rpoBand efpare stable candidate reference genes for quantitative real-time PCR analysis in Saccharopolyspora spinosa

AbstractSpinosad (spinosyn A and spinosyn D), the secondary metabolite produced by Saccharopolyspora spinosa,is a potent insecticide with low effects on the environment and mammals. Strategies such as metabolic engineering, mutagenesis and fermentation process optimization have been employed for its production enhancement. Quantitative real-time polymerase chain reaction(qRT-PCR) is one of the preferred methods for the evaluation of gene transcript levels, whose accuracy and sensitivity depend on the normalization using optimal reference genes. However, no single reference gene is universally appropriate for all strains under various conditions. In this study, the transcriptional stability of 35 candidate reference genes, including 23 traditionally used reference genes and 12 novel reference genes identified in S. spinosahomologous species, was analysed in S. spinosaATCC 49460 and spinosad high-yield strain S. spinosaS3-3 under three fermentation phases. The transcriptional stability of these genes was assessed by three statistical algorithms, geNorm, NormFinder and BestKeeper. The overall rankings suggested that rpoBand efpwere the most stable candidate reference genes, and they may be the most promising reference genes for further study on the measurement of expression levels of target genes involved in the biosynthetic process of spinosad.