Clonging and molecular characterization of ripening-related ACC synthase from papaya fruit (Carica papaya L.)

Papaya is a climacteric fruit and like other climacteric fruits in the tropics, one of the main problems is the significant post-harvest loss due to uncontrolled ripening. One molecular strategy to control ripening after the climacteric rise in ethylene is to down-regulate ethylene biosynthesis through antisense expression of the ripening-related ACC synthase gene. Total RNA was isolated from 100% ripe papaya fruit and reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify ripening-related ACC synthase cDNAs. Two pairs of degenerate primers (oligonucleotides) derived from conserved regions of ACC synthase from several plant sources were used for RT-PCR. Two different members of the ACC synthase multigene family were isolated and characterized from ripe tissues of papaya whose expression patterns differ greatly from each other. The two ACC synthase cDNAs, ACS1 and ACS2 differ from each other in terms of restriction digest patterns, DNA and amino acid sequence analysis and expression during ripening. ACS2 expression pattern in papaya cv Solo Kapoho exhibits a striking similarity to that of leacs2 in tomato whose transcript levels are high during ripening. ACS2 is a good candidate gene of ACC synthase for antisense strategies to regulate or control fruit ripening. ACS2 was inserted in an antisense orientation in two types of vector constructs for papaya transformation via particle bombardment