Cloning, characterization and expression of carnation (Dianthus caryophyllus L.) ubiquitin genes and their use as a normalization standard for gene expression analysis in senescing petals

We cloned seven cDNAs coding for ubiquitin (polyubiquitin) (DcUbq1-7) from carnation petals: DcUbq1, 2, 3 encoded polyubiquitins consisting of five ubiquitin monomers; DcUbq4, three monomers and DcUbq5, 6, 7, a monomer. The 3'-UTR nucleotide sequences were separated into three groups; two were specific to DcUbq1 and DcUbq2, respectively, and the third was almost always common to other genes (DcUbq3-7). The transcript levels of DcUbq1 and DcUbq2 in petals fluctuated during flower opening, whereas those of DcUbq3-7 remained unchanged except for an increase in the last stage. On the other hand, during flower senescence, the transcript levels of DcUbq1 and DcUbq2 increased at later stages, and those of DcUbq3-7 remained almost constant during the process. Based on these findings, we suggest an association of ubiquitin gene expression with petal growth during flower opening and petal wilting during the senescence of carnation flowers through the degradation of specific proteins by the ubiquitin-proteasome system. Furthermore, we showed the successful use of DcUbq3-7 transcripts as a normalizing standard in the determination of transcript levels of a target gene in senescing carnation petals, where massive degradation of RNA, such as actin mRNA and rRNA, usually occurs, causing inaccuracy in the estimation of transcript levels of interest.