Screening of Reference Genes for qPCR of Pseudomonas fluorescens under Exogenous AHLs Culture
Objective:Pseudomonas flluorescens is the dominant spoilage organism in frozen foods,and its spoilage-caus-ing genes are regulated by quorum sensing system.In order to accurately quantify the expression of spoilage genes and thus investigate the mechanism of quorum sensing regulation in food spoilage,it is required to screen reference genes of Pseudomonas fluorescens.Methods:Using Pseudomonas fluorescens PF-08 as the research object,eight common reference genes(dsbA,carA,rpsL,gyrB,atpD,rpoD,gltA,16S rRNA)were selected and their gene expression was measured by quantitative real-time PCR(qPCR)after incubation with different types of quorum sensing signal molecules.geNorm,Normfinder,BestKeeper and RefFinder were used to evaluate the expression stability of the candidate reference genes and to screen out the most appropriate reference genes.Results:Under different types of exogenous signal molecules incuba-tion,the least change in Ct value of Pseudomonas fluorescens was gltA and the Ct value of 16S rRNA was too low.The most stable reference genes by geNorm analysis were atpD and rpoD and the combination of the two could more accu-rately quantify the expression levels of target genes.gltA was the most stable reference gene analyzed by Normfinder and BestKeeper.Further comprehensive evaluation with RefFinder showed rpoD and gltA were the most stable reference genes.Conclusion:Both rpoD and gltA were stably expressed in Pseudomonas fluorescens after incubation with different QS sig-nal molecules.They could be used for the subsequent study of Pseudomonas fluorescens spoilage gene expression,and could also provide reference genes for studying the expression of QS-related genes in other spoilage bacteria.
NETL
NSTL
万方数据
