Quantitative expression analysis of geranylgeranyl pyrophophate synthase gene in Oenococcus oeni under different ethanol stresses.

Background Oenococcus (O.) oeni has long been reported as the agent most commonly associated with the MLF and the LAB species most resistant to the presence of ethanol in wine. Objectives The effect of ethanol stress on the expression level of geranylgeranyl pyrophophate synthase gene (GGPPs) was investigated by real-time RT-qPCR to obtain a clearer picture of its function and role in the metabolism of O. oeni. Methods O. oeni cells treated with different ethanol concentrations (7%, 12%, 13%, 15%) and cells grown in absence of ethanol (control) were used in this study. The identification and evaluation of a panel of ten reference genes for real-time PCR normalization was performed. The ten control genes were ranked according to their stability of gene expression measure (M) and the coefficient of variation (V) using geNorm provided by qbase_plus software (Biogazelle). Once stable internal control genes were identified, the expression level of the target GGPPs gene was analyzed by using the geometric mean of copy numbers as normalization factor. Conclusions The quantitative expression gene analysis showed that various changes in the transcription level of the GGPPs gene appeared in response to different ethanol concentrations. Particularly, a significant increase of the expression level in the sample stressed with ethanol 12%, 13% and 15% occurred, while there was no transcriptional increase in the presence of 7% ethanol if compared to the control.