Gene expression profiling of key enzymes in azalea flower colour biosynthesis

Author(s): De Keyser E, De Riek J, Van Bockstaele E


Azalea flower colour is mainly determined by two groups of pigments, anthocyanins and flavonols. Genes coding for two key enzymes in the biosynthesis of these pigments, chalcone synthase (chs) and dihydroflavonol-4-reductase (dfr) were isolated from an azalea cDNA library and fully characterised. The expression of these two genes in the petals of 8 azalea flower colour sports of the ’Hellmut Vogel’ sporting series will be used as a model for the development of a real-time RT-PCR protocol for gene expression analysis in azalea. Real-time RT-PCR is currently the most sensitive method, especially suitable because very little amounts of RNA are sufficient. Optimisation was needed at all crucial steps from RNA isolation up to the final quantification. The most critical step for the whole quantification process was the selection of the appropriate housekeeping genes. In previous analysis, only GAPDH was applied for this purpose, but at least two housekeeping genes are recommended. Starting from the ‘Flamenco’ cDNA library, twelve candidate housekeeping genes were selected and primers suited for real-time PCR were developed. All genes were tested together with GAPDH in real-time PCR. By using the geNorm software we could state that the use of three housekeeping genes will be sufficient for expression analysis in the ‘Hellmut Vogel’ sporting series. In the end the expression of chs and dfr was determined and the qBase software was used to find a correlation between gene expression and azalea flower colour.

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