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

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％。

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

The plasmids of lepidopteran-specific Bacillus thuringiensis strain from our laboratory were hybridized with RNA probe of cry 1Aa EcoR I-F fragment labelled using DIG. Cry1 gene was located in 39.3 MD plasmid. The plasmid was digested with Hind III and analysed by southern blot. It appeared both 7.1 kb and of 6.5 kb positive bands. The 7.1 kb fragment was ligated to broad-host-range vector pSUP106 and transformed into Pseudomonas fluorescens Pfx-18. The cloned strain, LZP-1 was obtained. The plasmids of LZP-1 were analysed by PCR. The results showed that gene-type is cry1Ab. SDS-PAGE analysis demonstrated that LZP-1 could express 66 kD insecticidal crystal protein and some small molecular weight peptides. Bioassay showed that motality of 1000-fold diluted fermentation broth was 33% to 3rd instar plulella xyloslelly larvae.