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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/2633 |
| Date | 12 1900 |
| Creators | Samodien, Mugammad Ebrahim |
| Contributors | Lloyd, J. R., Kossmann, J. M., University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB) |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | Unknown |
| Type | Thesis |
| Rights | University of Stellenbosch |
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