CLONING AND CHARACTERIZATION OF FRUIT-EXPRESSED ACC SYNTHASE AND ACC OXIDASE FROM PAPAYA (CARICA PAPAYA L.)

Abstract

Papaya is a climacteric fruit. Like other climacteric fruit, ethylene plays an important role in the final stages of ripening. To investigate the role of ethylene in fruit ripening and to produce altered ripening fruit we have isolated fruit-expressed ACC synthase and ACC oxidase cDNAs. Messenger RNA was prepared from approximately 30% yellow fruit and the reverse transcriptase polymerase chain reaction (PCR) was used to amplify fruit-expressed ACC synthase and ACC oxidase present in this mRNA. Several oligonucleotides specific for conserved regions of ACC synthase or ACC oxidase were synthesized based on a comparison of ACC synthase and ACC oxidase sequences present in GenBank. Gene fragments produced using these PCR primers were radioactively labeled and used to screen a cDNA library constructed using mRNA isolated from approximately 30% ripe fruit. Near full-length cDNA clones were isolated for ACC synthase and ACC oxidase. ACC oxidase mRNA is present at significantly higher levels during all stages of fruit development than ACC synthase mRNA. However, both messages increase during ripening. There appears to be only a single gene for fruit-expressed ACC synthase, whereas fruit-expressed ACC oxidase exists as a multi gene family. We have cloned the fruit-expressed ACC synthase gene using our cDNA as the probe and are currently examining its structure and expression. We have also transformed papaya with a sense and an antisense ACC synthase cDNA. Regenerated plants will be examined for alterations in fruit ripening.