多杀菌素低剂量继代处理对小菜蛾受体基因mRNA的差异表达

Mechanism of insecticide hormesis is based on the level of receptor. But its specific mechanism is still not very clear. The most likely solution is to be analyzed the expression of the target gene. In preliminary studies, we found that Selected with LC25 of spinosad, the susceptibility of the third instards of Plutella xylostella to spinosad is decreased with the sublethal cycles increased. However, the fitness of P. xylostella over multi-generation exposure has gradually increased. After the strain was selected by LC25 of spinosad for multi-generations, its biological characteristics were restored to near-normal levels and were even greater in the later stages of treatment. This enhanced population growth is defined hormesis and is viewed as a major factor in recurrent pest outbreaks and resurgences. In the present study, therefore, sensitivity strain, low-dose spinosad selected strain and resistance strain of P．xylostella are chosen as the tested materials to investigate the toxicity level under the long-term low dose selection pressure. And the relative expression abundance of receptor gene from larvae treated continuously and discontinuously with low dose of spinosad are analyzed by quantitive RT-PCR method. And the receptor gene mRNA expression abundance from P. xylostella that resistant to spinosad was also analyzed．The objectives of this work are (i) to study and reveal the sensitivity change on the larva of P. xylostella to spinosad, (ii) to definitize the effects of spinosad on expression abundance of GABARα and nAChRα receptor gene mRNA from P．xylostella on the level of receptor, and (iii) to determine the quantitative relationship among low-dose treated, sensitive level and the expression abundance of receptor gene mRNA. This study aimed to develop theories and study methods of pesticides toxicology, to richen research contents of sublethal effect. In addition, the results of this work will have great important significance for rational assessing the possibility of the hormesis or the resistance risk on P．xylostella long-term exposed to low-dose of spinosad, exploring the mechanism of spinosad, and guiding the rational use of pesticides.

杀虫剂对昆虫低剂量兴奋效应的作用机制是基于受体水平，但其具体作用机制尚不很清楚，进行作用位点表达量分析是解决的最可能途径之一。笔者前期研究发现在多杀菌素长期低剂量作用下，小菜蛾出现敏感性下降，而种群参数呈现先降低后恢复趋势，且在处理后期，有刺激增殖现象。本项目以小菜蛾敏感种群、多杀菌素低剂量继代处理种群和抗性种群为供试材料，研究多杀菌素低剂量处理对小菜蛾幼虫毒力的影响；通过小菜蛾受体水平的敏感性变化，明确多杀菌素低剂量处理对小菜蛾可能靶标基因GABARα亚基和nAChRα亚基mRNA表达量的影响，探讨低剂量处理与小菜蛾敏感水平、受体基因表达之间的量化关系。本项目旨在发展农药毒理学的理论和方法、丰富亚致死研究内容。同时，项目成果对合理评估小菜蛾长期暴露于多杀菌素低剂量下可能出现Hormesis效应或产生抗药性的风险、探讨多杀菌素对小菜蛾的可能作用机制及指导农药的合理使用均具有重要意义。

杀虫剂对昆虫低剂量兴奋效应的作用机制是基于受体水平。为探索多杀菌素对小菜蛾长期胁迫下的亚致死效应，采用低剂量LC25（0.1122mg/L）进行继代处理，测定不同处理世代小菜蛾种群Sub对多杀菌素的敏感性水平，并运用实时定量PCＲ法进一步检测低剂量多杀菌素处理后小菜蛾4 龄幼虫GABA α受体和nAChＲα 受体mRNA的表达量变化。结果表明：随着处理世代的增加，小菜蛾对多杀菌素的敏感性下降，至第10代(Sub10)其抗性倍数为2.70 倍;同时多杀菌素LC25处理1代后，小菜蛾GABAR α亚基相对表达量最高，与抗性种群处于同一水平，5代后显著抑制，10代后与敏感无差异；而对nAChＲ α亚基基因表达呈现一定的抑制作用，Sub10中nAChＲα亚基基因的相对表达量较敏感种群SS下降了32.04%，且差异显著;而抗性种群ＲＲ（抗性倍数17.85）nAChＲ α亚基基因的相对表达量仅为SS的0.37 倍。多元相关性分析表明小菜蛾对多杀菌素敏感性可能与GABA α亚基和nAChＲα亚基基因的表达量有关。本研究可为探究多杀菌素长期低剂量处理对小菜蛾抗药性发展的潜在影响提供一定依据。