Isolation and characterisation of a melon cDNA clone encoding phytoene synthase

A cDNA clone (MEL5), encoding a protein homologous to phytoene synthase (PSY), has been isolated from a climacteric melon fruit cDNA library, using the tomato cDNA clone TOM5 [34] as a heterologous probe. MEL5 hybridised to a transcript of 1.65 kb which suggested that the 1.36 kb clone, isolated originally, was not full-length. The missing 5' end was isolated by a reverse transcriptase-polymerase chain reaction (RT-PCR)-based method. This enabled the full sequence of the protein to be deduced and the cleavage site of the transit peptide for chromoplast import to be predicted. Northern analysis of RNA extracted from fruit samples of different ripening stages as well as from roots, leaves and flower petals was used to examine the expression pattern of the corresponding mRNA. The corresponding mRNA. The transcript corresponding to MEL5 is present at low quantities in unripe (green) fruit, reaches its highest levels when the fruit turns from green to orange and persists at lower levels during later ripening stages. A similar transcript was also detected in flower petals and in trace amounts in leaves and roots. Genomic Southern analysis indicates that the clone is homologous to a low-copy-number gene family. Sequence analysis showed a high degree of conservation among plant PSYs.