cDNA cloning, expression and characterization of a putative glucosyltransferase (GT) from grapefruit (Citrus paradisi) leaves

Flavonoids are plant secondary metabolites that are integral to our lives. Grapefruits are well-known for production of unique glucosylated products and the enzymes responsible are UDP-glucose: glucosyltransferases (GTs). The objective of this research was to obtain full-length clones of putative grapefruit GTs, express them, and characterize them. Previously, gene specific primers (from conserved PSPG box) and clone specific primers (from partial 5 ' clones) were designed, and a compiled sequence attained using SMART RACE RT-PCR. A full-length clone was obtained using primers designed from the extreme ends of the compiled sequence. The full-length clone was inserted into expression vector (pET32a) and transformed into expression host BL21(DE3)RIL. Expressed protein was tested for GT activity using different flavonoid aglycones and UDP-14C-glucose as glucose donor. Results indicated that the expressed protein was probably not a flavonoid GT. A directionally cloned grapefruit leaf cDNA library is undergoing EST mining to identify additional GT candidates.