Gene expression profiling is increasingly applied to investigate molecular mechanisms for which, normalization with suitable reference genes is critical. Previously we have reported several suitable reference genes for laticifer samples from rubber, however, little is known about reference genes in leaf.
The main objective of this current study was to identify some reference genes with stable expression patterns in leaf at various developmental stages, as well as during abiotic (temperature extremes) and biotic stresses. Gene expression profiling experiments in rubber tree leaf identified the ubiquitin-proteasome system as having excellent potential as reference genes. Among a total of 30 tested genes investigated, 24 new (including 11 genes involved in the ubiquitin-proteasome system), 4 previously identified and 2 specific genes, were further evaluated using quantitative real-time PCR. Our results indicated that the new genes had better stability of expression when compared with others. For instance, an ubiquitin conjugating enzyme (RG0099) and three ubiquitin-protein ligases (RG0928, RG2190 and RG0118) expressed stably in all samples, and were confirmed to be suitable reference genes in rubber tree leaf in four different conditions. Finally, we suggest that using more than one reference gene may be appropriate in gene expression studies when employing different software to normalize gene expression data.
Our findings have significant implications for the reliability of data obtained from genomics studies in rubber tree and perhaps in other species.