在大肠杆菌TG1中克隆和表达苏云金芽胞杆菌cryI基因  

Cloning and Expression of CryI Gene of Bacillusthuringiemsis 8010 Strain in Escherichia coli

作　　者：关雄[1] 黄志鹏[1] 高日霞[1] 

机构地区：[1]福建农业大学生物技术中心

出　　处：《农业生物技术学报》1997年第2期173-177,共5页Journal of Agricultural Biotechnology

基　　金：国家计委和国家科委"九五"攻关资助

摘　　要：本研究分离并提纯苏云金芽胞杆菌（Ｂａｃｉｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ）８０１０菌株的质粒ＤＮＡ，经ＢａｍＨＩ／ＰｓｔＩ双酶解后与ＤＩＧ标记的ｃｒｙＩ基因ＥｃｏＲＩ－Ｆ片段的ＲＮＡ探针杂交，显示出分子量分别为６．５６ｋｂ和４．４ｋｂ的阳性片段。用Ｇｌａｓｓｍｉｌｋ回收４．４ｋｂ的阳性片段并与双功能载体ｐＲＩＴ５重组，转化感受态大肠杆菌ＴＧ１细胞，通过斑点杂交和快检获得含４．４ｋｂ片段的克隆株Ｔ２，ＳＤＳ－ＰＡＧＥ电泳表明它表达了１３０ｋＤ的晶体蛋白。生物测定结果表明Ｔ２对小菜蛾有毒杀活性。The plasmids isolated from Bacillus thuringiensis 8010 were digested with restriction endonucleases BamHI/PstI andwere hybridized with DIG labelled cryI gene EcoRI F RNA probe.Two positive fragments, 6.56 kb and 4.4 kb, were obtained. The latter one was then recovered with Glassmilk protocol,ligated with cohesive ends of expression vector pRIT5,transformed into competence cells E.coli TG1. Transformants were selected on media containing ampicillin at 50 μg·mL -1 .The cloned strains harboring the fragment of 4.4 kb were obtained by dot blot analysis and were designated T2. Cell extracts of recombinant strain of E.coli TG1 could produce full length 130 kD insecticidal crystal protein by SDS PAGE. Bioassay also showedthat T2 was toxic to Plutella xylostella larvae.

关 键 词：苏云金芽胞杆菌 cryI基因 克隆和表达 

分 类 号：S482.3[农业科学—农药学]