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Investigation of exopolysaccharide producing bacteria isolatedWillard, Kyle 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The deterioration of harvested sugarcane as a result of bacterial growth causes
major losses of sucrose and a build-up of exopolysaccharides (EPS).
Polysaccharides present during production increase the massecuite viscosity, which
negatively influences evaporation and crystallisation. In this study 38 culturable EPSproducing
bacteria were isolated from milled sugarcane. Analysis of the EPS showed
the ubiquitous presence of glucose, however, 14 polysaccharides also contained
mannose, fructose or galactose. In vitro treatment using Chaetomium erraticum
dextranase to evaluate is effectiveness indicated that 37 of the EPS were hydrolysed
to some extent. There were 21 polysaccharides that were only partially digested. The
capacity of the isolates to produce EPS on different sugars indicated a correlation
between sucrose and polysaccharide formation in 37 isolates. The results indicate
there are more species involved in EPS production than previously thought as well
as the presence of non-dextran polysaccharides. / AFRIKAANSE OPSOMMING: Bakteriële groei veroorsaak ‘n afname in gehalte, sukrose en ‘n verhoging in die
hoeveelheid van eksternepolisakkeriede (EPS). Die verhoogde konsentrasie van
polysakkariede gedurende die verwerkingsprosses veroorsaak ‘n verhoging in
“massecuite” viskositeit. Hierdie verskynsel het ‘n nadelige uitwerking op die
verdamping en kristalvorming van die produk. In gemaalde skuikerriet was 38
groeibare EPS-produserende bakterieë geisoleer. Die geanaliseerde EPS van
hierdie bogenoemde bakterieë was daar in almal glukose teenwoordig. In 14 van
hulle was mannose, fruktose en galaktose ook gevind. Die in vitro effektiwieteit van
Chaetomium erraticum dekstranase op die EPS het gewys dat 37 het tot ‘n mate
gehidroliseer maar 21 was net gedeeltelik verteer. As gevolg van die bo-genoemde
resultate was daar gevind dat sukrose was ‘n noodsaaklike subtraat vir EPS
produksie in die geisoleerde bakterieë. In hierdie studie was bevestig ‘n groter
verskiedenheid EPS-produserende bakterieë gevind was en dat hulle assosiasie aan
sukierriet prossering meer kompleks is as wat vooreen gedink was.
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Production of libraries to study biopolymer metabolism in Arabidopsis thaliana and Tylosema esculentumSwart, Corne 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Biopolymers and bio-degradable polymers are of utmost importance to ensure a sustainable economy. Industry depends on raw material, which in many cases are derived from fossil fuels, but in light of looming energy crises and green revolutions attention is being directed at cellulose and starch biopolymers. This study was therefore set forth to investigate novel genetic key elements of cell wall metabolism in Arabidopsis thaliana and starch synthesis in an under-utilized southern African crop plant, Tylosema esculentum.
In the first section of the study a cDNA library of good quality was constructed from regenerating A. thaliana protoplasts as it was expected to be enriching for genes involved in cell wall biosynthesis. Small scale EST sequencing of the library confirmed that a few sequences were similar to genes identified to be highly expressed during protoplast regeneration. The library was to be screened by expression in a microalgae as it is anticipated that cell wall metabolising genes would change the wall structure and visibly alter the colony morphology. An attempt was made at establishing a high-throughput transformation system in the unicellular algae Chlorella protothecoides in which the library was proposed to be screened. Conventional microalgal transformation techniques do not appear to be effective in this strain as the study produced no transgenic algae. Alternative studies into a screening system within another species could still lead to the identification of cell wall biosynthetic genes, which was the first objective in the study.
The second objective in the study was to investigate the potential of the orphan crop T. esculentum as starch-producing cash-crop in developing southern African countries. In this section of the study a cDNA library of good quality was produced form the tuber of T. esculentum. The library was transferred to an expression vector and screened functionally in E. coli for the presence of sequences with starch synthase activity. No sequences have been identified yet and screening procedures are still on-going. The starch content in the tuber has also been determined for the first time. The relatively high starch content in combination with low agricultural inputs indicate the potential of the plant as an industrial starch source. Further investigations into the nature of the starch are proposed to identify prospective buyers within the industry. / AFRIKAANSE OPSOMMING: Biopolimere en bio-afbreekbare polimere is van kardinale belang om ‘n volhoubare ekonomie te ontwikkel. Industriële toepassings maak op die oomblik hoofsaaklik staat op fossielbrandstof verwante bronne, maar met die oog op ‘n groen revolusie en energie krissise wat dreig word meer belangstelling getoon in sellulose en stysel biopolimere. Hierdie studie is daarom onderneem om genetiese elemente te identifiseer wat betrokke is by die sintese van die selwand in Arabidopsis thaliana en stysel sintese in die suider Afrikaanse gewas Tylosema esculentum wat grotendeels onderbenut is.
In die eerste deel van die studie is ‘n cDNA biblioteek, van goeie kwaliteit, geskep vanuit A. thaliana protoplaste wat besig was om hulle selwande te herbou. Dit word verwag dat die protoplaste gedurende die tydperk aktief besig sal wees om gene uit te druk wat betrokke is by selwandsintese. DNA volgordebepaling het bevestig dat ‘n klein aantal volgordes ooreengestem het met gene wat voorheen gevind was om in ‘n oormaat uitgedruk te word tydens die herbou van protoplas-selwande. Daar was beoog om die biblioteek in ‘n mikroalge uit te druk en sodoende die morfologie op kolonievlak waar te neem vir verandering wat in die selwand meegebring is. Om hierdie rede was die doel om ‘n hoë opbrengs transformasie sisteem te ontwikkel in die mikroalge Chlorella protothecoides. Algemene mikroalge transformasie tegnieke blyk om nie effektief in die spesie te wees nie aangesien geen transgeniese alge waargeneem is nie. Die ontwikkeling van ‘n soortgelyke proses in ‘n ander spesie kan steeds lei na die ontdekking van gene betrokke by selwandsintese in A. thaliana wat die eerste uitkoms van die projek as geheel was.
Die tweede uitkoms van die projek was om te ondersoek wat die waarskynlikheid was om T. esculentum te kommersialiseer as ‘n stysel gewas en sodoende ‘n inkomste te skep vir arm boere in ontwikkelende lande in suider Afrika. In hierdie gedeelte van die projek was daar ‘n goeie cDNA biblioteek geskep uit die knol van T. esculentum. Die biblioteek is oorgedra na ‘n plasmied waarop dit aktief uitgedruk kon word in Escherischia coli G6MD2 en daar is gesoek na volgordes wat lei na die sintese van stysel in hierdie bakterieë. Tot op hede is geen sulke volgordes gevind nie, maar die ondersoek gaan steeds voort. Die styselinhoud van die knol is ook vir die eerste keer bepaal in hierdie ondersoek. ‘n Styselinhoud wat relatief hoog is en die lae moeite wat geverg word om die gewas te verbou toon dat die plant potensieel het as ‘n kommersiële bron van stysel. Verdere ondersoeke in die aard van die stysel word ook voorgestel om toekomstige industriële kopers te identifiseer.
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Investigating the role of Brettanomyces and Dekkera during winemakingOelofse, Adriaan 12 1900 (has links)
Thesis (PhD (Genetics. Plant Biotechnology))--Stellenbosch University, 2008. / Wine quality is greatly influenced by the number of microorganisms, which occur
throughout the winemaking process. These microorganisms are naturally present on
the grapes and in the cellar from where they can be introduced to the winemaking
process at any given time and consequently impart specific contributions to the wine
quality. However, these microorganisms can be seen either as beneficial or as wine
spoilage microorganisms, depending on the conditions under which they can
proliferate during the winemaking process. Wine yeasts (Saccharomyces spp.) are
typically responsible for the alcoholic fermentation; lactic acid bacteria (LAB) are
responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) and
other wild yeasts (non-Saccharomyces spp.) are typically associated with the
formation of off-flavours under poorly controlled winemaking conditions.
In recent years, evidence from the wine industry has highlighted a specific group
of non-Saccharomyces yeast species as a serious cause for wine spoilage that
required more research investigations. Yeast of the genus Brettanomyces or its
teleomorph Dekkera has been identified as one of the most controversial spoilage
microorganisms during winemaking as they can produce several compounds that are
detrimental to the organoleptic quality of wine. This has triggered the research
initiative behind this doctoral study on the significance of Brettanomyces and Dekkera
yeasts during winemaking.
In this dissertation, various aspects of the detection, isolation and identification
methods of Brettanomyces yeast from the winemaking environment were
investigated. As a first objective, a culture collection of Brettanomyces bruxellensis
wine isolates had to be established. This followed after the isolation of
Brettanomyces yeasts from various red wine cultivars from South African wineries
from different stages of the winemaking process. Different conventional
microbiological methods such as plating on selective agar media and microscopy
were investigated along with molecular identification techniques such as the
polymerase chain reaction (PCR) in this regard.
Other focus areas of this study aimed at performing genetic characterisation and
differentiation studies of B. bruxellensis wine isolates. For this purpose, different
intraspecific identification methods were investigated on several strains, including
strains of European origin. The application of molecular techniques allowing strain
identification aided in the selection of specific strains that were evaluated for volatile
phenol production in synthetic media and wine. The results obtained from this work
indicated that a large degree of genetic diversity exists among B. bruxellensis strains
and that the volatile phenol production differed between the strains after evaluation in
synthetic media and wine.
In addition to the molecular intraspecific strain identification techniques that were
investigated, a feasibility study was also performed that focused on evaluating Fourier transform infrared (FTIR) spectroscopy combined with chemometrics as an
alternative approach for differentiating between B. bruxellensis strains.
The two approaches of FTIR spectroscopy that were investigated involved the
use of firstly, Fourier transform mid-infrared (FTMIR) spectroscopy to obtain spectral
fingerprints of spoiled wines by different B. bruxellensis strains; and secondly,
Attenuated total reflectance (FTIR-ATR) to obtain spectral fingerprints from whole
cells of B. bruxellensis on microbiological agar media. The results of this study
illustrated the potential of FTIR spectroscopy to become a reliable alternative to
molecular based methods for differentiating between B. bruxellensis strains and for
characterisation studies.
The formation of volatile phenols in wine by species of the genera Brettanomyces
and Dekkera is one of the primary reasons for their classification as wine spoilage
yeasts. The enzymatic activities of this reaction have been identified and involve a
phenyl acrylic (phenolic) acid decarboxylase (PAD) and a vinyl phenol reductase
(VPR). However, only a limited amount of information is available about these
enzymes from Brettanomyces/Dekkera yeasts and no genetic data have been
described. It was therefore imperative that this dissertation should include a genetic
investigation into the phenylacrylic (hydroxycinnamic) acid decarboxylase from the
species B. bruxellensis involved in the formation of volatile phenols. Strategies that
were investigated included various molecular DNA techniques and protein purification
procedures to obtain either genetic or protein sequence data. The decarboxylase
activity of this yeast species towards p-coumaric acid was demonstrated and
substantial genetic sequence data was obtained.
The results from this dissertation made a substantial contribution to the current
available knowledge about Brettanomyces/Dekkera spp. and led to a better
understanding of this wine spoilage yeast. This research developed a platform from
which further investigations could follow and the knowledge gained will be invaluable
for future Brettanomyces research projects at the Institute for Wine Biotechnology at
Stellenbosch University.
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Isolation and evaluation of the sugarcane UDP-glucose dehydrogenase gene and promoterVan der Merwe, Jennie 12 1900 (has links)
Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006. / The young internodes of sugarcane are ideal targets for altering metabolism, through genetic
manipulation, to potentially control known fungal diseases such as Smut or to increase sucrose
yields in these regions that are currently being discarded. At present, no regulatory sequences
that specifically drive transgene expression in young developing sugarcane tissues are available.
The objective of this study was therefore to isolate and evaluate such a sequence. The promoter
targeted for isolation in this study regulates the expression of UDP-glucose dehydrogenase (EC
1.1.1.22), an enzyme which catalyses the oxidation of UDP-glucose to UDP-glucuronic acid, a
precursor for structural polysaccharides which are incorporated into the developing cell wall. A
strong correlation between the expression of UDP-glucose dehydrogenase and a demand for
structural polysaccharides in developing tissues could therefore be expected.
The first part of this study addressed the general practicality of promoter isolation from
sugarcane, a complex polyploid. A gene encoding UDP-glucose dehydrogenase was isolated
from a sugarcane genomic library. The gene contains an open reading frame (ORF) of 1443 bp,
encoding 480 amino acids and one large intron (973 bp), located in the 5’-UTR. The derived
amino acid sequence showed 88 – 98% identity with UDP-glucose dehydrogenase from other
plant species, and contained highly conserved amino acid motifs required for cofactor binding
and catalytic activity. Southern blot analysis indicates a low copy number for UDP-glucose
dehydrogenase in sugarcane. The possible expression of multiple gene copies or alleles of this
gene was investigated through comparison of sequences amplified from cDNA prepared from
different tissues. Although five Single Nucleotide Polymorphisms (SNP) and one small-scale
insertion/deletion (INDEL) were identified in the aligned sequences, hundred percent identity of
the derived amino acid sequences suggested the expression of different alleles of the same gene
rather than expression of multiple copies. The finding that multiple alleles are expressed to
provide the required level of a specific enzyme, rather than the increased expression of one
dominant allele, is encouraging for sugarcane gene and promoter isolation.
In the second part of the study the suitability of UDP-glucose dehydrogenase as a target for the
isolation of a developmentally regulated promoter was investigated. The contribution of UDP glucose dehydrogenase to pentan synthesis, as well as the expression pattern and subcellular
localisation of the enzyme in mature sugarcane plants was studied at the tissue and cellular level.
Radiolabelling with positionally labelled glucose was used to investigate the relative
contributions of glycolysis, the oxidative pentose phosphate pathway and pentan synthesis to
glucose catabolism. Significantly (P=0.05) more radiolabel was released as CO2 from [6-14C]-
glucose than [1-14C]-glucose in younger internodes 3, 4 and 5, demonstrating a significant
contribution of UDP-glucose dehydrogenase to glucose oxidation in the younger internodes. In
addition, there was significantly (P=0.05) more radiolabel in the cell wall (fiber) component
when the tissue was labelled with [1-14C]-glucose rather than [6-14C]-glucose. This also
demonstrates a selective decarboxylation of glucose in position 6 prior to incorporation into the
cell wall and is consistent with a major role for UDP-glucose dehydrogenase in cell wall
synthesis in the younger internodes.
Expression analysis showed high levels of expression of both the UDP-glucose dehydrogenase
transcript and protein in the leafroll, roots and young internodes. In situ hybridisation showed
that the UDP-glucose dehydrogenase transcript is present in virtually all cell types in the
sugarcane internode, while immunolocalisation showed that the abundance of the protein
declined in all cell types as maturity increased. Results obtained confirmed that this enzyme
plays an important role in the provision of hemicellulose precursors in most developing tissues of
the sugarcane plant, indicating that UDP-glucose dehydrogenase was indeed a suitable target for
promoter isolation.
Lastly, the promoter region and first intron, located in the 5’-untranslated region (UTR) of this
gene, were isolated and subsequently fused to the GUS reporter gene for transient expression
analysis and plant transformation. Transient expression analysis showed that the presence of the
intron was essential for strong GUS expression. Analysis of stably transformed transgenic
sugarcane plants, evaluated in a green house trial, showed that the isolated promoter is able to
drive GUS expression in a tissue specific manner under these conditions.
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Development of a transformation system for sugarcane (Saccharum spp. hybrids) in South Africa using herbicide resistance as a model systemSnyman, Sandra Jane 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT:
Please refer to fulltext for abstract / AFRIKAANSE OPSOMMING:
Sien asb volteks vir opsomming
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Nitric oxide-mediated signaling in legumes and its role in maize responses to salt stressKeyster, Marshall 03 1900 (has links)
Thesis (PhD (Genetics))--University of Stellenbosch, 2011. / Includes bibliography. / Please refer to full text to view abstract.
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Isolation of grapevine promoters with special emphasis on the vacuolar pyrophosphataseVenter, Mauritz 04 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Understanding the complex nature of grapevine molecular biology is of great importance for
viticulturists. Progress in the elucidation of key events on a genetic level could provide further
insight into the underlying cues responsible for the precise control of physiological and
metabolic changes during a specific condition such as fruit development. The use and analysis
of molecular ‘tools’, such as promoters controlling the site and level of gene activity, could
assist in the understanding of grapevine biology and serve as a platform for the future design
and development of recombinant DNA protocols and strategies for Vitis vinifera L.
A high-throughput gene expression system, cDNA-AFLPs, was successfully used to analyse
large-scale transcriptional activity during berry ripening. Candidate cDNA fragments were
selected on the basis of desired expression patterns and/or known gene function for subsequent
promoter isolation. From three candidate cDNAs selected, the promoter of a gene encoding
vacuolar pyrophosphatase (V-PPase) was isolated for computational and comparative analyses.
Promoter activity was evaluated on a transient level using the green fluorescent protein (GFP)
reporter gene. Comparative integration has allowed for putative correlation of cis-elements,
acting as receptors within promoter regions, to regulate V-PPase gene expression in response to
development, environmental stress and tissue-specificity.
In this study, integration of genetic data have advanced the understanding and transcriptional
role of a key enzyme (V-PPase) during grape ripening. Although never a replacement for
experimental verification, this integrative strategy of combining gene expression profiles with
bioinformatics and regulatory data will greatly assist in further elucidation of various other key
components and regulatory cues associated with grapevine molecular biology. This study has
allowed us to use molecular tools that could assist in gaining further insight into genetic
complexities and could serve as a platform for a more refined genetic manipulation strategy in
Vitis vinifera L. / AFRIKAANSE OPSOMMING: Begrip van die komplekse aard van wingerd molekulêre biologie is van groot belang vir wingerdkundiges. Vooruitgang in die begrip van belangrike gebeurtenisse op ń genetiese vlak behoort verdere insig in die onderliggende instruksies vir die noukeurige beheer van fisiologiese en metaboliese veranderinge tydens ń spesifieke kondisie soos vrug rypwording te bevorder. Die gebruik en analise van molekulêre ‘instrumente’ soos promoters, wat die posisie en vlak van geen aktiwiteit beheer, kan bydra tot n beter begrip van wingerd biologie en sodoende dien as ń platform vir die toekomstige ontwerp en ontwikkeling van rekombinante DNS (deoksiribonukleiensuur) protokolle en strategieë vir Vitis vinifera L. ń Hoë-kapasiteit geen uitdrukkings sisteem, nl. kDNS-AFLPs (komplementêre deoksiribonukleiensuur- geamplifiseerde fragment lengte polimorfisme), is suksesvol gebruik vir die analise van grootskaalse transkripsionele aktiwiteit tydens druif rypwording. Kandidaat kDNS fragmente is geselekteer, gebaseer op verlangde uitdrukkings-patrone en/of bekende geen funksie vir daaropvolgende promoter isolering. Van drie geselekteerde kandidaat kDNS fragmente, is die promoter van ń geen wat vakuolêre pirofosfatase (V-PPase) kodeer geïsoleer vir rekenaar- en vergelykende analise. Promoter aktiwiteit is op ń nie-stabiele vlak deur die gebruik van ń groen-fluoresserende proteien (GFP) verklikker geen geëvalueer. Vergelykende integrering het dit moontlik gemaak om veronderstelde korrelasies van cis-elemente, wat as reseptore binne ń promoter area dien, en die regulering van V-PPase geen uitdrukking, in reaksie tot ontwikkeling, omgewings stres en weefsel-spesifisiteit, te maak. Tydens hierdie studie, het die integrering van genetiese data gehelp om die transkripsionele rol van ń belangrike ensiem (V-PPase) tydens druif rypwording beter te verstaan. Alhoewel dit nooit ń plaasvervanger vir eksperimentele bewyse sal wees nie, kan hierdie gëintegreerde strategie, wat die kombinasie van geen-uitdrukkingsprofiele met bioinformatika en regulatoriese data behels, grootliks bydra om verskeie ander belangrike komponente en regulatorieseaanwysings geassosieërd met wingerd molekulêre biologie te ontrafel. Hierdie studie het verdere insig in genetiese kompleksiteite verleen, en kan nou dien as ń platform vir ń meer presiese genetiese manipulering strategie in Vitis vinifera L.
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Genetic engineering of sugarcane for increased sucrose and consumer acceptanceConradie, Tobie Tertius 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Sugarcane is a crop that is farmed commercially due to the high amounts of sucrose that
is stored within the mature internodes of the stem. Numerous studies have been done to
understand sugar metabolism in this crop as well as to enhance sucrose yields. Until now
sugarcane improvement strategies have been implemented through either breeding
programs or transgenic manipulation. Public mistrust and regulatory hurdles, however,
have made the commercialisation of transgenic crops difficult, expensive and timeconsuming.
In this thesis two projects will address issues relating to the above. The first will address
an effort to increase sucrose accumulation within the sugarcane culm. This was attempted
via the expression of an Arabidopsis thaliana vacuolar pyrophosphatase (AtV-PPase)
gene, linked to the maize ubiquitin promoter, in sugarcane callus. It was anticipated that
increased activity of the tonoplast-bound AtV-PPase will result in increased sucrose
accumulation in the vacuole. Transgenic sugarcane callus lines were tested for soluble
sugar content which suggested no significant increase in sucrose content. However, this
may change upon further assessment of sugarcane suspension cultures and glasshouse
plants.
The second project was concerned with the development of a novel sugarcane
transformation technology that utilises only sugarcane sequences. This ‘cisgenic’
approach to sugarcane transformation will require a native sugarcane promoter,
terminator, vector backbone and selection marker. It was attempted to first isolate a functional promoter as well as developing a selection system based on an endogenous
selection marker.
A promoter was amplified from sugarcane, using primers designed on a sorghum
template, and its expression assessed using a GFP reporter gene. Unfortunately
expression could not be confirmed in transgenic sugarcane callus. Currently, an alternative
approach is followed by using short fragments of constitutively expressed genes to screen
sugarcane Bacterial Artificial Chromosome (BAC) libraries to isolate their corresponding
promoters.
Lastly, it was attempted to develop a selection system for transgenic sugarcane based on
resistance to the herbicide chlorosulfuron. A mutant acetolactate synthase (alsb) gene
from tobacco, which has shown to confer resistance to the tobacco, was transformed into
sugarcane callus. It was anticipated that this gene will confer chlorosulfuron resistance to
transgenic sugarcane. If resistance is achieved, the corresponding sugarcane gene will be
mutated via site-directed mutagenesis and checked if it also confers resistance to
sugarcane. Results showed that although transgenic lines were generated, resistance
development is still inconclusive. / AFRIKAANSE OPSOMMING: Suikerriet is ‘n kommersiële gewas wat verbou word as gevolg van die hoë hoeveelhede
sukrose wat gestoor word in die volwasse tussenknope van die stam. Verskeie studies is
al gedoen om suiker metabolisme in die gewas te ondersoek, sowel as om die sukrose
opbrengs te verhoog. Huidige strategieë vir suikerriet verbetering word beywer deur
middel van teel-programme of transgeniese manipulasie. Die kommersialiseëring van
transgeniese gewasse word egter bemoeilik deur publieke wanpersepsies, sowel as
regulatoriese uitdagings.
Hierdie tesis beoog om boenoemde kwessies aan te spreek, deur middel van twee
projekte. Die eerste projek poog om sukrose akkumulasie in sukerriet te verhoog. Dit was
onderneem om die Arabidopsis thaliana vakuolere pirofosfatase (AtV-PPase) geen, wat
verbind is met die mielie ubiquitien promoter, uit te druk in suikerriet kallus. Daar was
verwag dat die verhoogde aktiwiteit van die tonoplast-gebonde AtV-PPase sal veroorsaak
dat meer sukrose in die vakuool akkumuleer. Oplosbare suiker inhoud was getoets in
transgeniese suikerriet kallus lyne, maar geen merkbare verhoging in sukrose inhoud was
waargeneem nie. Hierdie mag egter verander met verdere ondersoeke in suikerriet
suspensie-kulture en glashuis-plante.
Die tweede projek het beywer om ‘n nuwe suikerriet transformasie tegnologie te ontwikkel,
wat slegs van suikerriet genetiese materiaal gebruik maak. Hierdie ‘cisgeniese’ benadering
tot suikerriet transformasie sal ‘n inheemse suikerriet promoter, terminator, vektor ruggraat
en seleksie-merker, benodig. Dit was eers beoog om ‘n funksionele promoter te isoleer,
sowel as om ‘n seleksie sisteem, gebasseer op ‘n inheemse seleksie merker, te ontwikkel. Deur gebruik te maak van primers wat op ‘n sorghum templaat gebasseer is, was ‘n
promotor geisoleer vanuit suikerriet; die uitdrukking hiervan is bepaal deur gebruik te maak
van ‘n GFP verklikker geen. Ongelukkig kon uitdrukking nie bevestig word in transgeniese
suikerriet kallus nie. Tans word suikerriet Kunsmatige Bakterieële Chromosoom (KBC)
biblioteke geskandeer, deur gebruik te maak van geen-fragmente van globaal-uitgedrukte
gene, om ooreenstemmende suikerriet promoters te isoleer.
Die tweede deel van die cisgeniese projek het beoog om ‘n seleksie sisteem vir
transgeniese suikerriet te ontwikkel, wat gebasseer is op weerstand teen die plantdoder
chlorosulfuron. Suikerriet kallus was getranformeer met ‘n mutante tabak geen –
asektolaktaat sintase (alsb) – wat chlorosulfuron weerstand in tabak meebring. Daar was
verwag dat die geen chlorosulfuron weerstand aan transgeniese suikerriet sou oordra.
Indien weerstand ontwikkel, sal die ooreenstemende suikerriet geen deur gerigte
mutagenese gemuteer word; dan sal dit kan bepaal word of weerstand ook oorgedra word
aan suikerriet. Daar is bevind dat alhoewel transgeniese lyne gegenereer is, daar steeds
nie ‘n konklusiewe bevestiging van weerstand ontwikkeling is nie.
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Analysis of the effects of the plant growth promoting substances GR24 and smoke water on abiotically stressed Nicotiana benthamiana seedlingsSteenkamp, Letitia Elizabeth 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Almost all processes during the life of a plant are affected by the environment.
Changes in phytohormone, metabolite and protein levels follow in response to
changes in the environment. Plant growth promoting substances can stimulate
changes at these levels to facilitate increased plant growth and yields above what
the plant would normally establish. In this study, the effects of two growth promoting
substances, smoke water (SW) derived from bubbling smoke from the burning of
plant material through water, and a synthetic strigolactone analogue, GR24, on plant
growth and architecture, as well as the proteome and metabalome of salt stressed
Nicotiana benthamiana seedlings were investigated. Physiological studies were
conducted to identify the effects of the growth substances on salt stressed seedlings
in a tissue culture system. Under non-stress conditions, SW treatment increased
seedling fresh mass, root length and leaf area. Under salt stress conditions (100
mM and 150 mM NaCl), SW increased fresh mass, root length, leaf number and
lateral root number significantly. Under non-stress conditions, GR24-treated
seedlings showed increased fresh mass, leaf number and area and root length.
When GR24-treated seedlings were placed under salt stress, the seedlings showed
significant increases in fresh mass, leaf number and lateral root number, but only
marginal increases in root length and leaf area. Despite these similarities, slight
differences were observed in the metabolomes and proteomes of smoke water and
GR24-treated seedlings, both with and without the addition of salt stress. Relatively
few of the differentially expressed proteins could be identified with the instruments
available. Changes in the metabolome indicated that photoassimilation and
photosynthesis could be affected in response to smoke water and GR24 treatment.
Our results suggest that smoke water and GR24 both promote growth under salt
stress conditions in seedlings and we furthermore conclude that, although there are
distinct overlaps between treatments, this is accomplished via slightly different
mechanisms. / AFRIKAANSE OPSOMMING: Gedurende ‘n plant se lewe word omtrent alle prosesse deur die omgewing
geaffekteer. Veranderinge in die omgewing word gevolg deur veranderinge in
hormoon, metaboliet en protein vlakke. Plant groei stimulante affekteer hierdie
vlakke om plant groei en -opbrengs na bo normalle vlakke te verhoog. In hierdie
studie word die effek van twee groei stimulante, rook water verkry deur rook van
plant materiaal deur water te borrel en ‘n sintetiese strigolaktoon, GR24, ondersoek
op ‘n morfologiese, metaboliese en ‘n proteomiese vlak in Nicotiana benthamiana
saailinge. ’n Studie is onderneem om die veranderinge as gevolg van die
onderskeie groei stimulante te ondersoek in ‘n weefsel kultuur sisteem. Rook water
het onder normale groei omstandighede vars en droeë massa, blaar aantal asook
wortel en blaar lengte verhoog. Rook water het na sout behandeling (100 en 150
mM NaCl) steeds vars massa, wortel lengte, blaai aantal en laterale wortel aantal
beduidend verhoog in vergelyking met die sout stres kontrole. Behandeling met
GR24 het ook vars massa, wortel lengte, blaar aantal en grootte verhoog en onder
sout stres met GR24 is ‘n beduidende vergroting opgemerk in vars massa, blaar
grootte en laterale wortel aantal. Ongeag van die veranderinge in groei is klein
verskille opgemerk in die metaboliet en protein studies. Net ‘n paar proteine kon
positief geidentifiseer word met die apparaat beskikbaar. Verandering in die
metaboloom wys na veranderinge in fotoassimilasie en fotosintese in reaksie tot rook
water en GR24. Hierdie resultate lei tot die gevolgtrekking dat rook water en GR24
beide groei verbeter in saailing behandel met sout en ook dat alhoewel daar sekere
ooreenkomste is tussen die reaksies as gevolg van die plant groei stimulante, dit wel
geskiet deur geringe verskillende meganismes.
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Isolation and characterisation of genes encoding biopolymer manufacturing enzymesRapp, Telana 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Biopolymers exhibit the required material properties to replace conventional, non-biodegradable, petroleum-based polymer products. They have a closed carbon cycle, making them carbon neutral and environmentally friendly. Biopolymers are produced from non-toxic substrates during in vivo enzymatic reactions. Biosynthesis of the most commercially important biopolymers is too complex to be reproduced in in vitro reactions. Identification of the genes responsible for their biosynthesis has been under investigation, with some pathways already elucidated. The genes involved in the biosynthesis of these polymers have been targeted for genetic manipulation to increase productivity, as well as create tailor-made polymers. Novel biopolymers and the genes responsible for their synthesis are of interest for their potential commercial applications. Bacteria produce a wide range of biopolymers and are being implemented as the bio-factories for biopolymer production. They are capable of utilising easily accessible and renewable carbon sources such as sucrose for polymer biosynthesis. Bacteria thus allow for economical production of these environmentally beneficial polymers.
In this study, the gene responsible for the production of an unknown biopolymer from an unknown bacterium was identified. The biopolymer producing bacteria were grown on media enriched with sucrose as carbon source, during an expression library screening in a previous study. Expression library technology was used to search for the gene and it was identified as a 424 amino acid levansucrase which had a 100% homology to Leuconostoc mesenteroides M1FT levansucrase (AAT81165.1). Biopolymer analysis revealed that the biopolymer was a levan, a polysaccharide consisting of only fructose molecules with a molecular weight of ± 5 kDa. Analysis of a 516 bp fragment of the 16S rRNA determined that the unknown bacteria were a Pseudomonas species. / AFRIKAANSE OPSOMMING: Bio-polimere besit noodsaaklike materiële eienskappe wat toelaat dat dit konvensionele, nie bio-afbreekbare, petroleum-gebasseerde polimeer produkte kan vervang. Hulle het n geslote koolstof kringloop en is dus koolstof neutraal en omgewingsvriendelik. Bio-polimere word vervaardig van nie-toksiese substrate, gedurende ensiematiese reaksies in vivo. Die belangrikste kommersiële bio-polimere se ensiematiese produksie is te kompleks om in ʼn in vitro reaksie te herproduseer. Ondersoeke tot die identifikasie van die gene wat verantwoordelik is vir die produksie van die polimere is onderweg, en sommige produksie paaie is reeds bekend. Die bekende gene word geteiken vir genetiese manipulasie om hulle produktiwiteit te vermeerder en om unieke polimere te produseer. Unieke bio-polimere en die gene wat vir hul produksie verantwoordelik is, is van belang vir hulle potentiële implimentering in komersiële toepassings. Bakteria produseer ʼn verskeidenheid bio-polimere en word as die bio-fabrieke vir polimeerproduksie geimplimenteer. Hulle kan maklik bekombare koolstofbronne, soos sukrose, gebruik om bio-polimere te produseer. Bakteria laat dus die ekonomiese produksie van hierdie omgewingsvriendelike polimere toe.
In hierdie studie word die geen wat verantwoordelik is vir die produksie van ʼn onbekende bio-polimeer van ʼn onbekende bakteria, geidentifiseer. Die bakteria was gevind op media, wat verryk was met sukrose as koolstofbron, tydens ʼn vorige studie, waartydens ʼn uitdrukkingsbiblioteek gesif was op hierdie media. Uitdrukkingsbiblioteek tegnologie was gebruik om die geen te vind. Die geen was geidentifiseer as ʼn 424 aminosuur, homo-fruktose-polimeer produseerende geen, ʼn “levansucrase”. Die geen het ʼn 100% homologie met die M1FT “levansucrase” geen (AAT81165.1) van Leuconostoc mesenteroides gehad. Analise van die bio-polimeer het bepaal dat die polimeer ʼn polisakkaried was, wat slegs uit fruktose molekules bestaan het. Die molekulêre gewig van die polimeer was ± 5 kDa. Analise van ʼn 516 bp fragment van die 16S rRNS het bepaal dat die bakteria van die Pseudomonas spesie afkomstig was.
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