CrylA毒素与二化螟中肠刷状缘膜囊泡受体蛋白配基结合分析

Ligand blot analysis of Bt CrylA toxin binding with the midgut brush border membrane vesicle receptors of Chilo suppressalis(Lepidoptera:Pyralidae)

分离和鉴定二化螟Chilo suppresalis幼虫中肠刷状缘膜囊泡(BBMV)中Cryl A毒素的受体蛋白,对于阐明Cryl A毒素作用机理和二化螟抗性机理具有十分重要的意义。为此,本文就Cryl A毒素对二化螟杀虫活性及Cryl Ac与二化螟中肠受体的配基结合进行了研究。结果表明:Cryl Ab对二化螟室内品系(CN)的毒力高于Cryl Ac,而Cryl Ac高于Cryl Aa。配基结合分析表明二化螟CN品系幼虫中肠BBMV中有6个Cryl Ac结合蛋白(分子量分别为50,70,90, 120,160和180 kDa),其中180,160和90 kDa结合蛋白的条带颜色明显深于其他结合蛋白的条带,表明这3个受体蛋白具有较高的结合浓度。同源竞争结合研究表明,180和90 kDa结合蛋白为Cryl Ac的低亲合性结合蛋白,其他4个为高亲合性结合蛋白。为了研究Cryl Ac和Cryl Ab受体结合部位的相互作用,进行了异源竞争结合研究。Cryl Ab可以与Cryl Ac所有的6个结合蛋白进行竞争性结合,与180,120,70和50 kDa结合蛋白具有高亲合性,而与160和90 kDa结合蛋白具有低亲合性。结果显示,Cryl Ac与Cryl Ab在二化螟幼虫中肠BBMV上拥有多个共享的结合位点,但对每个结合位点的亲合性有差异。基于毒素结合部位的相似性,Cryl Ac和Cryl Ab不宜同时用于转基因Bt水稻来控制二化螟。

Isolation and identification of the binding proteins of Cry1A toxins in the midgut brush border membrane vesicles （BBMV） of Chilo suppressalis is very important for understanding the mode of action and resistance mechanism of Cry1A toxins. In the present study, insecticidal activity and ligand binding of Bacillus thuringiensis Cry1A toxins to the midgut BBMV of striped rice stem borer C. suppresalis were investigated. The results indicated that Cry1Ab was more toxic than Cry1Ac, and Cry1Ac was more toxic than Cry1Aa against the 3rd instar larvae of the laboratory strain （CN） of C. suppresalis. Ligand blot analysis showed that there were six major proteins （50, 70, 90, 120, 160 and 180 kDa） binding to Cry1Ac in the midgut BBMV of the CN strain. Bands of 180, 160 and 90 kDa binding proteins were much darker than the others, suggesting that these proteins had higher binding concentrations. Results of homologous competition binding indicated that the 180 and 90 kDa protein bands were of low binding affinity, while the other four protein bands （160, 120, 70 and 50 kDa） were of high binding affinity. Heterologous competition binding assays were conducted to study cross-reactivity of the binding sites between Cry1Ab and Cry1Ac. Cry1Ab competed for all binding sites recognized by Cry1Ac, with high affinity to 180, 120, 70 and 50 kDa proteins and low affinity to 160 and 90 kDa proteins. These data suggest that Cry1Ac and Cry1Ab share several common binding sites in the midgut BBMV of C. suppressalis, but they have different binding affinity with respect to each binding site. Considering the similarity in binding sites, Cry1Ab and Cry1Ac should not be used simultaneously in the transgenic Bt rice to control the target pest C. suppressalis.