Cloning and Characterization of UDP-dependent Glycosyltransferases (UGTs) in Pomegranate

Graduate #6
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Session: 1
Room: Virginia B

Sheriff-Seedy Phaal - Fort Valley State University
Co-Author(s): Alexander Cain, University of California, Davis; Li Tian, University of California, Davis

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Pomegranate tissues are abundant in bioactive metabolites, many of which are glycosylated. Glycosylation by UDP-dependent glycosyltransferases (UGTs) modifies the solubility, reactivity, and bioactivity of the core compounds. The objective of this research is to clone and characterize selected pomegranate UGTs that belong to the UGT phylogenetic group G. Several candidate group G UGTs were previously identified. cDNA was synthesized from RNA extracted from pomegranate tissues. We performed polymerase chain (PCR) reactions using cDNA as template, separated the PCR products on agarose gels, and purified the target PCR products using a gel extraction kit. We cloned the genes into the Gateway pENTR vector and then subcloned into the Gateway expression vector pDEST17 using LR reactions. We transformed the pDEST17-UGT plasmid into the E. coli strain Rosetta 2 for expression of recombinant proteins. To date, we have successfully cloned several group G UGTs from pomegranate into the entry and expression vectors. We have also successfully expressed group G UGTs in E. coli. We will continue to optimize the expression of soluble proteins in E. coli, which will be purified using Ni-NTA beads and used for enzyme assays.

Funder Acknowledgement(s): Funder Acknowledgement: Special thanks to the Plant Agricultural Biology Graduate Admission Pathway (PABGAP) at UC Davis funded by the University of California HBCU Initiative programs.

Faculty Advisor: Alexander Cain, abcain@ucdavis.edu

Role: I was involved in every part of the research after the cDNA was synthesized from the RNA. From performing PCR onwards.