HaSNPV几丁质酶基因在大肠杆菌和昆虫细胞中的表达及其产物对Bt杀虫剂的增效作用  

Expression of HaSNPV Chitinase Gene in E.coli and Insect Cells andSynergism of Expressed Products with Bt in Killing Insects

作　　者：王芳[1] 顾奇伟[1] 武家才[1] 张传溪[1] 

机构地区：[1]浙江大学应用昆虫学研究所分子生物学实验室,杭州310029

出　　处：《中国生物化学与分子生物学报》2004年第6期792-797,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目 (No .3 0 0 70 0 3 2 );高等学校全国优秀博士学位论文作者专项资金 (No .2 0 0 0 5 5 )资助~~

摘　　要：为了探索杆状病毒几丁质酶对微生物杀虫剂的增效作用及其利用途径 ,分别在大肠杆菌和昆虫细胞中表达棉铃虫单粒包埋型核型多角体病毒 (HaSNPV)几丁质酶 .用PCR方法扩增出不含N端信号肽编码序列的几丁质酶基因片段 ,并分别克隆至原核表达载体pET2 8a和重组到杆状病毒BactoBac表达系统 ,在大肠杆菌 (E .coli)BL2 1和粉纹夜蛾 (Trichoplusiani)细胞系Tn 5B1 4中分别进行了表达 .在大肠杆菌中表达量约占细菌总蛋白 15 % ,在昆虫细胞中表达量约占细胞总蛋白10 % .将含有几丁质酶的大肠杆菌和昆虫细胞表达产物添加到苏云金杆菌 (Bt)菌液中一起喂食 2龄家蚕 .结果显示 ,HaSNPV几丁质酶基因的 2种表达产物和Bt杀虫剂的混合物使处理的家蚕的致死时间较对照处理均明显缩短 .昆虫细胞和大肠杆菌表达产物与Bt混合物处理的LT50 分别从 93 5h和 95 1h缩短到 5 6 2h及 6 7 2h ,并且供试家蚕的生长速度明显缓慢 .研究结果表明 。To investigate the synergism of baculovirus chitinase with Bacillus thuringiensis(Bt) against insect pests and to develop a new Bt synergist,the chitinase gene of Helicoverpa armigera single-nucleopolyhedroviruses(HaSNPV) was expressed in bacteria and insect cells,respectively.The PCR product of the mature chitinase encoding region was cloned into the prokaryotic expression vector pET28a and the baculovirus expression vector Bac to Bac,and then expressed in E. coli BL21 and Trichoplusia ni cell line Tn-5B1-4,respectively.The expressed target protein accumulated up to about 15% and 10% of the total cellular proteins,respectively.The total cellular proteins with expressed chitinase were mixed with Bt and fed to the second instar larvae of the silkworm(Bombyx mori).The results showed that the lethal time of the silkworm larvae fed with Bt supplemented with the chitinase produced in Tn-5B1-4 cells and in E.coli was much shorter than that of the corresponding controls; LT 50 were shortened from 93.5 hours and 95.1 hours to 56.2 hours and 67.2 hours,respectively.The growth rates of the tested individuals also decreased when compared with their corresponding controls.These results suggested that the recombinant HaSNPV chitinase was a promising Bt synergist.

关 键 词：HASNPV 几丁质酶 原核表达 真核表达 BT杀虫剂 增效作用 

分 类 号：S482.91[农业科学—农药学] S482.39[农业科学—植物保护]