Selection and verification of candidate reference genes for gene expression by quantitative RT-PCR in Hibiscus hamabo Sieb.et Zucc.

Abstract

Ten candidate reference genes were examined in Hibiscus hamabo Sieb. et Zucc. ACT and SKIP are proposed as good reference genes for gene studies in Hibiscus hamabo Sieb. et Zucc. Because of its sensitivity and rapidness, quantitative real-time PCR (qRT-PCR) is currently extensively used to analyze gene-expression patterns. Selecting suitable reference genes to normalize qRT-PCR results is essential. Hibiscus hamabo Sieb. et Zucc. (H. hamabo) is a semi-mangrove plant that is widely used for the ecological restoration of saline-alkali land and coastal afforestation owing to its excellent salt tolerance. However, suitable reference genes used for the normalization of H. hamabo qRT-PCR data have not been selected or verified. Here, we tested the expression stabilities of ten candidate reference genes in different H. hamabo tissues under a set of abiotic stresses (salt, drought, high temperature, and low temperature) and hormonal treatments (methyl jasmonate, abscisic acid, and salicylic acid) using three statistical algorithms, i.e., NormFinder, geNorm, and BestKeeper. Actin (ACT) and ski-interacting protein (SKIP) can be regarded as good choices as reference genes in studying gene expression of H. hamabo. In addition, the qRT-PCR analysis of the NAC (NAM/ATAF1/2/CUC2) target gene’s expression pattern under NaCl-treated conditions confirmed the suitability of selected reference genes. Here, we used qRT-PCR technology to provide a stable reference gene list for H. hamabo gene-expression studies.