Validation of reference genes for real-time quantitative polymerase chain reaction analysis in Lactobacillus plantarum R23 under sulfur dioxide stress conditions

Abstract

Background and Aims Malolactic fermentation (MLF), accomplished by lactic acid bacteria (LAB), is indispensable for the production of some red wines. As the key additive for wines, sulfur dioxide (SO2) frequently affects LAB growth and MLF development. We aimed to investigate suitable reference genes in Lactobacillus plantarum R23, an excellent LAB with good tolerance to SO2, for a real-time quantitative PCR analysis of SO2 tolerance. Methods and Results The expression of eight candidate reference genes under SO2 stress conditions for L. plantarum R23 was analysed, and geNorm and BestKeeper software packages were employed to assess the reference genes. The expression of mleR1 and mleP1 under SO2 stress conditions was used to confirm the results. The geNorm analysis indicated that the stability ranking of reference genes was rpoB > rpoC > recA > ldh > cfal > mtlR > asd2 > gpp. Both software packages suggested that four genes should be selected for accurate normalisation, and the validated experiment further confirmed the suitability of the reference genes revealed in this study. Conclusions The genes rpoB, rpoC, recA and ldh were the most stably expressed reference genes and thus represent the reference gene combination that should be used to obtain the most accurate results for different SO2 stress conditions in L. plantarum R23. Significance of the Study This is the first detailed study to evaluate selected reference genes in L. plantarum R23 under SO2 stress conditions, which should allow the quantification of bacterial gene expression levels under SO2 stress conditions and also provide a foundation for the more accurate use of real-time quantitative PCR in L. plantarum.