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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 construct a transposon suicide plasmid carrying chloramphenicol acetyltransferase (CAT) and obtain a Salmonella paratyphi A promoter library by conjugative transposition. Methods The gene of CAT was amplified by PCR and cloned into pMD-18T vector. After identified correctly, the transposon suicide plasmid pSC189 was digested with restriction endonuclease to obtain a new transposon vector pSC-CAT. The donor vector carrying pSC-CAT The parathyroid receptor bacteria were transplanted, the chloramphenicol plate was coated on the transplanted bacteria, and the randomly selected bacterial genome was used as a template to amplify the flanking sequence of the insertion site by thermal asymmetric PCR and perform sequencing and comparison analysis. Results About 1000 mutants were screened and 6 bacteria were picked randomly. The upstream of transposon insertion site corresponded to 6 suspicious promoter regions respectively. Conclusion The promoter library of Paratyphillus can be obtained efficiently by suicide transposon vector pSC-CAT, which lays the foundation for further study on the expression and regulation of Paramyxobacteria.