苏云金芽孢杆菌cry1基因在荧光假单胞菌Pfx-18中的表达特性

EXPRESSION CHARACTERISTIC OF BACILLUS THURINGIENSISCRY1 GENE IN PSEUDOMONAS FLUORESCENS Pfx-18

用DIG标记的cry1Aa基因EcoRI-F片段的RNA探针，对筛选的鳞翅目高毒力菌株的质粒进行Southern分析，将cry基因定位在39．3MD质粒上。该质粒经HindⅢ酶解，用同样探针进行杂交，呈现6．5kb和7．1kb两条阳性带，其中7．1kb片段的杂交强度明显高于6．5kb片段。将7．1kb片段与多寄主质粒pSUP106连接，转化荧光假单胞菌Pfx－18，获得克隆子LZP-1。克隆基因用PCR鉴定，显示典型的cry1Ab谱带。经SDS-PAGE分析，克隆株表达66kD杀虫晶体蛋白和一些小分子多肽。其发酵液稀释1000倍对三龄小菜蛾幼虫的致死率为33％。

The plasmids of lepidopteran-specific Bacillus thuringiensis strain from ourlaboratory were hybridized with RNA probe of cry 1Aa EcoR I-F fragment labelledusing DIG. Cry 1 gene was located in 39.3 MD plasmid. The plasmid was digestedwith Hind Ⅲ and analysed by southem blot It appeared both 7.1kb and of 6.5kbpositive bands. The 7.1kb fragment was ligated to broad-host-range vector pSUP106and transformed into Pseudomonas fluorescens Pfx-18. The cloned strain, LZP-1 wasobtained. The plasmids of LZP-1 were analysed by PCR The results showed thatgene-type is cry1Ab. SDS-PAGE analysis demonstrated that LZP-1 could express66kD insechcidal crystal protein and some small molecular weight petides. Bioassayshowed that motality of 1000-fold diluted fermentation broth was 33% to 3rd instarplulella xyloslelly larvae.