ISOLATION AND SEQUENCING OF A GENOMIC DNA ENCODING FOR ASCORBATE OXIDASE, A KEY ENZYME INVOLVED IN THE BIODEGRADATION OF ASCORBIC ACID IN MELON

Abstract

A melon genomic library was used to isolate and characterize a clone of genomic DNA coding for ascorbate oxidase (AO) which is considered a key enzyme in the biodegradation of ascorbic acid (AA). The screening of genomic library was performed by using two probes: AO-PCR product and αE/CMAO3. Seven clones of recombinant phage, with positive signal, were isolated. The extracted DNA was digested with EcoRI, Sal I and Bam HI and the combinations among these restriction enzymes. Four clones of AO gene were isolated: AO1; AO2; AO3 and AO4. Since only clone AO4 had not been characterized so far, the sequencing of this clone was performed. The isolated sequence of DNA has a length of 3072 bp being bordered at both ends by EcoRI enzymes. The comparison between genomic DNA for AO4 in melon and DNA for AO in cucumber revealed a high similarity (94.45%).

Keywords

• isolation
• sequencing
• genomic dna
• ascorbic acid
• melon