副溶血弧菌lux报告基因系统的建立与应用

Construction and application of the lux reporter assay platform in Vibrio parahaemolyticus

目的:建立副溶血弧菌的lux报告基因融合实验平台,为后续研究基因转录调控机制奠定基础。方法:采用PCR扩增opaR的启动子区序列,并将其克隆入pBBRlux质粒中,构建重组质粒opaR-lux。将opaR-lux重组质粒分别转入野生株(WT)、opaR突变株(ΔopaR)和aphA突变株(ΔaphA)中,检测特定培养条件下三者发出的冷光值(Lux)以及在600 nm处的吸光度值(OD600),以“Lux/OD600”表示单位OD600下的相对平均冷光单位(relative light unit,RLU)。通过比较各菌株的RLU大小,判断OpaR和AphA对opaR的调控关系,以检验实验平台的稳定性。结果:成功构建出带有opaR的pBBRlux重组质粒;在低密度(OD600<0.4)时,AphA负调控opaR的转录,当细菌达到高密度(OD600=0.8)时,AphA则对opaR的转录不起调控作用;在细菌从低密度生长到高密度(即OD600<0.8)中,OpaR对自身的转录存在负调控作用。结论:成功建立了副溶血弧菌的lux报告基因融合实验平台,用于后续转录调控机制的研究。

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.