获得四环素抗性的温敏同源重组载体质粒pRNT15的构建

Construction of Temperature Sensitivity Recombinant Plasmid pRNT15 with Tetracycline Resistance Gene

苏云金芽孢杆菌工程菌株TnX对蚊幼虫具有高毒力,但红霉素抗性基因的存在限制了其商品化。根据转座子Tn917载体的基因序列,利用重叠延伸PCR法克隆同源重组序列到pRN5101上得到pRN15,根据pBU4载体上的四环素抗性基因序列,对pRN15进行改造,使其失去红霉素抗性,获得四环素抗性,载体命名为pRNT15。重组载体四环素抗性的获得,使之扩大了使用范围,也为工程菌株染色体中红霉素抗性基因的敲除奠定了基础。

The engineered strain Bacillus thuringiensis （Bt） TnX, which has high virulence to mosquito larvae, was confined in commercial application due to the existence of erythromycin gene in its chromosome. Plasmid pRN15 was constructed by ligating a homologous recombination sequence, primers of which were synthesized according to the sequence of transposon Tn917, to plasmid pRN5101 by means of overlap polymerase chain reaction （PCR）. According to the sequence of plasmid pBU4, a tetracycline resistance gene sequence was synthesized and ligated to pRN15, with the previous erythromycin gene deleted at the meantime, constructing a recombinant plasmid pRNT15 by PCR. The characteristic of tetracycline resistance expanded the application of pRNT15. The recombinant plasmid was significant for its expanding use in genetic engineering, such as for knocking out the erythromycin resistance gene from the chromosome of engineered strain Bt Tnx.