Expression And Regulation Of EF-P And Cloning Analysis Of Ef-p Promoter In Kocuria Rhizophila

EF-P(Elongation factor P)is a translation elongation factor commonly conserved in bacteria.Due to its L-shaped structure similarly to tRNA,EF-P can rescue polyproline-mediated translational pausing.Although it is not an essential protein in bacteria,it is crucial for bacterial fitness and virulence of some pathogens.At present,the research of EF-P focuses on its origin,structure,function,mechanism and biological significance.Until now,little literature has been found on expression regulation of EF-P.Based on the commonly drug sensitive target bacteria Kocuria rhizophila as test materials,the expression of EF-P and its promoter sequence under environmental factor stress are studied by modern molecular microbiology methods,such as real time fluorescence quantitative PCR in this study.The main contents and results are as follows:1 Based on qRT-PCR,we analyzed the effects of environmental factors(temperature,pH and some antibiotics)on the expression of EF-P in K.rhizophila.The results showed that the expression of EF-P was adjustable.It was significantly influenced by environmental factors.The expression of EF-P was steadily up-regulated under the optimal growth conditions,while the expression of EF-P was up-regulated at a short period and down-regulated followed when it on the stress state.2 We were used bioinformatics method to analyze the whole genome sequence of the K.rhizophila DC2201,and results show the promoter of ef-p which region was located within the upstream 210 bp of the ef-p gene.3 Using AcGFP1 as the reporter gene and pET-32a(+)as the skeleton plasmid,the prokaryotic promoter probe vector pET-32a(+)*:AcGFP1 was constructed by DNA in vitro recombination method.The ef-p gene promoter fragment obtained from the clone was transformed into the host cell Rosetta(DE3),then detected the fluorescent expression of reporter gene.Analysis and determination according to the results,the upstream 210 bp is the ef-p promoter.The results may provide the experimental basis for elucidating the molecular mechanism of EF-P's transcriptional regulation in K.rhizophila.