Identification and validation of reference genes for qPCR of Pseudomonas aeruginosa L10 under varying n-hexadecane concentrations.

Pseudomonas aeruginosa can efficiently degrades petroleum hydrocarbons, but its degradation rate is affected by hydrocarbon pollutant concentrations, which must be neither too high nor too low. To quantitatively analyze P. aeruginosa's gene expression and assess factors affecting it, reference genes stably expressed under varying concentrations of n-hexadecane stress need to be identified. In this study, we used P. aeruginosa L10 as the test strain and identified eight candidate reference genes: rpsL, nadB, clpX, anr, rpoS, gyrA, tipA, and recA. The cultures were cultured under different concentrations of n-hexadecane (the concentrations were 0%, 0.1%, 0.5%, 1.0%, 1.5%, 2.0%,), and then the gene expression was detected by quantitative Real-time PCR, that is, the changes of Ct values. Using geNorm, Normfinder, BestKeeper and RefFinder to conduct analysis. Among, geNorm measures the expression stability of a candidate gene by calculating the stability value of gene expression. Normfinder calculates gene stability values taking into account within- and between-group variation in gene expression. BestKeeper evaluates the stability of individual genes through correlation analysis. RefFinder provides a comprehensive assessment of reference gene stability by integrating four methods. The results indicated that among the reference genes of P. aeruginosa L10 cultured under various concentrations of n-hexadecane stress, anr had the highest Ct value and expression abundance, while tipA had the lowest. GeNorm analysis showed that nadB, anr and rpsL were more stable, while tipA and gyrA were less stable. Normfinder analysis showed that nadB, anr, gyrA and rpsL were more stable, while tipA was less stable. The analysis results of BestKeeper were different from the previous two analysis tools. Further comprehensive analysis using RefFinder showed that nadB and anr were the most stable parameters, and tipA was the most unstable. In different treatments, the most stable internal parameters are not same. After verification and analysis, nadB and anr were the two most stable internal reference gene, and tipA was the least stable. The excavation of stable internal reference can help the application of qPCR in the determination of gene expression of P. aeruginosa L10, and thus lay a foundation for the study of related genes or metabolic pathways.