Selection and Validation of Suitable Reference Genes for Gene Expression Studies in Callerya speciosa(Champ. ex Benth.) Schot under Different Experimental Conditions

The accuracy of reverse transcription quantitative real-time PCR (RT-qPCR) strongly depends on the stability of reference gene. It is essential to select a suitable reference gene in order to obtain reliable RT-qPCR results when gene expression profiles were evaluated. Callerya speciosa, a traditional Chinese medicine, has a long cultivation history in south China. However, suitable reference genes in C. speciosahave not yet been investigated for accurate gene expression quantification under different experimental conditions. In this study, eight candidate reference genes, including GAPDH(glyceraldehyde-3-phosphate dehydrogenase), 60S(60S ribosomal protein L34), ACTIN(actin), TUA2(tubulin alpha chain), TUB1(tubulin beta-1 chain), TIF5(eukaryotic translation initiation factor 5A), UBQ(polyubiquitin), and EF2(elongation factor 2), were selected from the transcriptome databases, and their expression stability under six different experimental conditions (developmental stages, tissues, MeJA treatment, GA3treatment, CPPU treatment, and PP333treatment) was evaluated using ΔCT, geNorm, NormFinder, BestKeeper, and RefFinderprograms. The results showed that GAPDHwas the optimal reference gene for all different experimental conditions, whereas ACTINshowed the best stability under most of the hormone treatments. TUA2and EF2were the two least suitable ones as reference genes in C. speciosa. The expression pattern of CsMYB36, a gene associated with the regulation of isoflavonoid biosynthesis in C. speciosa, verified the accuracy of our experimental results. These results provided a theoretical basis for subsequent research on the regulation of functional gene expression in C. speciosaand other Calleryaspecies in the future.