Analysis of enzymes involved in starch phosphate metabolism

Samodien, Mugammad Ebrahim (Stellenbosch : University of Stellenbosch, 2009-12)

Thesis (MSc (Genetics. Plant Biotechnology)) --University of Stellenbosch, 2009.


ENGLISH ABSTRACT: This project examined the role of proteins in starch phosphate metabolism. The first part was aimed at the functional characterization of the SEX4, LSF1 and LSF2 genes in both plants and bacteria. Constructs were produced to allow for expression of the three proteins in E. coli with the SEX4 and LSF2 proteins being successfully purified and used to produce antibodies. Immunoblot analysis indicated that the antibodies recognised the repective proteins in extracts, but it was not clear if they actually recognised the proteins or the GST tags they were fused to. Virus induced gene silencing constructs were also produced to allow repression of these three genes in Nicotiana benthamiana. This resulted in a starch excess phenotype being observed in the leaves of silenced plants which is consistent with the known or presumed roles for the genes. The antibodies produced were not specific enough to confirm that the respective protein were actually repressed, but it is likely that this was the case as plants infiltrated at the same time with a VIGS vector designed to repress phytoene desaturase exhibited a chlorophyll bleaching phenotype. These data confirm that SEX4 and LSF1 probable play the same role in N. benthamiana as in Arabidopsis, and provide evidence that LSF2 is also necessary for starch degradation. It was also attempted to characterise these proteins with respect to their substrate utilization by setting up a glyco-array experiment. Various potato starches from genetically modified plants were subjected to hydrolytic attack by starch degrading enzymes and fractionated by anion exchange chromatography to produce a multitude of glucans. These will be spotted onto glass filters and probed with the purified proteins to see if they bind to specific starch breakdown products preferentially. iv The project also involved investigating the effect the SEX4 protein has on E. coli glycogen contents. SEX4 was expressed in wild type and glgX mutant E. coli strains as it has been shown that this stops glycogen accumulation in the wild type, but not the glgX mutant. The cells were grown in liquid culture and glycogen contents measured. In liquid cultures SEX4 had no effect on glycogen contents in the wild type, possible because of problems with plasmid stability in the strain used. This final part of the project investigated the effect that a gwd mutation has on carbohydrate metabolism in leaves and fruits of the Micro-tom tomato cultivar. Starch and soluble sugar contents were measured in leaves and ripening fruits. A starch excess phenotype was found in the leaves, but no change in starch contents was determined in either the placenta or pericarp of the fruit. Soluble sugar contents were reduced in the fruit tissues, although the reason for this in unclear.

AFRIKAANSE OPSOMMING: Hierdie projek het die rol van proteine in stysel-fosfaat metabolisme ondersoek. Die eerste deel handel oor die funksionele karaktiseering van die SEX4, LSF1 en LSF2 gene in beide plante en bakteriee. Vektore is gekonstrueer om die uitdrukking van die drie proteine in E.coli toe te laat terwyl die SEX4 en LSF2 proteine suksesvol gesuiwer is vir die gebruik vir teenliggaam produksie. Immunoklad analises het getoon dat die teenligame die spesifieke proteine in die ekstrak herken het, maar dit was nie duidelik of dit die onderskeie proteine was of die GST-verklikker waaraan die onderskeie proteine verbind was nie. Virus geindiseerde geen onderdrukking konstrukte is ook geproduseer om toe te laat vir die onderdrukking van hierdie drie gene in Nicotiana benthamiana. Dit het ‘n stysel oorskot fenotipe tot gevolg gehad in die blare van onderdrukte plante wat konstant is met die bekende of voorgestelde rolle van die gene. Die teenliggame wat geproduseer is was nie spesifiek genoeg om te bewys dat die onderskeie proteine wel onderdrukis nie. Dit kon wel die geval gewees het want plante geinfiltreer op dieselfde tyd met ‘n VIGS vektor wat ontwerp is om phytoene desaturase te onderdruk het ‘n chlorofil bleikings fenotipe getoon. Hierdie data bevestig dus dat SEX4 en LSF1 moontlik dieselfde rol speel in N. benthamiana as in Arabidopsis, en toon bewyse dat LSF2 ook nodig is vir stysel afbreek. Karakterisasie van die onderskeie proteine met respek tot hul substraat gebruik is ondersoek deur ‘n gliko-array eksperiment. Verskillende aartappel stysels van genetiese gemodifiseerde plante was geonderwerp aan hydrolitiese afbreek deur stysel afbrekende ensieme en geskei deur anioon uitruilings chromotografie om veelvuldige glukans te vi vervaardig. Dit is geplaas op glas filters en is ondersoek saam met die gesuiwerde proteine om te sien of dit mag bind aan spesifieke stysel afbreek produkte. ‘n Verdere ondersoek is onderneem na die effek van die SEX4 protein op E. coli glikogeen inhoud. SEX4 was uitgedruk in die E .coli wildetipe en glgX mutant omdat dit reeds bewys is dat SEX4 glikogeen ophoping veroorsaak in die wildetipe maar nie in die glgX mutant. Die selle is opgegroei in vloeibare media en glikogeen inhoud is gemeet. In vloeibare media het SEX4 geen effek op die wildetipe se glikogeen inhoud nie wat moontlik kan wees as gevolg van plasmied stabiliteit in die E. coli ras wat gebruik is. Die finale deel van die projek was om die effek van ‘n gwd mutasie op koolhidraat metabolisme in blare en vrugte van die Micro-tom tamatie kultivar te ondersoek. Stysel en oplosbare suikers is gemeet in blare en rypwordende vrugte. ‘n Oortollige stysel fenotipe is in die blare gevind maar geen verandering in stysel inhoud is waargeneem in die plasenta of perikarp van die vrug nie. Oplosbare suiker inhoud het afgeneem in die vrugweefsel dog is die rede hiervoor nie te verstane.

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