Browsing by Author "Hartzenberg, Lionel"

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    Identification and manipulation of starch synthases from marama bean and potato
    (Stellenbosch : Stellenbosch University, 2016-12) Hartzenberg, Lionel; Lloyd, James Richard; Kossmann, Jens; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Starch is a major storage carbohydrate in plants and algae. It consist of amylose and amylopectin and provides energy during heterotrophic growth. The accumulation of starch differs between certain families. Viridiplantae (plants and green algae) utilize ADP-glucose for starch synthesis and store it within the chloroplast, whereas Rhodophyceae (red algae) utilize UDP-glucose and both synthesize and store starch in the cytoplasm. Starch is a commercial product of worldwide importance and research into improving current starch sources and examining alternatives may provide benefits to the starch industry. This study consisted of two parts. The first involved increasing the physical and genetic information available for the southern African legume Tylosema esculentum. This involved obtaining and analysing transcriptomic data in order to find genes relating to starch synthesis. The second part aimed to randomly mutagenize a Solanum tuberosum SSI gene for the utilization of UDP-glucose instead of ADP-glucose for starch synthesis. Tylosema esculentum plants were grown for approximately one year before the leaf and tuber material was harvested. RNA extraction and cDNA synthesis was performed and the samples were sent for next-generation sequencing at the Agricultural Research Council (Pretoria). Reads were compiled and trimmed to produce three contigs related to starch metabolism. Using the Phytozome soybean database, we selected three genes (granule-bound starch synthase (GBSS), starch synthase 2 (SSII), starch synthase 4 (SS4)), and soybean-like actin, relating to starch synthesis within soybean. We matched them to open reading frames within the marama bean transcriptome and designed primers for their amplification from cDNA. All were successfully amplified. We also obtained additional information on the physical properties of T. esculentum starch granules. Two methods were performed in order to accurately obtain data on the marama bean starch granule size distribution. Both showed that the starch granule size is predominantly between 15-35 μm, with no particles above 50 μm. A reducing end assay was also performed to determine the amylose and amylopectin average chain length of marama bean starch. The amylose chain was found to be 67.5% longer than that of harvested potato starch, and 31.8% longer than that of commercial grade potato starch (Sigma-Aldrich). The amylopectin chain length was found to be 34.8% lower than that of harvested potato starch, but similar to that of commercial grade potato starch. The higher accumulation of long amylose chains could account for previous Rapid Visco Analyser (RVA) analyses performed that showed marama bean starch having unique viscosity properties, and may in turn lead to several other novel uses for marama bean. In the second part of this study, we amplified the UGPase (UDP-glucose pyrophosphorylase) gene from Arabidopsis thaliana to facilitate the production of UDP-glucose. The UGPase gene was ligated into the pACYC 184 vector and transformed into G6MD2, an E. coli strain lacking the glg operon necessary for glycogen metabolism. The S. tuberosum SSI gene within pBluescript.SK was randomly mutagenized using XL1-red E. coli cells, which are deficient in 3 of the primary proof reading mechanisms. Of these mutagenized plasmids, 33 were pooled and analysed against the unaltered SSI gene as template. We obtained 192 mutations in total, 4 per kb, with 98 point mutations, 57 insertions and 37 deletions. The SSI library was transformed into G6MD2::pACUG and screened for activity via iodine vapour. 150 000 colonies were screened but no expressing colonies were found. In the future alternative mutation methods as well as larger colony screenings may yield better results.