Fuctional Study Of LuxR’Solo’Homologue Gene And Prokaryotic Expression, Purification Of Expr Protein From Erwinia Amylovora Strain Ncppb1665

Fire blight, caused by Erwinia amlyvora, is one of the most devastating and difficult-to-control diseases.until now,The fire blight bacteria pathogenic Mechanism has been extensively studied, laid a solid foundation for further effective prevention and control of fire blight.Bacteria are social microbes that can interact with the adjacent bacteria in the same niche.QS (Quorum sensing) is an intricate regulatory system depend on cell density controlling the virulence biosynthesis of exoenzymes,motility,nutritional intake, and biofilm formation.QS represents a major mode that gram-negative bacteria regulate genes expression.LuxR is a widespread and functional diverse transcription factors and belongs to TetR protein superfamily. It could both activate and inhibit the expression of many genes contingent on the contexts, thereby involving in many crucial physiological events, such as virulence factors production, biofilm formation, quorum sensing (QS), acetate metabolism, motility, bioluminescence, and ecological competition.We summarized the function diversity of LuxR and underlying mechanisms. Transcription factors are proteins specifically binding to DNA sequences to ensure that the optimum spatiotemporal expression of target genes via enhancement or attenuation.LuxR is a member of TetR protein superfamily transcriptional factor widespread among gram-negative bacteria containing a helix-turn-helix in C-terminal of the DNA binding domain.In this paper, a luxR solo homologous gene was amplified by PCR from Erwinia amylovora, A luxR disruption mutant EaAluxR was successfully generated by homologous recombination,and studied the function preliminarily.The experimental results show that, compared to wild-type strain, the EPS, anti-oxidative stress, growth, and pathogenicity of mutant EaAΔluxR decrease;However,still EaΔluxR cause tobacco hypersensitive response,and no significant difference in antibiotic tolerance level and biofilm formation comparing to wild-type strain.These findings demonstrated that luxR gene in Erwinia amylovora is involved in EPS, growth, and anti-oxidative stress and play an important role in virulence.Quorum sensing has been widely employed by a variety of bacterial species to coordinate communal behavior.The LuxI-LuxR protein is the most importment protein of Gram-negative bacteria.LuxR protein is the receptor of AHLs and also causes express of a series of relevant genes.In this study,based on existing research work in this laboratory, EaexpR gene coding region was amplified by PCR.With restriction endonuclease and sequence analysis, and cloned into the prokaryotic expression vector pET28a.The recombinant plasmid pET28a22801,was transformed into E.coli Rosepta (DE3) pLysS competent cells and induced by IPTG to express the fusion protein about28kDa.The protein exists inclusion bodies and supernatant.