Selection of reference genes for qRT-PCR normalization in Cestrum nocturnum during flowering

To screen stable reference genes for studies on gene expression of aroma release and the circadian clock changes in Cestrum nocturnum,six traditional reference genes,Actb7,EF-1A,GAPDH,TUA,TUB2,and UBQ,were isolated from flowers and leaves in the blooming stage by homology cloning and RACE technology.The expression patterns of 18s rRNA and the six reference genes were analysed by real-time quantitative PCR (qRT-PCR) and the expression stabilities were analysed by Bestkeeper,geNorm,and NormFinder.Results showed that the expression of Actb7 was stable in flowers;EF-1A and UBQ were stable in leaves;and EF-1A was stable in both tissues.According to the analysis of the three groups by geNorm,the optimal number of reference genes was two and the best combination was Actb7 and EF-1A.The stable reference genes screened in this study provide a basis for accurate detection of the expression of genes related to petal rhythm movement,aroma release,and circadian clock changes.