Construction And Preliminary Study On The Biological Function Of MarC Gene In Trueperella Pyogenes

Trueperella pyogenes is a conditional pathogen that can cause diseases such as mastitis and endometritis in domestic animals,leading to threaten to the breeding industry.Antibacterial drugs are normally used to treat diseases caused by pathogenic bacteria.However,due to the unreasonable use of antibacterial drugs,the drug resistance emerged in bacteria,such as T.pyogenes.The multidrug resistance family protein(Mar C)is a transmembrane protein that plays an important role in the transport of substances.Studying the biological function of the cryptic bacillus mar C gene can be useful in revealing the resistance mechanism and pathogenic bacterium.This can provide a basis for the prevention and treatment of diseases caused by T.pyogenic.In this study,the wild strain of T.pyogenes isolated and identified by researchers in our laboratory was used.The upstream and downstream fragments of the mar C gene were used as homologous fragments for homologous recombination in the shuttle vector p BAV1K-T5-gfp to construct the recombinant plasmid p BAV1K-T5-gfpΔmar C;The recombinant plasmid p BAV1K-T5-gfpΔmar C was transferred into the competent cells of T.pyogenes by electroporation,and the lost gene was screened by passage after the plasmid was lost,and PCR and whole genome sequencing were used.The mar C gene mutation strain was identified;the changes of growth,drug resistance,biofilm and morphology,and transcription level of T.pyogenes between parent strain and the mutant were compared.PCR and digestion of the recombinant plasmid indicated that the recombinant plasmid was successfully constructed.The sequencing results showed that there was no base mutation between the homologous fragment on the recombinant plasmid and the upstream and downstream fragments of the mar C gene in the isolate.PCR identified the mar C gene of the suspected T.pyogenes knockout strain,PCR amplification and sequencing with the upstream primer of the upstream homologous arm and the downstream primer of the downstream homology arm,and the result was a 1818 bp target band.Compared with the 2439 bp of the original strain,the lack of the 621 bp mar C gene,the whole genome sequencing of the suspected mar C gene deletion strain,and no mar C gene was amplified,so the mar C gene mutation strain of the wild strain of T.pyogenes was successfully constructed.The results indicated that the mar C gene had no effect on the growth and minimum inhibitory concentrations of T.pyogenes.The biofilm formation ability of T.pyogenes was quantitatively analyzed by crystal violet staining.The results showed that the biofilm formation ability of the mutant was significantly reduced.Staining of living bacteria in the formed biofilm revealed that the total amount of bacteria in the mar C mutation strain was reduced.The proportion of dead bacteria increased;transcriptome analysis showed that the m RNA expression levels of rbs B,mar R and mox R genes related to biofilm formation were significantly up-regulated,and the energy-dependent ABC transport family in multidrug resistant efflux family proteins The expression levels of protein and SNF family transporters were significantly up-regulated.It is speculated that the significant decrease in biofilm formation ability in the mutant may be due to a significant up-regulation of m RNA expression levels of the rbs B,mar R and mox R genes.Transcriptomic differential expression gene analysis showed 66 differentially expressed genes,23 differentially expressed genes were down-regulated,and 43 differentially expressed genes were down-regulated.Enrichment resulted in four differentially expressed metabolic pathways.