Ubiquitin-conjugating enzyme 2b (HbUBC2b) is an appropriate reference gene for gene expression analysis by reverse transcription - quantitative PCR (RT-qPCR) in the rubber tree (Hevea brasiliensis) under mechanical wounding and methyl jasmonate treatment

Abstract

The rubber tree (Hevea brasiliensis) is one of the most valuable members of the genus Hevea because it has high latex production and the latex exploited from the rubber tree is the primary source of natural rubber. Rubber trees are often damaged and exposed to pathogen attacks through wounds during and after tapping. Gene expression studies help us to gain insights into molecular mechanism underlying the responses of this plant species to stress signals such as wounding and pathogenicity. To ensure the reliability of gene expression measurements, quantitative procedures, particularly reverse transcription - quantitative PCR (RT-qPCR), require the normalization of the expression level of a gene of interest (target gene) to that of a stably expressed internal reference gene. Selecting and validating an appropriate reference gene is thus an essential step before conducting RT-qPCR experiments to look for differences in transcriptional expression of the target gene in H. brasiliensis under specific experimental conditions. Here, the transcriptional expression of ubiquitin-conjugating enzyme 2b (HbUBC2b) was measured by RT-qPCR in a set of 12 RNA samples derived from the bark of the rubber tree H. brasiliensis (clone RRIV 209) exposed to mechanical wounding, and stress signal molecules like methyl jasmonate (MeJA). With the aim of estimating PCR efficiency, and thus to include it in further analysis procedures, a standard curve assessment has been developed using HbUBC2b-specific primers and a 5-fold dilution of cDNA as template for qPCR reactions. The amplification efficiency (102,9%), as well as the R2 value (0,9992) are within the recommended range (90-110%). The HbUBC2b gene was shown to be stably expressed across control and treatment groups, and therefore could be used as a reference gene for quantitative gene expression normalization in H. brasiliensis (clone RRIV 209) under mechanical wounding or methyl jasmonate treatment.