苏云金芽孢杆菌vip3A基因的检测及保守性分析  

Screening of Bacillus thuringiensis Strains Containing vip3A Genes and Analysis of Gene Conservation

作　　者：陈建武[1] 唐丽霞[1] 宋少云[1] 袁美妗[1] 庞义[1] 

机构地区：[1]中山大学生物防治国家重点实验室昆虫学研究所,广州510275

出　　处：《生物工程学报》2003年第5期538-544,共7页Chinese Journal of Biotechnology

基　　金：国家 973计划 (No .G2 0 0 0 0 162 2 0 9);863计划 (No .2 0 0 1AA2 14 0 11);广东省自然科学基金项目~~

摘　　要：Vip3A蛋白是苏云金芽孢杆菌 (Bacillusthuringiensis,Bt)在营养期分泌的一类新型杀虫蛋白。用PCR方法从 114个Bt菌株和 4 1个Bt标准菌株中筛选到 39株即约 2 5 %的菌株含有vip3A基因。利用所制备的Vip3A蛋白的多克隆抗体对以上含有vip3A基因的Bt菌株进行Western印迹分析 ,发现多数PCR反应为阳性的菌株都产生 89kD大小的蛋白 ,其中有 4株没有Vip3A蛋白的表达。从以上菌株中挑选 2个对夜蛾科害虫具有较高和较低毒力的菌株 ,即S10 1和 6 11,并分别进行vip3A基因的克隆和测序 ,再与GenBank上所登录的其它 6个全长vip3A基因和 2个已报道的但未登录GenBank的vip3A基因进行核苷酸和氨基酸序列比较 ,结果表明 ,vip3A是一个极其保守的基因。将以上所克隆的 2个vip3A基因即vip3A S10 1和vip3A 6 11分别插入表达载体pQE30构建了表达质粒pOTP S10 1和pOTP 6 11,转化到大肠杆菌M15 ,经 1mmol LIPTG诱导后均表达 89kD大小的Vip3A蛋白。蛋白可溶性试验表明 ,Vip3A S10 1和Vip3A 6 11分别有 4 8%和 35 %的蛋白是可溶的。将Vip3A S10 1和Vip3A 6 11蛋白和已报道的Vip3A S184蛋白对初孵斜纹夜蛾 (Spodopteralitura)幼虫进行生物测定 ,结果表明 ,3个Vip3A蛋白对斜纹夜蛾幼虫毒力没有显著性差异 ,这说明了Vip3A个别氨基酸的?Vip3A, a novel insecticidal protein, is secreted by Bacillus thuringiensis (Bt) during vegetative growth. Vip3A protein possesses insecticidal activity against a wild spectrum of lepidopteran insect larvae. Since the first cloning of vip3A gene from Bt, many other vip3A genes have been isolated. To investigate vip3A genes contribution to Bt and reflect the revolution relationships, the strains containing vip3A genes were screened and gene similarity was analyzed. 114 wild type Bacillus thuringiensis (Bt) strains isolated from different regions and 41 standard Bt strains from the Institute of Pasteur were screened for the vip3A genes using PCR amplification. 39 strains including B. thuringiensis subsp. kurstaki (Btk) HD 1 were found to contain the vip3A genes. Because acrystallerous strain Cry -B derived from Btk HD 1 was proved not to contain vip3A gene, it suppose that the vip3A gene may be located at the plasmids. Vip3A proteins expressed in these strains were detected with polyclonal antibody by Western blot and 4 strains among them were shown not to express the Vip3A proteins. The vip3A genes amplified from wild type Bacillus thuringiensis strains S101 and 611 with different levels of activity against lepidopteran insect larvae were cloned into pGEM T Easy vector. Alignment of these 2 putative Vip3A proteins with 6 others (Vip3A(a),Vip3A(b),Vip3A S,Vip3A S184, Vip83 and Vip3V) in the GenBank data base and 2 reported Vip3A proteins (Vip14 and Vip15) showed that vip3A genes are highly conservative. The plasmids pOTP S101 and pOTP 611 were constructed by inserting 2 vip3A genes ( vip3A S101 and vip3A 611) into the expression vector pQE30 respectively and were transformed into E. coli M15. E. coli M15 cells harboring the pOTP plasmids were induced with 1 mmol/L IPTG to express 89 kDa protein.Experiments showed that the level of soluble proteins of Vip3A S101 in E. coli M15[pOTP S101] and Vip3A 611 in E. col

关 键 词：苏云金芽孢杆菌 VIP3A基因 检测 保守性 克隆 表达 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]