棉铃虫中肠Bt毒素受体蛋白(APN)的分离与纯化  

Isolation and Purification of Aminopeptidase N (APNs), the Main Receptor Proteins for Bacillus thuringiensis toxin in Midgut of Helicoverpa armigera Larva

作　　者：梁革梅[1] 徐广[1] 王桂荣[1] 吴孔明[1] 郭予元[1] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100094

出　　处：《农业生物技术学报》2005年第1期102-107,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.30200182);国家重点基础研究发展规划(973)项目(No.001CB109004)资助

摘　　要：利用Mg/EGTA方法,以不同的速度进行离心,成功地分离了棉铃虫(Bacillusthuringiensis)中肠刷状缘膜囊泡(BB-MV0,BBMV中保留了大部分氨肽酶N(aminopeptidaseN,APN)活性,BBMV中APN的特异活性较粗匀浆液相比提高了3.29倍;CHAPS3-[(3-chlor-amidopropyl)dimethylammonio]-1-propanesulphonate可以增加BBMV的溶解,加入1%(CHAPS)之后APN特异活性增加至5.21倍;利用磷酸酰肌醇特异性的磷脂酶(PI-PLC)能将APN从膜上解离下来,BBMV经PI-PLC处理后,SDS-PAGE电泳结果仅有120kD的1条带;Mono-Q和FPLC结合的方法可以纯化部分APN,SDS-PAGE电泳结果显示,经Mono-Q柱处理后的BBMV中分子量约100kD的蛋白条带加粗;棉铃虫3个抗性品系与敏感品系相比,APN特异活性差异不显著,说明棉铃虫对Bt产生抗性与APN活性的变化无关,抗性的产生可能与Bt毒素和APN结合能力的改变有关。Using Mg/EGTA method and differential centrifugation, the brush border membrane vesicle (BBMV) in midgut of Helicoverpa armigera (Hübner) larva was separated and most of the activities of aminopeptidase N (APNs) in BBMV were preserved. The specific APN activity in BBMV was enhanced to 3.29 times as much as that in roughly homogeneous fluid. While after addition of 1% CHAPS (3-[(3-chlor-amidopropyl) dimethylammonio]-1-propane- sulphonate), the solubility of BBMV was further increased and the specific APN activity attained to 5.21 times. The PI-PLC (phosphatidylinositol - specific phosopholipase C) could cleave the APN from midgut membrane, after digestion by PI-PLC, only one 120 kD band appeared on the figure of SDS-PAGE of BBMV. The results of SDS-PAGE showed that after passing through Mono-Q column, the band around 100 kD of BBMV was more thickened, indicating that the combination of Mono-Q and FPLC could purify partial APN. There were no significant differences among the three Bt-resistant strains and the susceptible strain, i.e. the change of APN activities was no directly associated with Bt resistance of H. armigera, suggesting that the Bt resistance of cotton bollworm would relate to the change of binding ability between Bt toxin and APN.

关 键 词：APN 活性 受体蛋白 中分子 处理 改变 中肠 棉铃虫 BT毒素 抗性 

分 类 号：S435[农业科学—农业昆虫与害虫防治] R192.6[农业科学—植物保护]