| An ACC synthase fragment was isolated from 30% yellow papaya (Carica papaya L.) fruit using reverse a transcriptase polymerase chain reaction (RT-PCR). Six degenerate oligonucleotides, specific to highly conserved domains of ACC synthase sequence, were used as primers for the RT-PCR. A fruit-expressed ACC synthase cDNA was isolated, from a cDNA library of 30% yellow-fruit, using the RT-PCR product as a random labelled probe. The cDNA (pACSC-2) was 1888 bp long with a deduced peptide of 487 amino acids. It showed up to 72% sequence identity to published ACC synthase sequences.;Northern blot analysis of seven ripeness stages (immature green, mature green, color break, 20% yellow, 30% yellow, 70% yellow and 100% yellow) revealed that the mRNA expression imitated an inside outwards pattern of fruit ripening. In mesocarp tissues, ACC synthase induced between mature green and color break and peaked at 70% yellow stages. However, in endocarp tissues ACC synthase began earlier (immature green) and peaked earlier (color break stage). Genomic southern blot analysis indicated the presence of a single ripening-related gene.;A general transformation vector, pKR1, was developed by inserting two loxP sequences, in direct orientations, surrounding the nptII gene of the binary vector pBI121. The GUS sequence of pKR1 or pBI121 were replaced by ACC synthase cDNA in sense (pBIACS-S and pKR3) or antisense (pBIACS-A and pKR4) orientation. Different constructs were transformed into "Kapoho" or "Sunrise" cultivars.;Somatic embryos generated from immature zygotic embryos were used as target tissue for transformation. Both Agrobacterium-mediated or particle gun-mediated gene transfers were performed. Putative transgenic lines (two from the Agrobacterium-mediated transfer of pBIACS-A and three from particle gun-mediated transfer of pKR4) were regenerated. Leaves of two plants, AA-1-1 and AA-1-2, expressed antisense message in northern blot.;Additionally, bacteriophage P1 Cre recombinase gene was expressed in papaya. Somatic embryos generated from immature zygotic embryos were bombarded with pHA11, using a particle delivery system. The transgene was confirmed by northern blotting and a GUS expression assays. |
