Molecular Analyses of the Soybean Glabrous Mutation (P1) and of Transgenic Lines Designed to Modify Seed Composition

225 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.The second project aimed to reduce the expression of the pyruvate dehydrogenase kinase (PDHK) gene by RNAi. The PDHK gene regulates the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of the E1alpha subunit of the PDC. The PDC catalyzes the oxidative decarboxylation of pyruvate into acetyl-CoA and thereby controls the entrance of carbon subunits into the TCA cycle. The activity of the PDHK protein is enhanced during periods of active photosynthesis, which leads to downregulation of the PDC in the light. The goal of this project was to decrease expression of the PDHK gene(s) in soybean seeds specifically by introduction of a PDHK RNAi construct transgenically into soybean. The insertion of the PDHK RNAi construct was confirmed in the soybean sublines by PCR and Southern blot analysis. The dry seeds from the transgenic lines were significantly different than non-transformed plant seeds in that they had statistically significant increases in both average 100 seed weight and protein/oil ratios. These increases in average seed weight and protein/oil ratios from the transgenic lines were associated with decreased yield in both seed weight and total seeds yielded per plant. Unexpectedly, the overall PDHK seed expression levels of the transgenic seeds do not appear to be different from the non-transformed seeds as determined by both Northern blot and QRT-PCR analysis. The altered seed traits of the transgenic lines may be associated with a reduction in certain members of the PDHK gene family (with up to five members) that are not detected in the blots or QRT-PCR.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD