Selection of appropriate reference genes for quantitative real-time PCR in Clerodendrum trichotomum

AbstrsctClerodendrum trichotomum, an important medicinal plant, has excellent salt tolerance and beautiful ornamental character. However, reliable reference genes for quantitative real-time PCR data (qRT-PCR) in C. trichotomum have not been investigated. Using our previous transcriptome data, 17 reference genes were selected in different tissues (leaves, flowers, fruits, stems, and roots) and under various abiotic stresses (salt, drought, flood, and heat) for C. trichotomum, using four different reference gene analysis software types: GeNorm, NormFinder, BestKeeper and ReFinder. The results identified RPL, ACT and HSP70 as the three most suitable reference genes for tissues. Genes ACT and AP-2 were most stably expressed under drought stress; MDH and UBCE2 were stable under flooding stress; RPL and UBCE2 were most stable under salt stress; and MDH and EF-1A were most appropriate under heat stress. For abiotic treatments, RPL, MDH and AP-2 were the most stable reference genes; and AP-2, RPL and ACT were stably expressed in all examined samples. The expression profile of the genes for Na+/H+ Exchanger1 (ClNHX1) and laccase (ClLAC) were selected to validate the stability of the determined reference genes. Our study provided reliable normalization for gene expression analysis and ensured more accurate data for further molecular mechanism research in C. trichotomum.