Investigation of starch metabolism genes and their interactions

Claassens, Adrianus Petrus (2013-12)

Thesis (MSc)--Stellenbosch University, 2013.

ENGLISH ABSTRACT: Starch is widely used in industries around the word, some of these are food, oil drilling, paper milling and cosmetics. It is a polymer which has two components, amylose and amylopectin. The production and degradation if starch in plants is fairly well studied and a sizable number of enzymes have been identified which play critical roles in its metabolism. There are still remaining questions, namely if there are more unidentified enzymes that play roles and how the enzymes interact with each another. To study the effect on starch metabolism possible novel starch metabolic genes were studied by analysing Arabidopsis T-DNA insertion mutants for two genes, designated SP1 (At5g39790) and CBD1 (At5g01260). cDNAs for these two was used to produce recombinant protein and investigated potential activities. The cbd1 mutant plants had a starch excess phenotype with iodine staining but this could not be confirmed with quantitative starch measurements. The sp1 mutants did not have a significant difference in all the lines and time points when compared to the Wt plants. No link could be established between the SP1 kinase domain and glucan phosphorylation. From my data a clear involvement of these two genes could not yet be elucidated. To study the interactions of starch metabolic proteins (BEI, BEII, GWD and ISA2) chimeric RNAi constructs was built and transformed into potato. Only StBEI and StBEII lines could be analysed and it was found that the G6P content was increased in both StBEI and StBEII. The BEII leaves and tubers had increased amylose contents. Intriguingly it would appear that starch isolated from both the tubers and leaves of StBEI lines demonstrated a reduction in amylose, with the leaves showing a much bigger decease than the tubers. This needs to be confirmed and the remaining lines need to be analysed. Gaining knowledge about starch metabolism is critical in producing engineered crops that can produce more starch in a smaller agricultural area. With the population growing beyond 8 billion individuals it will be one of the best routes to enhance cop yields through biotechnology.

AFRIKAANSE OPSOMMING: Stysel word reg oor die wereld benut in ‘n verskyndenheid van industiee. Dit is divers en sluit die voedsel, oliebooring, papiermeule en die kosmetiese bedryf in. Dit is ‘n polimeer wat uit twee komponete: amylose en amylopektien bestaan. Stysel metabolisme, wat die vervaardiging en afbreek van dit insluit, is al baie goed bestudeer. Die ensieme wat ‘n kritiese rol speel is al gevind, maar daar bly nogsteeds ‘n paar vrae wat moet beantwoord word. Is daar nog ensieme wat ‘n rol speel wat nog nie geidentifiseer is nie? Wat is die manier hoe die bekende ensieme met mekaar ‘n interaksie het? Om die invloed van twee moonlike nuwe stysel metabolisme gene te bestudeer, is T-DNA insersie mutante ondersoek. Hulle word na verwys in die studie as SP1 (At5g39790) en CBD1 (At5g01260). cDNAs vir hierdie twee was gemaak vir die vervaardeging van rekombonante proteine. Hierdie rekombinante proteine was dan ondersoek vir moonlike aktiwiteite. ‘n Oormaat stysel was wel gevind in die cbd1 mutant plante wanner n jodium vlek tegniek gebruik was. Ongelukkig kon hierdie oormaat die bevestig word wanner n kwantitatiewe metode gebruik was nie. Daar was nie ‘n beduidende verskil in stysel wanner die sp1 mutante plante vergelyk was met die wilde tiepe nie. Daar kon ook geen verbintenis gevind word tussen die kinase area en die fosforilasie van stysel nie. Volgens hierdie data kon daar die n duidelike verbintenis gevind word tussen die twee gene en stysel metabolisme nie. Om die interaksies tussen bekende stysel metabolisme proteine (BEI, BEII, GWD en ISA2) te bestudeer was chimeriese RNAi konstrukte gebou en toe in aartappels in getransformeer. Slegs die StBEI and StBEII kon geanalisser word en daar was bevind dat die G6P hoeveelheid in beide hoër was. Amilose was in groter hoeveelheide teenwoordig in beide BEII blare en knolle. ‘n Onverwagse obserwasie was gemaak toe die BEI lyne ondersoek was. Daar was gevind dat in die blare en knolle daar ‘n laer hoeveelheid amilose was. Die blare het wel baie laer amilose gehad as die knolle. Die obserwasie moet bevestig word met n ander tegniek en die orige RNAi lyne moet nog bestudeer word. Om al die fasette van stysel metabolisme te ken is uiters belangrik vir die vervaardiging van gewasse wat groter opbrengste lewer in n kleiner area. Met die wereld bevolking wat al verby 8 biljoen individue gestyg het is dit moontlik al hoe almal voor gesorg kan word in terme van voeding.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/85863
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