质粒拯救法克隆细菌基因组大片段

Recombinational Cloning of Large DNA Fragments of Bacterial Chromosome by Plasmid Rescue

摘要:

【目的】细菌基因组大片段尤其是基因簇的克隆与操作, 是细菌基因功能分析的一个难点。基因组测序工作的不断完成和序列信息的大量积累, 为细菌基因组DNA的操作提供了方便。本文报道了利用细菌的全基因组信息和质粒拯救法的原理建立的一种克隆细菌基因组大片段的方法。【方法】首先, 根据基因组序列信息, 在待克隆片段的一侧扩增一段DNA, 并将其克隆到自杀载体上构建打靶质粒, 然后, 将打靶质粒整合到细菌的基因组中构建重组菌, 提取重组菌的基因组DNA, 酶切, 自连, 转化, 将自杀质粒与待克隆的目的片段一起拯救出来。最后, 根据需要将拯救的DNA片段亚克隆到新的载体中。【结果】我们利用该方法克隆了布鲁氏菌中长度为11kb的virB操纵子, 并构建了互补质粒。将该质粒导入到virB的突变株中后使virB操纵子的转录活性得到了恢复, 表明该策略切实可行。【结论】这种重组克隆策略给我们提供了一种新的对细菌基因组大片段进行操作的方法。

Abstract:

[Obiective]To develop a new method to cline large DNA fragment by combining whole genome sequence information and the principle of plasmid rescue. [Methods]First, we amplified a fragment downstream the region to be cloned and cloned the fragment into a suicide plasmid to construct targeting plasmid, which was then targeted into chromosome by homologous recombination. Then, genomic DNA was isolated, digested with appropriate enzyme, re-ligated and transformed into host bacteria to rescue the plasmid and the large DNA fragment. The rescued DNA fragment was sub-cloned into new plasmids for special purposes. [Results]Using this method, we successfully cloned the 11kb virB operon of Brucella and constructed complementary plasmid. The transcription of the disrupted virB operon was restored with the complementary plasmid, validating the feasibility of the strategy. [Conclusion] this recombinational cloning strategy provides us a new method to modify large DNA fragment of bacteria.