CsGSTε1在桃小食心虫对阿维菌素解毒代谢中的作用

    Role of CsGSTε1 in the detoxification metabolism of Carposina sasakii to avermectin

    • 摘要: 桃小食心虫(Carposina sasakii)是重要的落叶果树蛀果害虫,为分析谷胱甘肽S-转移酶(glutathione-S-transferase,GST)在桃小食心虫对阿维菌素解毒代谢中的作用,本研究采用点滴法分析了阿维菌素对桃小食心虫的毒力,研究了有效致死中浓度(LC50)阿维菌素处理桃小食心虫3龄幼虫后其GST活力变化,利用分子对接技术分析CsGSTε1蛋白与阿维菌素分子的结合模式;体外原核表达CsGSTε1蛋白,分析阿维菌素对重组GST蛋白的竞争活性;以1-氯-2,4-二硝基苯(CDNB)为底物测定重组蛋白CsGSTε1的酶动力学参数;利用超高效液相色谱分析重组CsGSTε1对阿维菌素的代谢作用。结果显示:阿维菌素对桃小食心虫3龄幼虫LC50值为10.19 mg/L,其3龄幼虫经阿维菌素处理后GST粗酶活性显著上升,CsGSTε1在14个GST基因中被诱导上调表达的水平最高(6.03倍);通过原核表达得到CsGSTε1重组蛋白,CsGSTε1重组蛋白的产量为0.36 μg/μL,Km值为(683.50 ± 47.9) μmol/L,Vmax值为(1538.0 ± 105.4) μmol/(min∙mg),具有良好的催化活性,阿维菌素对CsGSTε1的半抑制浓度(IC50)为60.4 μmol/L;代谢试验显示重组CsGSTε1对阿维菌素有代谢活性。本研究结果可为深入分析GST在桃小食心虫代谢抗药性中的作用奠定基础。

       

      Abstract: Carposina sasakii is a major fruit-boring pest in deciduous fruit trees. To investigate the role of GST in the detoxification metabolism of abamectin in C. sasakii, we employed a droplet method to analyze the toxicity of abamectin to third-instar larvae. The change in GST enzyme activity following treatment with the median lethal concentration (LC50) of abamectin was examined. Molecular docking was used to analyze the binding interaction between CsGSTε1 and abamectin. The CsGSTε1 protein was heterologously expressed in Escherichia coli, and its catalytic activity and inhibitory potential by abamectin were analyzed. The enzyme kinetics of the recombinant CsGSTε1 protein was determined using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, while the metabolic effect of CsGSTε1 towards abamectin was analyzed using ultra-high performance liquid chromatography (UHPLC). The results showed that the LC50 value of abamectin for third-instar larvae was 10.19 mg/L. After treatment with abamectin, the GST enzyme activity in larvae significantly increased. Among 14 GST genes, CsGSTε1 was the most highly induced and upregulated (6.03-fold) . The recombinant CsGSTε1 protein was successfully expressed in E. coli, yielding 0.36 μg/μL, with a Km of (683.50 ± 47.9) μmol/L and a Vmax of (1538.0 ± 105.4) μmol/(min∙mg), indicating good catalytic activity. The half maximal inhibitory concentration (IC50) of abamectin for CsGSTε1 was determined to be 60.4 μmol/L. The metabolic assays indicated that recombinant CsGSTε1 has metabolic activity toward abamectin. The findings of this study lay a foundation for further analysis of the role of GST in metabolic resistance to the insecticide in the peach fruit borer.

       

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