Validation of reference genes for quantitative gene expression analysis in Ralstonia pseudosolanacearum CQPS-1 under environment stress

Abstract

Quantitative real-time reverse transcriptase PCR (qRT-PCR) has become the method choice for quantification of gene expression changes, however, the accuracy of the method depends on the stability of reference genes. Ralstonia pseudosolanacearum (R. pseudosolanacearum) is an important plant pathogen, infecting >450 plant species and causing bacterial wilt. In order to identify stable reference genes in R. pseudosolanacearum CQPS-1 under different environment stresses. We used five tools (△Ct method, GeNorm, NormFinder, BestKeeper, and RefFinder) to evaluate the stability of seven candidate reference genes including phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 16S ribosomal RNA (16S), cell division protein ftsZ (ftsZ), DNA gyrase subunit A (gyrA), Ribosomal protein L13 (rplM), and phosphoserine aminotransferase (serC) under biotic (growth phases) and abiotic stress (temperature, hydroxycoumarins, nutrition). Overall, gyrA and serC were the most stable genes under different growth phases, while serC, gyrA and ftsZ during temperature stress, gyrA, ftsZ and 16S under hydroxycoumarins stress, and serC and 16S under nutrition stress conditions. This study provides useful resources for normalizing expression changes of target genes in R. pseudosolanacearum subjected to environment stress.

Keywords: Environmental stress; Gene expression; Normalization; Ralstonia pseudosolanacearum; Reference gene; qRT-PCR.