Browsing Masters Degrees (Genetics) by browse.metadata.advisor "Bauer, R."
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- ItemAnalysis of intermediate carbon metabolism in strawberry plants(Stellenbosch : Stellenbosch University, 2008-12) Basson, Carin Elizabeth; Groenewald, J.-H.; Bauer, R.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.Strawberry (Fragaria x ananassa) fruit quality is largely determined by the relative amounts of sugars and organic acids present, as well as soluble solid content. This study had three components: 1) Characterisation of cytosolic carbohydrate metabolism and carbon partitioning to sugars and organic acids in two commercial varieties, 2) analysis of transgenic strawberry fruit with increased pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP) activity and 3) analysis of transgenic strawberry fruit with increased ß-fructosidase (invertase) activity in either cytosol or apoplast. Analyses of transgenic strawberry may inform similar attempts in grape berries. Festival and Ventana, two popular commercial strawberry cultivars in South Africa, were fairly similar with respect to sugar and organic acid content. Twelve cytosolic enzymes were investigated. Temporal differences in maximum catalytic activity were observed for invertase, PFP, pyruvate kinase and ADP-glucose pyrophosphorylase (AGPase). Invertase, PFP and AGPase activity also differed between the cultivars. One enzyme, SuSy, could not be analysed effectively, due to the purification method employed. These analyses established methodology for the analysis of transgenic berries. Constructs were designed to constituitively express Giardia lamblia PFP (GL-PFP), or to express Saccharomyces cerevisiae invertase (SCI) in a fruit-specific manner. A second invertase construct was designed to target SCI to the apoplast. Strawberry (cv. Selekta) was transformed and the presence of each transgene confirmed by PCR. Untransformed Selekta was used as control in both transgenic studies. Transgenic lines were selected based on GL-PFP activity in leaves and total PFP activity in ripe fruit. Sugar and organic acid content of ripe berries with high PFP activity was determined. Although berries displayed marked changes in sugar composition, the total sugar content was similar to controls, in all except one line. Organic acid content was decreased, leading to a clear reduction in organic acid-to-sugar ratio. This points to a gluconeogenic role for PFP in strawberry fruit. Transgenic berries were screened for SCI activity. Berries containing untargeted SCI exhibited total invertase activity similar to controls and were not analysed further. Berries with apoplasttargeted SCI displayed three-fold increases in invertase activity compared to controls. Total sugar content was reduced and exhibited reduced sucrose content relative to hexoses. Despite the effect of increased invertase activity on metabolites, maximum catalytic activity of enzymes involved in cytosolic sucrose, hexose and organic acid metabolism were unchanged. Transgenic plants selected in these studies were subsequently vegetatively replicated and future work will include immature fruit.
- ItemBiopolymer gene discovery and characterization using metagenomic libraries(Stellenbosch : Stellenbosch University, 2008-12) Ohlhoff, Colin Walter; Lloyd, James Richard; Bauer, R.; Kossmann, J. M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.Traditional methods used for the discovery of novel genes have previously relied upon the ability to culture the relevant microbes and then demonstrate the activity of a specific enzyme. Although these methods have proved successful in the past, they severely limit our access to the genomes of organisms which are not able to be cultured under laboratory conditions. It was therefore the aim of this project to use metagenomic strategies for the identification of novel polymer-producing genes with the prospect of commercial exploitation. In this study, soil-derived metagenomic libraries were functionally screened for potential -glucan producing clones using aniline blue staining. Positive reacting clones were selected and sequenced. Initial sequencing revealed a gene with high homology to previously described glucan synthases, the products of these genes all having significant industrial value. The clone was transformed into a suitable bacterial host, cultured and allowed to produce the polymer of interest. The polysaccharide was purified and subjected to various chemical analyses so as to confirm its monosaccharide composition. Data suggests that this polymer is composed mainly of glucose units and that it may be secreted out of the cell. Purification of the active enzyme was attempted using classical protein purification methods with faint activity being detected using Native polyacrylamide gel electrophoresis (PAGE). Further attempts to demonstrate activity were made through the construction of a GST (glutathione S-transferase) tagged fusion protein. The second part of this study focuses on the construction and screening of a metagenomic DNA library from whey, a by-product of the cheese manufacturing process. It was envisaged that this could provide a resource for the identification of high value polymers when lactose is provided as a sole carbon source. The library was screened for function using Congo Red for the detection of extra-cellular polysaccharides.
- ItemManipulation of ascorbate biosynthesis in Solanum lycopersicum (cv Money maker)(Stellenbosch : University of Stellenbosch, 2010-12) Cronje, Christelle; Kossmann, J. M.; Bauer, R.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).ENGLISH ABSTRACT: Vitamin C (ascorbate or AsA) is a secondary metabolite produced in many eukaryotes including yeasts, plants and animals. It plays essential roles as an anti-oxidant and enzyme cofactor, functions as an electron donor and -acceptor and is involved in various developmental processes. This study was initiated with the aim of increasing vitamin C production in tomato. Three genes, namely GDP-mannose pyrophosphorylase (GMPase) from Saccharomyces cerevisiae, arabinono-1,4-lactone oxidase (ALO) from Saccharomyces cerevisiae and myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana were ectopically expressed in the tomato cultivar Money Maker. GMPase converts D-mannose-6-P to GDP-D-mannose. This reaction forms part of the well characterized, “Smirnoff-Wheeler” pathway. ALO catalyzes the terminal step in erythroascorbate synthesis in yeast. In situ it also metabolizes the plant and animal substrates for ascorbate manufacture. Myo-inositol (MI) is converted into D-glucuronate by the activity of MIOX. D-Glucuronate is a precursor to L-guluno-1,4-lactone synthesis which is the precursor to AsA in animals and thought to be present in plants. The genes were independently introduced with the aid of Agrobacterium tumefaciens mediated transformation and expressed under the control of the CaMV 35S promoter. Plants with increased GMPase activity consistently showed increased L-ascorbate levels in leaves and fruit of between 20- and 70% compared to the wild-type. Plants transcribing the ALO gene exhibited small increases in L-ascorbate in green fruit (p < 0.1). Leaf tissue from MIOX plants displayed significant activity increases (p < 0.05), and substantial decreases in MI. In green fruit two MIOX lines had increases in activity, cell wall uronic acids and AsA levels. Marginal increases in L-ascorbate would not warrant industrial application, but follow-up research with over-expression of other enzymes of the “Smirnoff-Wheeler” pathway should be explored.