利用DNA改组技术改造aac C1基因启动子活性的研究

Study on DNA Shuffling Method for Changing the Promoter Activity of aac C1 Gene

从表达质粒pYPX2 5 1 (GenBank登陆号 :AY1 780 4 6 )中获得aacC1基因启动子 ,采用DNA改组 (DNAshuffling)技术在体外获得突变体。以lacZ作为报告基因 ,筛选获得活性明显改变的启动子。经过验证 ,对其中活性变化明显的 7个启动子用邻硝基苯基 β半乳糖苷 (ONPG)作为底物进行表达活性测定。结果表明 ,获得的强启动子比原来的提高了 3～ 8倍 ,而弱启动子则活性下降明显 ,其中 3个几乎无活性。进一步对这

DNA shuffling was a method for in vitro homologous recombination of related genes by random fragmentation and polymerase chain reaction reassembly.In this study,this method was used to change the promoter activity of aminoglycoside 3 O acetyltransferase I gene ( aac C1 gene),which encoded gentamicin resistance for the host.The promoter of aac C1 gene was amplified from the plasmid pYPX251(GenBank number:178046)constructed by this lab.After restriction enzyme digesting,the shuffling DNA were inserted into the prokaryotic expression vector pYF5428 and the recombinant plasmid library was constructed,using lac Z as report gene. The library was primarily screened and some promoters with obviously changed activity were obtained. After recutting and recloning,seven promoters changed intensely had been detected the lac Z activity using o nitrophenyl beta D galactopyranoside(ONPG)as substrate.Results showed that the activity of strong promoters had been improved 3～8 fold while one weak promoter had been decreased 3 fold and three weak promoters showed almost no activity.Further nucleotide sequences analysis showed there were many types of mutations in these promoters,including transition, transversition, deletion and insertion.