棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解Cry1Ac毒素分析

Activity assay of midgut serine proteases and analysis of Cry1Ac protoxin activation and toxin degradation in Helicoverpa armigera and Spodoptera exigua.

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigera(Hübner)与甜菜夜蛾Spodoptera exigua(Hübner)的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。

The aheration of protoxin activation and degradation of activated toxin was considered as one of the main mechanisms of insect resistance to Bacillus thuringiensis. The activities of proteinases in midgut juice and BBMV from Helicoverpa armigera Hǔbner and Spodoptera exigua Hǔbner were assayed and the processes of protoxin activation and degradation of activated Cry1Ac toxin were analysed by SDS-PAGE. Serine proteases of midgut juice from cotton bollworm and beet armyworm were more active than those of BBMV. Midgut juice and BBMV could activate Cry1Ac protoxin and thereafter breakdown the activated Cry1Ac toxin, However the process of protoxin activation and toxin degradation by BBMV is much slower than by midgut juice, and the protoxin activation and toxin breakdown by cotton bollworm is much faster than by beet armyworm. Moreover, the inhibition of PMSF, TLCK, TPCK over midgut protease activities were also measured. Three inhibitors gave the same inhibition pattern over proteases from cotton bollworm and beet armyworm. TLCK gave strong inhibition on trysin activity, TPCK had weak inhibition over chymotrypsin.