Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Biology

Supervisor

Sangeeta Dhaubhadel

Abstract

GmMYB176 is an R1 MYB transcription factor that regulates isoflavonoid biosynthesis in soybean. The deletion of phosphorylation sites in GmMYB176 abrogates its interaction with 14-3-3 proteins and alters its intracellular localization, thus demonstrating the crucial role of phosphorylation in its regulation. While phosphorylation is undoubtedly crucial for GmMYB176 regulation, the identity of kinase(s) responsible remains unknown. Interestingly, a previous LC-MS/MS study using GmMYB176 as bait identified seven putative protein kinases. To further validate these putative GmMYB176-specific protein kinases, the interaction of all seven candidate protein kinases with GmMYB176 were assayed in planta using bimolecular fluorescence complementation assay. All of the candidate protein kinases: Gm02PK, Gm04PK, Gm08PK.1, Gm08PK.2, Gm14PK, Gm17PK and Gm17PPD interacted with GmMYB176 in the nucleus. In addition, Gm17PK also showed interaction with GmMYB176 in the cytoplasm. Furthermore, subcellular localization studies of the individual candidate protein kinases showed that Gm08PK.1 and Gm08PK.2 localize to the nucleus, Gm02PK, Gm04PK and Gm14PK to the endoplasmic reticulum, Gm17PK to the plasma membrane and Gm17PPD to the plastid. The nuclear proteins Gm08PK.1 and Gm08PK.2 were expressed in bacteria and successfully purified for in vitro kinase assays. The results from kinase assays suggested that both Gm08PK.1 and Gm08PK.2 phosphorylate GmMBY176 in vitro. This research will provide the necessary foundation for future studies into the regulation of GmMYB176 and isoflavonoid biosynthesis and thus will help in the metabolic engineering of isoflavonoid biosynthesis in plants.


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