Cloning and analysis of RcAG promoter in Rosa chinensis ‘Viridiflora’

Rosa chinensis ‘Viridiflora’ is a valued Chinese garden rose cultivar and represents an important rose germplasm resource, in which the petals, stamens, and pistils are converted into sepal-like organs (i.e., sepalody). Expression of the RcAG gene in flower tissues of the R. chinensis ‘Viridiflora’ and ‘Old Blush’ varieties was analyzed by real-time quantitative polymerase chain reaction (PCR). Results showed that the expression level of RcAG was down-regulated in R. chinensis ‘Viridiflora’. To explore the reason for its down-regulation, the promoter sequences of RcAG were cloned in ‘Viridiflora’ and ‘Old Blush’, respectively. Results showed that the two promoter sequences contained TATA-box, TATC-box, and MBS cis-elements. Notably, MRE and Circadian were specifically found in the RcAG promoter in ‘Viridiflora’, while the TCT motif was only found in ‘Old Blush’. Furthermore, double sulfite sequencing technology was used to analyze promoter methylation. Results showed that all four CpG sites in the promoter region of ‘Viridiflora’ were methylated, and the degree of methylation was much higher than that of ‘Old Blush’. Our findings suggest that the down-regulation of the RcAG gene in ‘Viridiflora’ may be related to its promoter cis-elements and methylation.