随机转座子插入构建甲型副伤寒杆菌启动子文库

Construction of a Promoter Library of Salmonella paratyphi A by Inserting Transposon

目的构建携带氯霉素乙酰转移酶(CAT)的转座子自杀性质粒,通过接合转座获得甲型副伤寒沙门氏菌(副甲)启动子文库。方法 PCR扩增CAT的基因亚克隆至pMD-18T载体,鉴定正确后酶切插入转座子自杀性质粒pSC189,获得新的转座子载体pSC-CAT,将携带pSC-CAT的供体菌与副甲受体菌进行接合转座,涂布氯霉素平板筛选转座的细菌,以随机筛选出来的细菌基因组为模板,热不对称PCR扩增插入位点侧翼序列,并进行测序、比对分析。结果筛选获得约1000个突变菌,随机挑取6个菌,转座子插入位点的上游分别对应6个可疑启动子区域。结论通过自杀性转座子载体pSC-CAT可高效获得副甲菌启动子文库,为进一步研究副甲菌基因表达与调控奠定了基础。

Objective To establish a promoter library of Salmonella paratyphi A by transposition with new suicide plasmid with transposon, which containing chloramphenicol acetyltransferase （CAT） as the report gene. Methods The CAT gene of was amplffed by PCR and cloned into pMD18- T vector. The above fragment was then inserted to suicide plasmid pSC189, and the new plasmid was named pSC-CAT. After conjugation between S17-1 λpir/pSC-CAT and Salmonella paratyphi A, the mixer was put on the plate containing chloramphenicol to screen the transductants. The ge- nome of random picked bacteria was extracted as template of the thermal asymmetric interlaced PCR （Tail- PCR） to amplify flanking sequence near insertion site. The PCR products were sequenced and analysed with blast. Results About 1 000 mutant strains were got. Six suspicious promoter regions were found in the front of inserted site from six random selected bacteria. Conclusion A promoter library of Salmonella pamtyphi A could be effectively established through suicide vector pSC-CAT, which laid a foundation for further study of gene expression and regulation of Salmonella paratyphi A.