利用苏云金芽胞杆菌转座子Tn4430构建含cry1Ac10基因的解离载体

将cry1Ac10基因和苏云金芽胞杆菌的复制起始区连 接在一起，并在其两侧按相同方向各连接一个来自苏云金芽胞杆菌转座子Tn4430的解离位点，构成转移单位。再将革兰氏阳性细菌的抗性标记基因和大肠杆菌克隆载体pUC19与之连接，获得含cry1Ac10基因的解离载体pBMB801。将其转化苏云金芽胞杆菌无晶体突变体，再导入含解离酶基因的辅助质粒pBMB1200。在解离酶作用下，两个解离位点间发生重组，消除基在操作中的抗性标记基因等非必需基因片段，获得仅保留有完整cry1Ac10基因和来 自苏云金芽胞杆菌质粒复制起始区，在无抗生素选择压力下能稳定遗传的重组质粒pBMB801B 。

A NEW RESOLUTION VECTOR WITH CRY1ACiO GENE BASED ON BACILL US THURINGIENSIS TRANSPOSON Tn4430

A new resolution vector with crylAclO gene based on Tnp I-mediated site-spe-cific recombination system of Bacillus thuringiensis (Bt) transposon Tn4430 was developed.The gene crylAclO, encoding a protoxin against plutella acylostella larvae, and the gene oril030, from a plasmid of wide type Bacillus thuringiensis, were inserted into two copy sets of RES sites, named pBMB801. When pBMB801 was introduced into crystal negative Bt host BMB171, antibiotic resistance genes and other non-Bt DNA can be selectively eliminated. This recombinant plasmid was found very stable without antibiotic selection. The resulting strain only contained Bt DNA and is free of antibiotic resistance genes. This strategy should facilitate regulatory approval for its developemnt as a commercial biopesticide.