Identification of stable reference genes for qPCR analysis of gene expression in Oocystis borgei under various abiotic conditions

The green microalgae Oocystis borgei has remarkable potential for improving aquaculture water quality, particularly in terms of dissolved nitrogen. However, the mechanisms underlying nitrogen assimilation in this species remain poorly understood. The glutamine synthase (GS2) gene plays a crucial role in the nitrogen metabolism pathway in plants. To accurately reflection GS2 expression levels in O. borgei under abiotic conditions, it is essential to select stable reference genes for qPCR analysis. This study assessed the expression stability of nine reference genes (EF1α, RPL7, UBCE, GAPDH, 18S, rbcL, β-TUB, UBQ, and RPS27) of O. borgei under five abiotic conditions (temperature, light intensity, salinity, nitrogen concentration, and carbon concentration) based on the analysis results calculated by the four algorithms of ΔCt, GeNorm, NormFinder, and BestKeeper, which were ranked by ReFinder. The results demonstrated that EF1α and UBCE for temperature treatment, UBCE and RPS27 for light intensity and salinity treatment, RPS27 and RPL7 for nitrogen concentration treatment and the pooled sample, and UBCE and UBQ for carbon concentration treatment were the ideal reference genes combination for O. borgei, respectively. Additionally, the relative expression levels of GS2 under different abiotic conditions was detected and compared to verify the validity of selected reference genes and the results showed that the expression of GS2 in O. borgei was significantly affected by all experimental factors. Our study was helpful to acquire the accurate data of nitrogen assimilation related gene expression during aquaculture water quality control based on microalgae.