不同启动子表达Cry1Ie蛋白的特性分析  

Analysis of Expressed Cry1Ie Protein Initiated by Different Promoters

作　　者：张春鸽[1,2] 赵灿[1,2] 束长龙[2] 孙冰[2] 宋福平[2] 高继国[1] 张杰[2] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]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,北京100193

出　　处：《生物技术通报》2012年第11期192-196,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31171911);国家"973"计划项目(2009CB118902);国家"863"计划项目(2011AA10A203)

摘　　要：苏云金芽胞杆菌启动子P1Ac与T7启动子表达的Cry1Ie蛋白对鳞翅目害虫小菜蛾(Plutella xylostella)幼虫的杀虫活性有较大差异。P1Ac启动子表达的Cry1Ie蛋白LC50为1.73μg/mL,T7启动子表达的Cry1Ie蛋白LC50为18.18μg/mL,后者是前者的10.5倍。主要从形态、碱溶性及抗胰蛋白酶稳定性等方面对其进行了初步的探索。结果显示,两者在形态上无显著差异,均有相对较为规则的颗粒存在;在碱溶性方面无显著差异,均有约20.0%的包涵体能溶解于pH10.5 50.0 mmol/L Na2CO3的溶液;在对抗胰蛋白酶的稳定性方面无明显差异,由此说明这三方面都不是二者活性差异的原因,推测是T7启动子表达的Cry1Ie蛋白折叠不正确导致其活性较差。Insecticidal activity against Plutella xylostellabetween two proteins expressed under the promoter of cry1Ac gene（P1Ac）from Bacillus thuringiensis（Bt）and T7 fromE.colirespectively was very different.LC50of Cry1Ie polypeptides expressed by P1Ac was 1.73 μg/mL,and that expressed by T7 Promoter was 18.18 μg/mL.The latter is 10.5 folds of the former.In this study,the analyzes of Cry1Ie polypeptides on shape,solubility in alkaline,resistance to trypsin were performed respectively.The results showed no significant difference in both shape and solubility,the regular particles of expressed polypeptides could be observed under electric microscope,and approximate 20% inclusion body of two expressed polypeptides could be resolved in 50.0 mmol/L Na2CO3solution with pH10.5.Western blot result demonstrated that both expressed Cry1Ie showed very poor stability against trypsin.All the results indicated that the reason of toxicity difference between two expressed Cry1Ie was not resulted from shape,solubility and stability,but incorrect folding of Cry1Ie polypeptides expressed under strong promoter T7.

关 键 词：cry1Ac基因启动子P1Ac T7启动子 大肠杆菌 Cry1Ie蛋白 表达 

分 类 号：S476[农业科学—农业昆虫与害虫防治]