Isolation, Cloning and Transformation of DFR from Citrus sinensis (Velencia) leaf tissue into Escherichia coli (TOP10)

Presenter(s)

Gloria Rose

Abstract or Description

Dihydroflavonol 4-reductase performs a late-stage reduction reaction on dihydroflavonols as part of the flavonoid metabolic pathway. This is a committed step into the production of anthocyanidins, a common plant pigmentation molecule that primarily give off purple, red, or blue hues. In blood oranges, this pathway is necessary for the coloration of the dark fruit. However, in blonde this is much less apparent, so is DFR actually expressed in blonde oranges? Genomic material was isolated from Citrus sinensis (Valencia) leaf tissue. Gene isolation was performed by polymerase chain reaction (PCR), followed by directional cloning into the entry vector pENTR D-TOPO, chemically transformed into Escherichia coli (TOP10), and grown on selective media. Forward and reverse primers for directional TOPO cloning were selected keeping the 4-nucleotide overhang in mind. The forward primer being 5'-CACCATGGGCTCTATAGCTGAGACT-3’ with a Tm of 62, and the reverse primer 5'-TTATATGGATACTTCGCTGACATG-3’ with a Tm of 66. Finally gel electrophoresis was performed on PCR product and on digested plasmid purified from overnight transformants for verification. Isolation, purification, and successful transformation into the entry vector pENTR/D-TOPO will be utilized for further downstream application in a protein-protein interaction assay known as Yeast-2-Hybrid with the end goal of creating an interaction map of core flavonoid metabolic enzymes as well as UDP-glucuronosyltransferase expressed in citrus.

Mentor

Dr. Daniel Owens