Construction and application of the lux reporter assay platform in Vibrio parahaemolyticus
Objective:To construct the lux fusion assay platform for investigating gene regulation in Vibrio parahaemolyticus,and lay a foundation for further research of gene transcriptional regulation mechanism.Methods:The entire promoter region of opaR was amplified by PCR and then cloned into the vector of pBBRlux,yielding the recombinant opaR-lux plasmid.The recombinant plasmid was introduced into the wild type(WT)strain,as well as the opaR mutant(ΔopaR)and aphA mutant(ΔaphA).Subsequently,luminescence(Lux)and the OD600 values were measured under specific culture conditions.The relative light unit(RLU)was calculated using the formula:Lux/OD600,which represents the change in the lux relative to the OD600 values of cells.Consequently,the stability of the lux fusion assay platform can be evaluated by evaluating the regulatory relationship between OpaR and AphA on opaR based on their respeactive RLU differences.Results:The recombinant plasmid pBBRlux with opaR was successfully constructed.At low cell density(OD600＜0.4),AphA negatively regulated the transcription of opaR;however at high cell density(OD600=0.8),AphA did not exert any regulatory effect on the transcription of opaR.In contrast,OpaR consistently exhibited a negative regulatory effect on its own transcription from low cell density to high cell density(OD600＜0.8).Conclusion:The lux fusion assay platform in Vibrio parahaemolyticus has been successfully established,providing a valuable tool for future studies on transcriptional regulation mechanisms.
万方数据
维普
