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为了明确Cry1Ac毒素对小菜蛾Plutella xylostella幼虫中肠蛋白酶和羧酸酯酶活性的影响,利用蛋白酶专性底物测定其中肠各蛋白酶活性在不同缓冲液中的最适pH,并用不同浓度Cry1Ac毒素饲喂小菜蛾幼虫后测定该毒素的致死中浓度LC_(50),观察中肠蛋白酶和羧酸酯酶活性的变化,分析Cry1Ac毒素与昆虫中肠酶活性间的关系。结果表明,小菜蛾幼虫中肠总蛋白酶在Tris-HCl、KH_2PO_4/NaOH和Glycine/NaOH三种缓冲液中最适pH均为11.0;强碱性胰蛋白酶的最适pH分别为10.0、10.5和10.5;弱碱性胰蛋白酶的最适pH均为8.5;胰凝乳蛋白酶的最适pH分别为8.5、9.0和9.0。Cry1Ac毒素对小菜蛾的致死中浓度LC_(50)为0.343μg/mL。用不同浓度Cry1Ac毒素饲喂小菜蛾幼虫后,其中肠各蛋白酶活性均随时间的变化而变化。弱碱性胰蛋白酶和胰凝乳蛋白酶的活性明显升高,且随浓度升高呈上升趋势;在取食36~72 h期间低浓度处理组的羧酸酯酶活性与对照组间无显著差异,而高浓度处理组羧酸酯酶活性显著高于对照组。表明Cry1Ac毒素能够显著影响小菜蛾的中肠蛋白酶和羧酸酯酶活性。
In order to clarify the effect of Cry1Ac to midgut protease and carboxylesterase activity in Plutella xylostella larvae, the optimum pH of each protease activity in the midgut was determined by protease-specific substrates and incubated with different concentrations of Cry1Ac toxin The lethal concentration of LC50 of the toxin was determined after feeding on the diamondback moth larvae. The changes of protease and carboxylesterase activities in midgut were observed. The relationship between Cry1Ac toxin and insect midgut enzymatic activity was analyzed. The results showed that the optimum pH of total protease in the midgut of the Plutella xylostella was 11.0 in Tris-HCl, KH 2 PO 4 / NaOH and Glycine / NaOH respectively. The optimum pH of the strongly alkaline trypsin was 10.0, 10.5 and 10.5 ; The best pH of weak alkaline trypsin was 8.5; the optimum pH of chymotrypsin were 8.5, 9.0 and 9.0 respectively. The lethal LC 50 of Cry1Ac toxin to Plutella xylostella was 0.343μg / mL. After feeding the Plutella xylostella larvae with different concentrations of Cry1Ac toxin, the protease activity in the midgut varied with time. Weakly basic trypsin and chymotrypsin activity was significantly increased, and with the increase in the concentration was an upward trend; feeding 36 ~ 72 h period low concentration treatment group carboxylesterase activity and the control group no significant difference , While the carboxylesterase activity of high-concentration treatment group was significantly higher than that of the control group. This indicates that Cry1Ac toxin can significantly affect midgut protease and carboxylesterase activities of diamondback moth.