Identification and evaluation of suitable reference genes for gene expression analysis in rubber tree leaf.

Gene expression profiles are increasingly applied to investigate molecular mechanism for which, normalization with suitable reference genes is critical. Previously we have reported several suitable reference genes for laticifer samples from rubber tree, however, little is known in leaf. The main objective of this current study was to identify some stable expression reference genes at various developmental stages of leaf, as well as during abiotic (high and low temperature extremes) and biotic stresses (pathogen stress). Gene expression profilings identified the ubiquitin-proteasome system as excellent potential as reference genes for rubber tree leaf. Among a total of 30 tested genes investigated, 24 new candidate (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 candidate genes had better expression stability comparing 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 for rubber tree leaf under 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.