验证了其中的2个在β-细辛醚处理后表达明显下调，酶活测定也验证其酶活明显下降，说明细胞色素P450酶参与了褐飞虱对β-细辛醚的响应。本项目将对这3个细胞色素P450酶参与褐飞虱对菖蒲活性物质β-细辛醚的响应机制进行研究，在mRNA转录、酶活、功能缺失、抗药性等方面阐明β-细辛醚对褐飞虱的杀虫机理和对化学农药的增效机理，为其作为植物源杀虫剂产品开发奠定理论基础，为生物合理设计创制新农药提供作用靶标和理论依据。

褐飞虱是我国水稻上一种主要害虫，每年对我国水稻生产造成巨大的损失。褐飞虱防治主要依赖于化学杀虫剂，导致褐飞虱产生越来越强的抗药性，急需寻求新型的相对环境友好的化学农药替代物。本研究以菖蒲植物的根茎提取物中的活性物质β-细辛醚为研究对象，采用转录组学和分子生物学技术，研究了4个差异表达的细胞色素P450酶基因的克隆与表达模式；通过RNA干扰技术验证了2个上调表达基因的功能；检测了亚致死剂量β-细辛醚处理褐飞虱若虫的细胞色素P450的酶活性；沉默上调表达基因后褐飞虱若虫生长发育参数和死亡率的变化；沉默褐飞虱抗噻嗪酮性种群若虫上调表达基因后褐飞虱若虫生长发育参数和死亡率的变化等。研究结果对阐明β-细辛醚对褐飞虱的杀虫机理奠定了科学基础，对开发β-细辛醚作为植物源杀虫剂具有重要的实践价值。