论文部分内容阅读
【目的】羧酸酯酶(carboxylesterases,Car Es)是昆虫重要的解毒代谢酶之一,可以介导靶标昆虫对多种杀虫剂的代谢抗性。本研究检测了羧酸酯酶对小菜蛾Plutella xylostella抗药性的介导功能,旨在阐明羧酸酯酶在小菜蛾代谢解毒中的生理生化和分子机理。【方法】采用点滴法测定氟虫腈对小菜蛾敏感种群和抗氟虫腈种群的毒力,以及羧酸酯酶抑制剂磷酸三苯酯(triphenyl phosphate,TPP)对氟虫腈的增效作用;以LC30和LC50浓度的氟虫腈处理抗性小菜蛾,测定药剂处理后Car Es酶活性的变化;利用qRT-PCR技术分析Pxae22和Pxae31两个基因在小菜蛾不同发育阶段、组织和种群的表达模式;利用dsRNA干扰Pxae22和Pxae31后观察基因的表达变化和小菜蛾3龄幼虫对药剂敏感性的变化。【结果】TPP可以削弱小菜蛾3龄幼虫对氟虫腈的抗性,增效倍数约为6倍;使用较低剂量(LC30和LC50)氟虫腈处理小菜蛾3龄幼虫后,处理组Car Es比活力明显高于对照,提示氟虫腈对小菜蛾Car Es活性具有诱导作用。对羧酸酯酶基因Pxae22和Pxae31在小菜蛾不同发育阶段、4龄幼虫不同组织和不同种群3龄幼虫中的表达模式分析发现,这两个基因在小菜蛾4龄幼虫中的表达量最高;在4龄幼虫中以中肠组织中的表达量较高,头、表皮、脂肪体中的表达量很低;抗性种群中的表达量显著高于敏感种群。通过干扰Pxae22和Pxae31后的qRT-PCR验证,两个基因的表达量均显著降低,进一步的氟虫腈毒力测定发现,干扰Pxae22和Pxae31后的小菜蛾3龄幼虫对氟虫腈的敏感性分别增加了1.63倍和1.73倍。【结论】羧酸酯酶在小菜蛾对氟虫腈解毒代谢中具有重要作用;Pxae22和Pxae31是小菜蛾的两个抗性相关基因,其表达水平的变化直接影响小菜蛾对氟虫腈的敏感性。
【Objective】 Carboxylesterases (Car Es) are one of the important detoxification enzymes in insects and can mediate the metabolic resistance of target insects to various insecticides. This study examined the carboxylesterase resistance to Plutella xylostella resistance of Plutella xylostella to elucidate the carboxylesterase metabolism and detoxification of Plutella xylostella in physiological and biochemical mechanism. 【Method】 The toxicity of fipronil to susceptible and insecticidal fipronil to Plutella xylostella was determined by dot-drip method and the synergistic effect of carboxylesterase inhibitor triphenyl phosphate (TPP) on fipronil The resistance to Plutella xylostella was determined by LC-MS and LC50 at a concentration of 50 mg / L, and the enzyme activity of Car Es after treatment was determined. The qRT-PCR was used to analyze the expression of Pxae22 and Pxae31 at different developmental stages of Plutella xylostella The expression pattern of dsDNA was observed by dsRNA interference with the expression of Pxae22 and Pxae31 and the sensitivity of the 3rd instar larvae to drug sensitivity. 【Result】 TPP could attenuate the susceptibility of fipronil to the 3rd instar larvae of Plutella xylostella at the efficiency of about 6-fold. After treatment of the 3rd instar larva of Plutella xylostella with lower doses of LC30 and LC50, Es specific activity was significantly higher than the control, suggesting that fipronil induced the Car Es activity of Plutella xylostella. The expression pattern of carboxylesterase genes Pxae22 and Pxae31 in different stages of diamondback moth, Plutella xylostella, 4th instar larvae and 3rd instar larvae of different populations showed that the expression levels of these two genes were the highest in the 4th instar larva of Plutella xylostella. In the fourth instar larvae, the expression level of midgut tissue was high, while the expression level in head, epidermis and fat body was very low. The expression level in resistant population was significantly higher than that in susceptible population. The results of qRT-PCR showed that the expression levels of both genes were significantly reduced after interference with Pxae22 and Pxae31. Further investigation of felodipamide toxicity showed that the susceptibility of the 3rd instar larvae of Pxae22 and Pxae31 to fipronil Respectively, an increase of 1.63 times and 1.73 times. 【Conclusion】 Carboxylesterase plays an important role in the detoxification and metabolism of fipronil in Plutella xylostella. Pxae22 and Pxae31 are two resistance-related genes in Plutella xylostella. The changes of their expression levels directly affect the susceptibility of Plutella xylostella to fipronil Sex.