Cloning and characterization of differentially expressed genes in ethylene-treated watermelon fruit

Exposure of watermelon fruit to ethylene leads to acute placental-tissue softening and watersoaking. mRNA differential display strategy was utilized to isolate and characterize genes associated with development of the disorder. Watermelon fruit were harvested at the full-ripe stage and exposed to 50 μL L−1 ethylene for 12 h, and 1, 3, and 6 d at 20 °C. DNA-free total RNA (0.2 μg) isolated from fruit treated with air or ethylene for 12 h was reverse transcribed using three different one-base-anchored oligo dT primers. High stringency PCR was performed using eight different 13-mer arbitrary primers in combination with the fluorescent-labeled one-base-anchored oligo dT primers. The PCR fluorescent-labeled products were separated on a 6% polyacrylamide sequencing gel and differentially expressed bands were isolated. Confirmation of true positive bands was performed by reverse northern blotting. Comparison of mRNA profiles of ethylene- and air-treated fruit revealed that 78 genes were differentially expressed, of which 15 partial cDNAs were isolated and characterized. BLAST search results revealed significant homologies to ethylene biosynthesis and signal transduction pathway genes, lipid-metabolizing enzymes, proteins involved in plant defense responses, radical scavenging enzymes, exonucleases, and expansins. Northern blot analysis of RNA isolated from air- and ethylene-treated fruit stored for 1, 3, and 6 d revealed an ethylene-dependent expression pattern of all corresponding genes. The data indicate that ethylene treatment of watermelon fruit results in a rapid up-regulation of oxidative and hydrolytic enzymes.