Division III

Abstracts Submitted: Division III - Graduate students (2 + years) - Natural Sciences & Mathematics


FLAVONOID GLUCOSYLTRANSFERASES: CLONING, SEQUENCING, AND EVALUATION OF PUTATIVE GLUCOSYLTRANSFERASES FROM CITRUS PARADISI (GRAPEFRUIT) LEAVES USING SMART RACE RT-PCR AND EST MINING

CHRISTY L. STRONG, LEE M. PIKE, AND CECILIA A. MCINTOSH, DEPT. BIOLOGICAL SCIENCES, ETSU, JOHNSON CITY, TN 37614

Flavonoids, plant secondary metabolites, play roles in plant coloration, plant/animal interactions, and also possess antioxidant properties. Nine different subgroups exist for categorizing flavonoids and they are further modified through various reactions including glycosylation, methylation, hydroxylation, etc. During glucoysylation, the sugar moiety from UDP-glucose is transferred to aglycone flavonoid substrates by glucosyltransferases (GTs). Grapefruit contains five different flavonoid glucosyltransferases that demonstrate differences in substrate and position specificity. These five flavonoid GTs include: flavonol 3-O-GT, flavonol 7-O-GT, chalcone GT, flavone 7-O-GT and flavanone 7-O-GT. Determining sequence information and biochemically characterizing a clone of any of the five flavonid grapefruit GTs would be worthwhile because of their unique characteristics, however one GT holds a special interest. Flavanone-specific 7-O-glucosyltransferase (7GT) catalyzes the first glycosylation reaction leading to production of bitter naringin in grapefruit. The 7GT enzyme is of particular interest because of its high product accumulation rate (40-70% of the dry weight of young fruit and leaves), and extremely low sensitivity to UDP inhibition (over 10,000 times less sensitive than other characterized GTs). Primary structure of 7GT could not be determined directly due to low yield and N-terminal blockage. Therefore, a research plan designed to obtain full-length grapefruit GT cDNA clones, express them, and characterize them was put into action. This research yielded five putative 5’ partial grapefruit GT clones using SMART RACE RT-PCR with a degenerate gene specific primer based on a highly conserved sequence area in the Plant Secondary Product Glucosyltransferase box (PSPG). Previously, two 5’ partial clones (duplicates) were obtained. One difference occurred between the clones, a stop codon interrupting the open reading frame. A full-length clone based on the partial 5’ clone containing the open readin.....