双交换同源重组法构建Thermus thermophilus基因无痕敲除突变体

Construction of gene clean deletion mutants in Thermus thermophilus by the method of double-crossover homologous recombination

为在噬热栖热菌(Thermusthermophilus)中开収无需筛选标记的基于双交换同源重组的基因无痕敲除手段,以大质粒及染色体上的TTP0042与TTC0340_0341为靶基因,构建含有其两侧同源臂的基因敲除载体幵转化T.thermophilusHB27细胞,将转化混合物涂布于不含筛选物质的培养基上,通过PCR及Southern杂交检验转化子的基因型。结果表明,采用线状的TTP0042基因敲除载体迚行转化,获取表观Δ0042突变体的概率为10–4,而使用闭合环状载体,该概率可达10–3;基因型及表现型分析显示,这些表观Δ0042均为TTP0042无痕敲除型突变体。该敲除手段在染色体上的基因TTC0340_0341中得到验证,从300个单菌落中成功鉴定出了1个Δ0340_0341突变株。

In order to develop a gene clean deletion method in Thermus thermophilus without using selectable markers, the megaplasmid and the chromosome encoded TTP0042 and TTC0340_0341were chosen as target genes. The gene deletion vectors were flanked two homology target genes arms and then transformed into T. thermophilus HB27 cells with .the transformation mixtures streaked on selection free medium. The genotype of the resulted transformants were determined using PCR and Southern blot. The frequency of obtaining apparent Δ0042 mutant was 10^-4 when using linearized TTP0042 gene deletion vector for transformation;while the frequency was 10^-3 when used closed circular vector. The genotype and phenotype determination results showed that the apparent Δ0042 were all true TTP0042 gene clean deletion mutants. Therefore, in T. thermophilus, gene clean deletion mutants could be generated using the developed method with the frequency up to 10^-3. The method was also verified in the chromosomal gene locus TTC0340_0341 and managed to generate the Δ0340_0341 mutant from 300 single colonies.