Selection of suitable reference genes for qRT-PCR normalization during leaf development and hormonal stimuli in tea plant (Camellia sinensis)

Tea plant (Camellia sinensis) leaf is an important non-alcoholic beverage resource. The application of quantitative real time polymerase chain reaction (qRT-PCR) has a profound significance for the gene expression studies of tea plant, especially when applied to tea leaf development and metabolism. In this study, nine candidate reference genes (i.e., CsACT7, CsEF-1α, CseIF-4α, CsGAPDH, CsPP2A, CsSAND, CsTBP, CsTIP41, and CsTUB) of C. sinensis were cloned. The quantitative expression data of these genes were investigated in five tea leaf developmental stages (i.e., 1st, 2nd, 3rd, 4th, and older leaves) and normal growth tea leaves subjected to five hormonal stimuli (i.e., ABA, GA, IAA, MeJA, and SA), and gene expression stability was calculated using three common statistical algorithms, namely, geNorm, NormFinder, and Bestkeeper. Results indicated that CsTBP and CsTIP41 were the most stable genes in tea leaf development and CsTBP was the best gene under hormonal stimuli; by contrast, CsGAPDH and CsTUB genes showed the least stability. The gene expression profile of CsNAM gene was analyzed to confirm the validity of the reference genes in this study. Our data provide basis for the selection of reference genes for future biological research in the leaf development and hormonal stimuli of C. sinensis.