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

Abstract

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.

Cloning and Expression of cDNAs for Biosynthesis of Very-long-chain Fatty Acids, the Precursors for Cuticular Wax Formation, in Carnation (Dianthus caryophyllus L.) Petals

“…DcHCD1 transcripts were detected faintly in petals, calyx, leaves, and stem. DcECR1 transcripts were detected in all the transcripts were used to ensure equal loading of DNA samples (Nomura et al, 2012).…”

“…The cDNAs were amplified with Taq DNA polymerase under the conditions of 96°C for 3 min followed by 38 cycles of 30 s at 96°C, 30 s at 53°C, and 1 min at 72°C. DcUbq3-7 cDNAs (Nomura et al, 2012) were also amplified as standard under the conditions of 94°C for 3 min followed by 36 cycles of 30 s at 94°C, 30 s at 53°C, and 1 min at 72°C. The primer sets for amplification were the same as those used for real-time RT-PCR.…”

Identification and Characterization of Plasma Membrane Intrinsic Protein (PIP) Aquaporin Genes in Petals of Opening Carnation Flowers

“…The transcript level was calculated using a dilution series of standard plasmids. The transcript level of ubiquitin genes (DcUbq3 to DcUbq7; Nomura et al, 2012) was also measured and used for normalization of the PIP transcript level. Analyses were conducted with three independent replicates for each sample, and data were given by the mean ± SD.…”

Role of ABA in Triggering Ethylene Production in the Gynoecium of Senescing Carnation Flowers: Changes in ABA Content and Expression of Genes for ABA Biosynthesis and Action

“…The conditions were 96°C for 3 min followed by 38 cycles of 30 s at 96°C, 30 s at 53°C, and 1 min at 72°C. DcUbq3-7 cDNAs (Nomura et al, 2012) were also amplified as a standard under the conditions of 94°C for 3 min followed by 36 cycles of 30 s at 94°C, 30 s at 53°C, and 1 min at 72°C. The amplificates were separated by agarose gel electrophoresis, stained with ethidium bromide and photographed.…”

“…5). The transcript level of DcUbq3-7 was almost constant throughout the stages and tissues, and was used to normalize the transcript level (Nomura et al, 2012).…”