山梨糖脱氢酶基因在大肠杆菌染色体上整合及表达

以质粒pKF3为模板，扩增出两翼与ptsG基因上下游序列同源，中间为氯霉素抗性基因的DNA片段，连至pMD18T载体，构建得到pMD18PC。以质粒pQE60SDH为模板，扩增山梨糖脱氢酶基因sdh，与pMD18PC连接，得到pMD18PCSDH。将其用PvuⅡ酶切，回收含ptsG1catsdhptsG2的目的片段，电转化至JM109/pKD46，在Red重组酶的作用下，外源DNA片段与染色体上对应区域发生同源重组，将基因ptsG敲除，替换为catsdh基因，获得整合sdh基因的JM109s。经检测JM109s具有山梨糖脱氢酶活性。以ptsG基因上下游序列为引物，JM109s基因组DNA为模板进行PCR，扩增产物测序结果表明sdh基因染色体整合成功。

Integration and expression of sdh gene in Escherichia coli

The chloramphenicol-resistant cassette with short shared sequences of ptsG gene on both ends was PCR-generated from plasmid pKF3 and ligated to pMD18-T to construct pMD18-PC. The sdh gene for sorbose dehydrogenase was generated from plasmid pQE60-SDH and inserted into pMD18-PC, then pMD18-PC-SDH was constructed. It was digested with Pvu II and the target fragment ptsG1-cat-sdh-ptsG2 was recovered and electroporated into Escherichia coli JM109/pKD46. Homologous-recombination between linear DNA cassettes and Escherichia coli chromosomes took place by Red recombination. The detection result showed that the integron JM109s was of sorbose dehydrogenase activity. The PCR products assay using the upstream and downstream sequences of ptsG gene as primers and JM109s genomic DNA as template, indicated that sdh gene had been integrated at the ptsG gene site in Escherichia coli.