Molecular analyses of the soybean glabrous mutation (P1) and of transgenic lines designed to modify seed composition

The tools commonly available for the study of developmental biology have increased rapidly for crop systems in the last decade. With new tools such as microarrays and RNAi producing hpRNAi constructs available for the study of crop species such as soybean it has become possible to study developmental questions from both a global gene expression perspective and by manipulation of expression of a single target gene or gene family. Two general biological approaches were taken in two projects presented in this study. First, a forward genetics approach was taken to study the classic soybean glabrous P1 locus using microarray technology to identify candidate genes for the glabrous mutation. Second, a reverse genetics approach was taken to study the effect of reducing the pyruvate dehydrogenase kinase gene expression by a transgenic hpRNAi approach.;The first project aimed to identify possible candidate genes for the glabrous mutation in soybean by comparing the Clark standard isoline (CS) with the Clark glabrous (CG) isoline. The glabrous soybean mutation is associated with a complete lack of non-glandular trichomes on above ground portions of the plant. The comparison between CS and CG isolines was carried out by comparing gene expression between the two isolines with a cDNA microarray approach. Gene expression was compared using total RNA extracted from both shoot tip and older leaf tissue in CS and CG isolines. The comparison highlighted four initial candidate genes including clones with homology to a hypothetical gene, a BURP domain containing gene, a protein kinase II gene, and a phosphofructokinase gene. These clones were compared using Northern and Southern blots and the candidate genes were narrowed to the hypothetical gene and the BURP gene as both clones had significant expression differences between CS and CG isolines and multiple RFLPs between the CS and CG isolines. The hypothetical gene does not have any functional annotation as is common with many thousands of genes of unknown function in plant genomes. The BURP gene has homology to the cotton gene RDL1 associated with cotton fiber (seed trichome) initiation. Both candidate genes were identified as members of multi-gene families through Southern blot analysis and the data released in the draft soybean genome sequence released in the last year.;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.