FUNCTIONAL GENOMICS OF TOMATO ß-GLYCOSIDASES (Β-GLUCOSIDASES [EC 3.2.1.21] AND Β-GALACTOSIDASES [EC 3.2.1.23]): PHYLOGENETIC ANALYSIS AND CLONING

O-Glycoside hydrolases (EC 3.2.1.-) are enzymes that catalyze the cleavage of chemical bonds between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety. Two major families are of special focus. Family 1 includes β-glucosidases (β-D-glucoside glucohydrolase, EC 3.2.1.21) and thioglucoside glucohydrolases (myrosinases; EC 3.2.3.1), which function in higher plants in chemical defense against herbivores and pathogens, lignin biosynthesis, and plant growth and development. Family 35 contains the β-galactosidases (EC 3.2.1.23), which play key roles in fruit ripening, flower senescence, mobilization of carbohydrate reserves and galactolipid turnover. Arabidopsis contains approximately 75 members of both families. However, in tomato, only a few have been cloned and characterized. We aligned several reported plant β-glucosidases with ClustalW and designed degenerate primers but with minimal success. We next explored the SOL Genomics Network database, where eight putative tomato β-glucosidases were available as ETS contigs. The sequences were exposed to six-way reading frames to get the most suitable open reading frame (ORF). However, the protein sequences did not align well using ClustalW, neither among each other nor with other β-glucosidases from other plant species. RT-PCR was conducted using total RNA from tomato leaves to amplify those eight genes but only one was confirmed.