Selection and verification of reference genes for real-time quantitative PCR in endangered mangrove species Acanthus ebracteatus under different abiotic stress conditions

Acanthus ebracteatus is an endangered true mangrove species with great ecological and medicinal values. Real-time quantitative PCR (RT-qPCR) has been widely used to investigate transcriptional responses in A. ebracteatus, which can facilitate its protection and medicinal usage. However, lack of prior knowledge on the optimal reference genes for RT-qPCR data normalization of A. ebracteatus, especially under stress scenarios, restricts gene expression investigations of this species. To address this issue, we evaluated the expression stability of seven candidate reference genes (ACT, PP2A, TUB, TUA, UBQ, EF-1α and RPS13) in leaves of A. ebracteatus upon heat, cadmium (Cd), drought, cold, flood and salt stress, respectively, using four state-of-the-art methods, GeNorm, NormFinder, BestKeeper and RefFinder. The results indicated that ACT was the most stably expressed in most scenarios, while EF-1α, PP2A and TUB ranked first under Cd, flood and salt stress, respectively. TUB was also the suboptimal reference gene for the samples exposed to drought and cold stress, and ACT was the second-best for Cd stress. For all the examined stress conditions, a combination of two reference genes was considered to be adequate enough for accurate expression standardization. A functional gene FLA17 was further employed to validate the performance of the candidate reference genes. The expression profiles of FLA17 displayed similar trends when using the top two stable reference genes, but were under- or overestimated when normalized by less stable genes, indicative of the importance of choosing the optimal reference genes for RT-qPCR normalization. Our findings provide a foundation for future gene expression studies of A. ebracteatus.