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Genetic manipulation of trehalose-6-phosphate synthase – a study on the functional activity and role in carbohydrate homeostasis in plants
Thesis (MScAgric)--Stellenbosch University, 2021.
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| Language: | English |
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Stellenbosch : Stellenbosch University
2021
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| _version_ | 1867613956324982784 |
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| access_status_str | Open Access |
| author | Gabriel, Casey Ashley |
| author2 | Van der Vyver, Christell |
| author_browse | Gabriel, Casey Ashley Van der Vyver, Christell |
| author_facet | Van der Vyver, Christell Gabriel, Casey Ashley |
| author_sort | Gabriel, Casey Ashley |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/110423 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:44:22.712Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/110423 Genetic manipulation of trehalose-6-phosphate synthase – a study on the functional activity and role in carbohydrate homeostasis in plants Gabriel, Casey Ashley Van der Vyver, Christell Lloyd, James Richard Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics & Institute of Plant Biotechnology. Sugarcane -- Genetics Trehalose Transgenic plants -- Genetic engineering Saccharomyces cerevisiae -- Molecular cloning Carbohydrates -- Metabolism -- Genetic aspects Homeostasis -- Genetic aspects UCTD Thesis (MScAgric)--Stellenbosch University, 2021. ENGLISH ABSTRACT: Trehalose is a non-reducing disaccharide formed by two glucose molecules and is widely distributed in nature. Over the years it has been detected and isolated from different species of bacteria, fungi, and plants, especially in plants which can survive in a dehydrated state (desiccation tolerant) for long periods and regain metabolic function upon rehydration. The trehalose intermediate, trehalose-6-phosphate (Tre6P) is an essential regulatory and signaling molecule involved in both regulation of carbon metabolism and photosynthesis. Trehalose-6-phosphate is synthesised via trehalose-6-phosphate synthase (TPS) and sequentially dephosphorylated by trehalose-6-phosphate phosphatase (TPP). To explore the role of Tre6P we modified the TPS enzyme responsible for its synthesis in two plant systems namely Saccharum officinarum (sugarcane) and Solanum tuberosum (potato). A previous study (Fernhout, 2015) had identified three sugarcane TPS genes that encode functional proteins. The first aim of this thesis was to develop a better understanding of the effect of silencing expression of these TPS isoforms on sucrose accumulation in sugarcane. Silencing constructs were developed to down-regulate the expression of TPS in sugarcane and silencing was confirmed using real-time quantitative PCR (RT-qPCR) analysis. The transgenic sugarcane plants were further analysed for expression of genes encoding both TPS and TPP isoforms in leaf, immature and mature internode tissues. A combination of enzyme linked analysis and liquid chromatography mass-spectrometry (LC/MS) were used to determine the concentrations of sucrose, glucose, fructose, Tre6P and trehalose contents. Results showed that one transgenic line had decreased Tre6P, accompanied by most lines with decreased trehalose levels and some transgenic lines showed significantly lower levels of soluble sugars. LC/MS data further revealed that there were no consistent significant differences, although most metabolites appeared to decrease with culm maturity. Furthermore, the three novel SoTPS variants were transformed into Saccharomyces cerevisiae for biochemical characterisation. The plant TPS cDNA’s were able to complement growth in tps1Δ and tps2Δ yeast mutants. Recombinant SoTPS proteins were purified to determine protein activity, however, although purification of TPS proteins were successful we were unable to detect TPS activity. Therefore, the second objective, determining kinetic properties such as pH optimum, Michaelis constant (Km) and substrate utilization was not examined. The second aim was to examine the role of the three TPS splice variants (SoTPS-A, B and C) in carbon partitioning by overexpressing them in the starch storing organ of potato (Solanum tuberosum). This was achieved by first confirming the integrity of the constructed recombinant plant expression vectors, pCambia2300::B33 containing each of the three SoTPS isoforms under control of the tuber specific B33 promoter. Putative transformed plants were analysed using PCR to identify plants with increased TPS expression. The soluble sugar and starch content in the transgenic plants were determined alongside molecular analysis of the effect of heterologous SoTPS expression on native potato TPS gene expression. A total of eight transgenic potato lines showing both transgene insertion and expression were selected. All the transgenic lines showed significantly lower levels of sucrose compared to the wild-type (WT) plants with SoTPS-B and C lines containing reduced glucose and fructose amounts. Starch contents appeared to be lower in SoTPS-A lines and higher in SoTPS-B and C lines. AFRIKAANSE OPSOMMING: Trehalose is 'n nie-reduserende disakkaried wat deur twee glukose molekules gevorm word en kom wyd verspreid in die natuur voor. Oor die jare is dit opgespoor en geïsoleer uit verskillende soorte bakterieë, swamme en plante, veral plante wat maande of jare lank in 'n gedehidreerde toestand (verdrogingsverdraagsaam) kan oorleef en die metaboliese funksie kan herwin na rehidrasie. Die trehalose-intermediêr (tussenganger), trehalose-6-fosfaat (Tre6P) is 'n noodsaaklike regulerende- en seinmolekule wat betrokke is by die regulering van koolstofmetabolisme en fotosintese. Trehalose-6-fosfaat word gesintetiseer via trehalose-6-fosfaat sintase (TPS) en opeenvolgend gedefosforileer deur trehalose-6-fosfaat fosfatase (TPP). Om die rol van Tre6P te ondersoek, het ons die TPS ensiem verander wat verantwoordelik is vir die sintese daarvan in twee plantstelsels, naamlik Saccharum officinarum hibried spesie (suikerriet) en Solanum tuberosum (aartappel). ʼn Vorige studie (Fernhout, 2015) het drie suikerriet TPS gene geïdentifiseer wat funksionele proteïene kodeer. Die eerste doelwit (mikpunt) was om 'n beter begrip te ontwikkel van die effek van onderdrukte TPS-aktiwiteit op sukrose-ophoping in suikerriet. Die onderdrukkings-rekombinante vektore is ontwikkel om die uitdrukking van TPS in suikerriet af te reguleer en gedeeltelike geen-stilte is bevestig met behulp van intertydse kwantitatiewe PKR (RT-qPKR) analise. Die transgeniese suikerrietplante wat onderdrukte transgeen aktiwiteit getoon het, is verder geanaliseer vir beide TPS en TPP uitdrukking in blaarweefsel en onvolwasse- en volwasse internodes. Toets-analise en vloeistofchromatografie-massaspektrometrie (LC/MS)-analise is gebruik om die konsentrasies van sukrose, glukose en fruktose en die algehele impak van transgeenonderdrukking op die Tre6P- en trehalose-inhoud te bepaal. Resultate het getoon dat die meeste van die transgeniese lyne verlaagde Tre6P- en trehalose-vlakke gehad het, en sommige transgeniese lyne het aansienlike laer vlakke van oplosbare suikers getoon. LC/MS-data het verder aan die lig gebring dat daar 'n geen konsekwente beduidende verskille nie, alhoewel die meeste metaboliete blykbaar afneem met die rypwording van die riet Verder is die drie nuwe SoTPS-splitsvariante gene, geïsoleer van suikerriet, ingevoeg in Saccharomyces cerevisiae vir biochemiese karakterisering. Die komplementêre TPS DNA plantgene was in staat om die groei in tps1∆ en tps2∆ gismutante te komplementeer. Die rekombinante SoTPS proteïene is geïsoleer en gesuiwer om proteïen-aktiwiteit te bepaal. Suiwering van TPS-proteïene was suksesvol, maar ons kon nie TPS-aktiwiteit bevestig nie. Daarom is die een van die voorgestelde take, naamlik die bepaling van kinetiese eienskappe soos pH-optimum, Michaelis-konstante (Km) en substraatbenutting, nie ondersoek nie. Die tweede doel was om die rol van die drie TPS-splitsvariante gene (SoTPS-A, B en C) in koolstofverdeling te ondersoek, deur die gene in die styselstoringsorgaan van aartappel (Solanum tuberosum) oormatig uit te druk. Dit is bereik deur eers die integriteit van die reeds gegenereerde rekombinante plantuitdrukkingsvektore, pCambia2300::B33, wat elk van die drie SoTPS-isovorme bevat onder beheer van die B33-knolspesifieke promotor, te bevestig. Vermeende getransformeerde plante is geanaliseer met behulp van PKR om te selekteer vir plante met verhoogde TPS-uitdrukking. Die oplosbare suiker- en styselinhoud in die transgeniese plante is bepaal en die molekulêre analise van die effek van heteroloë TPS-uitdrukking op die inheemse aartappel-TPS-geenuitdrukking, is verder uitgevoer. 'n Totaal van agt transgeniese lyne wat beide transgeen-invoeging en -uitdrukking toon, is geïdentifiseer. Al die transgeniese lyne het beduidende laer vlakke van sukrose getoon in vergelyking met die wilde-tipe (WT) plante, en slegs in SoTPS-B en C-lyne is die glukose- en fruktose-vlakke verlaag. Styselinhoud blyk egter laer in SoTPS-A-lyne en hoër in onderskeidelik SoTPS-B- en C-lyne te wees. Masters 2021-04-29T10:51:24Z 2021-04-29T10:51:24Z 2021-03 Thesis http://hdl.handle.net/10019.1/110423 en Stellenbosch University 146 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Sugarcane -- Genetics Trehalose Transgenic plants -- Genetic engineering Saccharomyces cerevisiae -- Molecular cloning Carbohydrates -- Metabolism -- Genetic aspects Homeostasis -- Genetic aspects UCTD Gabriel, Casey Ashley Genetic manipulation of trehalose-6-phosphate synthase – a study on the functional activity and role in carbohydrate homeostasis in plants |
| title | Genetic manipulation of trehalose-6-phosphate synthase – a study on the functional activity and role in carbohydrate homeostasis in plants |
| title_full | Genetic manipulation of trehalose-6-phosphate synthase – a study on the functional activity and role in carbohydrate homeostasis in plants |
| title_fullStr | Genetic manipulation of trehalose-6-phosphate synthase – a study on the functional activity and role in carbohydrate homeostasis in plants |
| title_full_unstemmed | Genetic manipulation of trehalose-6-phosphate synthase – a study on the functional activity and role in carbohydrate homeostasis in plants |
| title_short | Genetic manipulation of trehalose-6-phosphate synthase – a study on the functional activity and role in carbohydrate homeostasis in plants |
| title_sort | genetic manipulation of trehalose 6 phosphate synthase a study on the functional activity and role in carbohydrate homeostasis in plants |
| topic | Sugarcane -- Genetics Trehalose Transgenic plants -- Genetic engineering Saccharomyces cerevisiae -- Molecular cloning Carbohydrates -- Metabolism -- Genetic aspects Homeostasis -- Genetic aspects UCTD |
| url | http://hdl.handle.net/10019.1/110423 |
| work_keys_str_mv | AT gabrielcaseyashley geneticmanipulationoftrehalose6phosphatesynthaseastudyonthefunctionalactivityandroleincarbohydratehomeostasisinplants |