As a tool for breeding new floral colors into azalea, we obtained basic gene information for flavonoid synthesis in floral tissues of azalea (Rhododendron × pulchrum Sweet cv. Oomurasaki). The partial- or full-length cDNA sequences of eight structural genes involved in the flavonoid biosynthetic pathway were isolated: chalcone synthase (CHS), chalcone isomerase (CHI), flavonone-3-hydroxylase (F3H), flavonoid-3′hydroxylase (F3′H), flavonoid-3′,5′-hydroxylase (F3′5′H), dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS). These genes were isolated from petals of R. × pulchrum by PCR using degenerate and gene-specific primers and their expression levels were analyzed. Deduced amino acid sequences of obtained genes showed 76-80% identities with the corresponding flavonoid biosynthetic pathway sequences from other dicotyledonous species. Expression of these eight genes during azalea flower development was investigated. A real-time PCR analysis showed that transcripts of CHS, F3H and ANS genes decreased as flowering progressed, and those of CHI, DFR, F3′H, and FLS genes transiently increased at the stage of pigmentation initiation. In addition, maximum expression of the F3′5′H gene was expressed concomitant with anthocyanin synthesis. Spatial expression showed that the CHS, DFR, and FLS genes are more abundant in other floral organs or leaves than petals. The levels of CHI, F3H, ANS, and F3′H gene transcripts were highest in leaf tissue compared with floral organs. The highest level of F3′5′H expression was in the petal at stage 3. These results indicated that F3′5′H gene is strongly correlated to pigmentation in azalea petals, but expression of other genes are unclear for anthocyanin synthesis.
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