Screening and validation of reference genes in Dracaena cochinchinensis using quantitative real-time PCR

Dragon's blood, the red resin derived from the wounded Dracaena, is a precious traditional medicine used by different culture. Dracaena cochinchinensis is one of the main species of Dracaena, and is the endangered medicinal plants in China. The vulnerable status severely limits the medicinal value and wide application of dragon's blood. Therefore, it's essential to analyze the mechanisms that form dragon's blood in order to increase artificial production. To clarify the mechanisms forming dragon's blood, understanding gene expression in the flavonoid biosynthesis pathway is the foundation. However, reference genes of D. cochinchinensis haven't been analyzed. In this study, expression profiles of seven commonly used housekeeping genes (Actin, alpha-EF, UBC, beta-tubulin, 18S, GAPDH, His) were evaluated by using quantitative real-time PCR combined with the algorithms geNorm, NormFinder, BestKeeper, and RefFinder. On the basis of overall stability ranking, the best reference genes were the combinations beta-tubulin +UBC for wounded stems and alpha-EF +18S + Actin for different organs. Reliability of the recommended reference genes was validated by normalizing relative expression of two key enzyme genes PAL1 and CHI1 in the flavonoid biosynthesis pathway. The results provide a foundation to study gene expression in future research on D. cochinchinensis or other Dracaena.