Identification of differentially expressed genes in the flesh of blood and common oranges

The objective of this research was the identification of genes differentially expressed in blood oranges compared to common oranges and the identification of anthocyanin pathway genes that are up-regulated in flesh of blood oranges. A subtracted complementary DNA library of 1,248 clones was constructed using RNA from the flesh of a nucellar line (58-8D-1) of Moro (a blood orange) as tester and from Cadenera (a common orange) as driver. After screening by reverse Northern, a total of 230 clones were found to be up-regulated in blood orange, while 30 were up-regulated in the common blond one. Sequence analysis identified genes involved in the anthocyanin pathway including genes encoding biosynthetic enzymes like phenylalanine ammonialyase, chalcone synthase, dihydroflavonol- 4-reductase, anthocyanidin synthase, UDP: glucose flavonoid 3-O-glucosyltransferase, glutathione S-transferase, and a regulatory gene encoding a basic Helix- Loop-Helix protein, while others were related to primary metabolism, flavor biosynthesis, signal transduction mechanisms, and defense. Some sequences were classified as unknown and unnamed and some others were unclassified. Semiquantitative reverse transcription-polymerase chain reaction (PCR) and quantitative real-time PCR were used to confirm the differential expression patterns of selected candidate genes of different functional classes. Correlations between the expression of some genes and the processes involved in the ripening of blood oranges were identified.