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TARP Promoter-Based Prostate Cancer Gene Therapy : From Development to ApplicationCheng, Wing-Shing January 2005 (has links)
Prostate cancer is one leading cause of cancer-related death among men in Western countries. The standard therapies for localized prostate cancer include radical prostatectomy and radiation therapy. Such measures are relatively effective in the short term, but many patients ultimately relapse. These patients may benefit from a combination of standard therapy and oncolytic virus therapy. My work aimed to develop viruses for this purpose. TARP is a protein that in males is specifically expressed in prostate epithelial and cancer cells. In my thesis, I characterized the TARP promoter and showed that TARP expression is regulated at the transcriptional level by testosterone through binding of the androgen receptor in the proximal TARP promoter. I further developed TARP promoter-based regulatory sequences for prostate-specific gene expression. A sequence comprising a PSA enhancer, a PSMA enhancer and the TARP promoter was constructed and designated PPT. An adenoviral vector containing the PPT sequence shielded from transcriptional interference by an H19 insulator showed high prostate-specific transcriptional activity in human cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer the PPT sequence is not active. Therefore, a two-step transcriptional amplification (TSTA) system was used together with the PPT sequence to develop an adenovirus that confers prostate-specific transgene expression also in murine cells. I constructed a conditionally replicating adenovirus where the E1A gene expression is controlled by an H19 insulator-shielded PPT regulatory sequence, Ad[I/PPT-E1A]. This virus exhibited absolute prostate specificity in terms of E1A expression, viral replication and cytolysis in vitro and in vivo. Importantly, our virus is active both in the presence and absence of testosterone, which may prove beneficial for patients treated by hormonal withdrawal. Hopefully, my work will improve existing gene therapy strategies for prostate cancer and in the long term improve the prognosis for patients with prostate cancer.
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Industrial yeast strains engineered for controlled flocculationGovender, Patrick 03 1900 (has links)
Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / In many industrial fermentation processes, Saccharomyces cerevisiae yeast should ideally meet two partially conflicting demands. During fermentation a high suspended yeast count is of paramount importance to maintain a rapid fermentation rate, whilst efficient flocculation should ideally be initiated only on completion of the primary alcoholic fermentation, so as to enhance product clarification and recovery. Most commercial wine yeast strains are non-flocculent, probably because this trait was counter-selected to avoid fermentation problems. In this study, we assessed molecular strategies to optimise the flocculation behaviour of non-flocculent laboratory and wine yeast strains. For this purpose, the chromosomal copies of three dominant flocculation genes, FLO1, FLO5 and FLO11, of a non-flocculent S. cerevisiae laboratory strain (FY23) and two commercial wine yeast strains (BM45 and VIN13) were placed under the transcriptional control of the stationary phase-inducible promoters of the S. cerevisiae ADH2 or HSP30 genes.
Under standard laboratory media and culture conditions, all six promoter-gene combinations resulted in specific flocculation behaviours in terms of timing and intensity. The data show that the strategy resulted in the expected and stable expression patterns of these genes in both laboratory and industrial wine yeast strains. Most importantly, the data confirm that inducible expression of the native FLO1 and FLO5 open reading frames, albeit to varying degrees, are responsible for a quantifiable cell-cell adhesion phenotype that can be characterized as a Flo1 flocculation phenotype. On the other hand, we found that inducible expression of the native FLO11 ORF under these conditions resulted in flor/biofilm formation and invasive growth phenotypes. However, the specific impact of the expression of individual dominant FLO genes with regard to characteristics such as flocculation efficiency, cell wall hydrophobicity, biofilm formation and substrate adhesion properties showed significant differences between the commercial strains as well as between commercial and laboratory strains. These adhesion phenotype differences may at least in part be attributed to wine yeast FLO gene open reading frames containing significantly smaller intragenic repeat regions than laboratory strains.
The data show that the ADH2 regulatory sequences employed in this study were unsuitable for the purpose of driving FLO gene expression under wine-making conditions. However, HSP30p-based FLO1 and FLO5 wine yeast transformants displayed similar flocculent phenotypes under both synthetic and authentic red wine-making conditions, and the intensities of these phenotypes were closely aligned to those observed under nutrient-rich YEPD conditions. The fermentation activities of
HSP30p-based transgenic yeast strains were indistinguishable from that of their parental host wine yeast strains. The chemical composition of wines obtained using transgenic yeast strains were similar to those produced by parental strains. The BM45-derived HSP30p-FLO5 transformant in particular was capable of generating compacted or ‘caked’ lees fractions, thereby providing a distinct separation of the fermented wine product and lees fractions. Furthermore, in this study we report a novel FLO11 induced flocculation phenotype that seems to exclusively develop under authentic red wine-making conditions. This strong FLO11 flocculation phenotype was not wine yeast strain dependant, possessed both Ca2+-dependant and Ca2+-independent flocculation characteristics and was insensitive to inhibition by both glucose and mannose. A distinct advantage of this unique FLO11 phenotype was highlighted in its ability to dramatically promote faster lees settling rates. Moreover, wines produced by HSP30p-FLO11 wine yeast transformants were significantly less turbid than those produced by their wild type parental strains. The benefit of this attractive property is it facilitates simpler and faster recovery of wines and also promotes greater volume recovery of the wine product.
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A Christian bioethical perspective on pre-implantation Genetic Diagnosis (PGD) and Genetic Manipulation (GM)Kotze, Manitza 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: With the development and continued developing of medical technology, treatments become available without the time to reflect ethically on them. Given how fast things change in medical technology, it is important to constantly reflect anew. Ethical reflection, however, seems to be lagging far behind bio-technological developments. Pre-implantation Genetic Diagnosis (PGD) and Human Genetic Manipulation (GM) is fast becoming an everyday reality and must therefore be reflected upon. Few Christian bioethical studies have been done on the impact that this could have on the larger populace, especially the local population in South Africa, where only a small percentage would be able to access these possible treatments.
This study is motivated by the quest of ethicists in general and Christian ethicists in particular, to respond adequately and appropriately to the challenges posed by bio-technological developments. The study will outline and discuss the various Christian perspectives on PGD and GM. It will be shown that most Christian responses to bio-technological matters are done from within the framework of the doctrine of creation. In response, this study will then discuss a trinitarian perspective on the confession of God as creator and investigate whether this perspective might advance and enrich, and even amend, the quests of Christians to formulate ethical responses to the challenges posed by PGD and GM. I have made the decision to focus, for the most part, only on the work of one theologian, and will therefore be applying the trinitarian doctrine of creation as found in the work of Jürgen Moltmann to the development of a Christian bioethical perspective.
Seeing that Christian ethics in general is concerned with human dignity, social justice and wellbeing, as well as moral upliftment, the ethical implications of this type of medical technology in the South African context, with its uneven distribution of wealth and access to medical care, must also be addressed from the perspective of this study. In this regard, the concept of human beings created imago Dei (in the image of God), with inherent human dignity, is of particular importance. / AFRIKAANSE OPSOMMING: Met die ontwikkeling en voortdurende ontwikkeling van mediese tegnologie word behandelinge beskikbaar sonder dat daar tyd is om eties daaroor te reflekteer. Gegewe hoe vinnig dinge verander in mediese tegnologie is dit belangrik om voortdurend nuut te reflekteer. Pre-implantasie Genetiese Diagnose (PGD) en Menslike Genetiese Manipulasie (GM) is vinnig beter om ‘n alledaagse realiteit te word en daarom moet daar daaroor reflekteer word. Daar is min Christelike bio-etiese studies gedoen oor die impak wat dit op die groter samelewing kan hê, veral in die plaaslike bevolking van Suid-Afrika, waar slegs ‘n klein persentasie toegang tot hierdie moontlike behandelinge sal hê.
Hierdie studie word gemotiveer deur die poging van etici in die algemeen en Christelike etici spesifiek, om behoorlik en toepaslik te reageer op die uitdagings wat bio-tegnologiese ontwikkelinge bied. Die studie sal die verskillende Christelike perspektiewe op PGD en GM uiteensit en bespreek. Daar sal aangedui word dat die meeste Christelike antwoorde op die bio-tegnologiese kwessies gedoen word binne die raamwerk van die skeppingsleer. In reaksie hierop sal hierdie studie dan 'n trinitariese perspektief op die belydenis van God as Skepper bespreek en ondersoek of hierdie perspektief die poging om ‘n Christelike etiese antword te formuleer op die uitdagings wat PGD en GM bied kan bevorder en verryk, en moontlik selfs wysig. Ek het die besluit geneem om hoofsaaklik net op die werk van een teoloog te fokus, en sal dus die trinitariese skeppingsleer soos gevind in die werk van Jürgen Moltmann toepas tot die ontwikkeling van 'n Christelike bio-etiese perspektief.
Aangesien die Christelike etiek in die algemeen gemoeid is met menswaardigheid, maatskaplike geregtigheid en welstand, asook morele opheffing, moet die etiese implikasies van hierdie tipe mediese tegnologie in die Suid-Afrikaanse konteks, met sy ongelyke verspreiding van rykdom en toegang tot mediese sorg, ook aangespreek. In hierdie verband is die konsep van die mens geskep Imago Dei (na die beeld van God), met inherente menswaardigheid, van besondere belang.
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Over-expression and analysis of two Vitis vinifera carotenoid biosynthetic genes in transgenic ArabidopsisBrackenridge, Anika Elma 03 1900 (has links)
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006. / Plants have evolved photosynthetic systems to efficiently harvest sunlight energy for
the production of carbohydrates, but these systems also are extremely susceptible to
an excess of light. To combat the potential damaging effects of light, plants have
developed various mechanisms to control and cope with light stress. These
mechanisms include the movement of either leaves, cells (negative phototaxis) or
chloroplasts to adjust the light-capturing potential, the adjustment of the
light-harvesting antenna size through gene expression or protein degradation, the
removal of excess excitation energy either through an alternative electron transport
pathway or as heat. However, the latter mechanism based on thermal dissipation,
remains the most effective to rid the plant of damaging excess light energy. This
process involves several carotenoid pathway pigments, specifically the de-epoxidised
xanthophyll cycle pigments. The process and extent of thermal dissipation in plants
can be measured and quantified as non-photochemical quenching (NPQ) of
chlorophyll fluorescence by using well-established methodologies. Several
Arabidopsis and Chlamydomonas mutants affected in the xanthophyll cycle have
been isolated. These mutants have provided evidence for the correlation between
the de-epoxidised xanthophyll cycle pigments and NPQ as well as better
understanding of the operation of the xanthophyll cycle and the related carotenoid
biosynthetic enzymes. This key photoprotective role of the xanthophyll cycle is
therefore a promising target for genetic engineering to enhance environmental stress
tolerance in plants. Several genes from the carotenoid biosynthetic pathway of
grapevine (Vitis vinifera L.) were isolated previously in our laboratory. The main aim
of this study was to over-express two xanthophyll cycle genes from grapevine in
Arabidopsis and to analyse the transgenic population with regards to pigment content
and levels as well as certain photosynthetic parameters. The transgenic lines were
compared with wild type Arabidopsis (untransformed) plants and two xanthophyll
cycle mutants under non-limiting conditions as well as a stress condition, specifically
a high light treatment to induce possible photodamage and photoinhibition.
Transgenic Arabidopsis lines over-expressing the two V. vinifera xanthophyll
cycle genes, β-carotene hydroxylase (VvBCH) and zeaxanthin epoxidase (VvZEP),
were established following Agrobacterium transformation. In addition to the
untransformed wild type, two NPQ mutants, npq1 (lacking violaxanthin de-epoxidase)
and npq2 (lacking zeaxanthin epoxidase), were used as controls throughout this
study. The transgenic lines were propagated to a homozygous T3-generation, where
stable integration and expression of the transgenes were confirmed in only 16% and
12% for VvBCH and VvZEP lines, respectively. No phenotypical differences could be
observed for the transgenic lines compared to the wild type, but the npq2 mutant
showed a stunted and ‘wilty’ phenotype, as was previously described. To evaluate the pigment composition of the transgenic lines a reliable and
reproducible method was needed to analyse carotenoids from leafy material. To this
end a new high-performance liquid chromatography (HPLC) method was developed
for the quantitative profiling of eight major carotenoids and chlorophyll a and b.
Emphasis was placed on baseline separation of the xanthophyll pigments, lutein and
zeaxanthin as well as the cis- and trans-forms of violaxanthin and neoxanthin. The
method effectively distinguished Arabidopsis wild type plantlets from the two NPQ
mutant lines (npq1 and 2) and could possibly find application for green leafy tissue
samples in general.
The carotenoid content of the NPQ mutants were in accordance with previous
reports. The lack of zeaxanthin epoxidase activity in the npq2 mutant resulted in the
accumulation of zeaxanthin under both low and high light conditions. This high level
zeaxanthin was found to cause an initial rapid induction of NPQ at low to moderate
light intensities, but this difference disappeared at high light, where zeaxanthin
formation induced considerable NPQ in the wild type. Similarly, the npq1 mutant was
unable to de-epoxidise violaxanthin to zeaxanthin under high light conditions, which
resulted in severe inhibition of NPQ induction. Furthermore, these mutant plantlets
were shown to be more susceptible to photoinhibition compared to that of the wild
type.
The over-expression of VvBCH resulted in a marked increase in the
xanthophyll cycle pool pigments (violaxanthin, antheraxanthin and zeaxanthin) and
reduced β-carotene levels under both low and high light conditions compared to that
the wild type, indicating elevated β-carotene hydroxylase activity possibly due to
over-expression of the VvBCH gene. Similar to the induction of NPQ in the npq2
mutant, the increased levels of zeaxanthin in the VvBCH lines did not offer any
additional photoprotection. This would suggest that the heightened zeaxanthin levels
observed for the VvBCH lines do not necessarily enhance photoprotection, however
may protect the thylakoid membrane against lipid peroxidation as has been shown
previously. The VvZEP lines however, showed reduce levels of zeaxanthin in high
light conditions to that of the wild type, probably due to the competing epoxidation
and de-epoxidation reactions of the xanthophyll cycle. This reduction in zeaxanthin
synthesis in the VvZEP lines resulted in significant reduced NPQ induction compared
that of the wild type, a phenomenon also observed for the npq1 mutant. Similar to
the npq1 mutant, these lines displayed significantly increased photoinhibition, which
may be due to photodamage of the reaction centers if one considers the lowered
photosystem II photochemistry efficiency and reaction center openness of these lines
compared to the wild type. This may suggest that even small reductions in
zeaxanthin amounts can result in an increase in photoinhibition, under high light
conditions.
This study and its results provide fundamental information regarding two
grapevine-derived carotenoid pathway genes and their possible physiological roles.
Moreover, studies like these provide information that is essential when possible biotechnological approaches are planned with this central plant metabolic pathway in
mind. The results highlighted the complex regulation of this pathway, necessitating
attention to flux control, simultaneous manipulation of several pathway genes, and
the measurement of other compounds derived from this pathway when evaluating the
possible applications of the carotenoid pathway of plants.
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The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene, as an antifungal and biocontrol agentCarstens, Maryke,1976- 04 1900 (has links)
Thesis (MScAgric) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Fungi are an extremely diverse group of organisms and, by acting as pathogens, they can
colonise various other organisms, including humans, plants and animals. The effect of this is
usually detrimental, not only to agricultural crops and livestock, but also to human well-being.
The extensive farming of crops and livestock requires persistent control of fungal populations,
commonly through the use of chemical fungicides. However, the exclusive use of fungicides is
no longer a sustainable practice, as a result of serious problems, such as increasing fungicide
resistance in pathogen strains, the high costs of fungicides, as well as concern about the
environment. The search by producers and scientists for alternative control measures is an
ongoing process.
The fungal cell wall consists of polysaccharides that not only playa role in protection of the
fungi, but also in relaying signals for the invasion and infection of susceptible hosts. Chitin, a
polysaccharide composed of N-acteylglucosamine (GleNAc) residues linked by P-1,4 glucosidic
linkages, is one of the major components of the fungal cell wall, where it plays an important role
in the apical growth of the vegetative hyphae.
Chitinases (EC 3.2.1.14) are abundant proteins produced by a variety of microorganisms
and plants and are necessary for the hydrolysis of the chitin polymer. During the invasion of
many plant species by a pathogen, the production of a specific group of proteins, designated
pathogenesis-related (PR) proteins that include chitinases, is induced as part of their defence
response. Due to the facts that pathogenic fungi contain chitin in their cell walls and that plant
chitinases are induced upon pathogen attack, chitinases have been confirmed as an integral and
crucial part of the plant's natural defence response. Chitinases have increasingly been targeted
to upregulate plants' endogenous disease resistance mechanisms through transgenic
overexpression in a variety of hosts.
Several species of fungi, including various Trichoderma spp., are potent biocontrol agents
of plant pathogenic fungi and insects. The antagonistic activities of these biological control
agents towards phytopathogens are based on the secretion of extracellular hydrolytic enzymes,
such as cell wall-degrading chitinase enzymes. However, biological control is not restricted to
naturally occurring biocontrol agents. Through the process of genetic transformation, other
fungal or yeast species can be enhanced to produce their own chitinases or other antimicrobial
substances more effectively in order to yield potent biocontrol agents.
Various types of chitinases have been applied in the production of fungal resistant plants
and some research has been done on the application of chitinases, from a variety of
microorganisms, as biological control agents. In contrast, very little is known about the
antifungal activity of the Saccharomyces cerevisiae chitinase enzyme, encoded by the CTS1-2
gene. The CTS1-2 gene was utilised in this study as a candidate for overexpression in both
yeast and plant expression systems to analyse the ability of the encoding chitinase to inhibit
fungal growth.
The first objective of this study involved the high level expression and optimisation of the
secretion of the CTS1-2 gene in S. cerevisiae to render recombinant yeast with enhanced
antifungal abilities and with possible applications as a biocontrol agent to control plant pathogenic fungi. It was hypothesised that high-level expression and efficient secretion would
be prerequisites in a biocontrol yeast strain. To this end, two strong promoters and terminators
were included in the study and the secretion of the chitinase gene was evaluated by testing
three different secretion signals. The secretion signals included: the native CTS1-2 secretion
signal, the S. cerevisiae mating pheromone a-factor (MFa1) secretion signal, as well as the
Trichoderma reesei f3-xylanase 2 (XYN2) secretion signal. The phosphoglycerate kinase 1
(PGK1) and alcohol dehydrogenase 2 (ADH2) promoters and terminators were employed to
achieve high-level expression.
The results obtained from the analysis of the recombinant yeasts showed that the PGK1
promoter-terminator constructs yielded high level CTS1-2-expressing and chitinase-producing
strains of S. cerevisiae PRY488. The ability of the different secretion signals to efficiently
secrete the overexpressed chitinase was analysed and it was found that the non-native
secretion signals delivered significantly more protein to the extracellular environment. It was
thus evident that the performance of the MFa1 and XYN2 secretion signals was superior to that
of the native secretion signal. The antifungal activities of the recombinant chitinases produced
by these constructs were tested in in vitro assays against Botrytis cinerea. The enzymes led to
a significant reduction in hyphal development, caused by extreme structural damage to the
hyphal tips, the hyphal cell walls as well as the ability of the fungus to form reproductive and
survival structures, thereby confirming the antifungal abilities of this enzyme. The ADH2
promoter-terminator constructs yielded CTS1-2 transcripts, but no chitinase activity could be
detected with any of these strains. The reasons for this still remain unclear.
The second objective of this study was to assess the potential of the yeast chitinase gene to
upregulate defence against fungal infection in planta. In order to elucidate this, the CTS1-2
gene was constitutively overexpressed in tobacco plants, targeting the chitinase both to the
intra- and the extracellular environment. The results obtained showed that the transgenic
tobacco lines regenerated in this study stably integrated the transgene, exhibiting transgene
expression as well as the production of a biologically active yeast chitinase enzyme. The F,
progeny were rigorously tested for resistance to B. cinerea, and both in vitro and in planta
assays confirmed that the yeast chitinase increased the plant's tolerance to fungal infection;
some of the lines showed disease resistance of 65 and 70%. The plants expressing an
extracellularly targeted chitinase gene are still under evaluation. Interesting results are expected
relating to the effect of the chitinase on the plant surface with regards to disease resistance to
fungal pathogens.
In conclusion, the combined set of results from both the yeast and plant overexpression
studies has confirmed the strong antifungal effect of yeast chitinases. The yeast CTS1-2
chitinase could be instrumental in the development of a new generation of yeast strains with
improved antifungal capabilities. This enzyme could also play an important role in genetic
transformation technologies aimed at enhanced disease resistance. / AFRIKAANSE OPSOMMING: Swamme omsluit 'n uiterste diverse groep organismes wat mense, plante en diere deur
patogeniese aksie kan koloniseer. Die uitkoms hiervan op landbougewasse, die veebedryf en
menslike gesondheid is gewoonlik skadelik. Uitgebreide gewas- en veeboerderye benodig
voortdurende beheer van fungiese populasies, tipies deur van chemiese swamdoders gebruik te
maak. Die uitsluitlike gebruik van swamdoders is egter nie meer 'n lewensvatbare praktyk nie,
hoofsaaklik as gevolg van probleme soos die opbou van weerstand van patogeniese rasse teen
swamdoders, die hoë kostes van die middels, asook besorgheid oor die omgewing. Die soektog
na alternatiewe beheermaatreëls deur produsente en wetenskaplikes bly 'n aaneenlopende
proses.
Die swamselwand bestaan uit polisakkariede wat nie net In rol in die beskerming van die
swam speel nie, maar ook betrokke is in die oordrag van aanvals- en infeksieverwante seine in
'n vatbare gasheer. Chitien, 'n polisakkaried bestaande uit N-asetielglukosamien (GlcNAc)
residu's gekoppel deur 13-1,4glukosidiese bindings, is een van die hoofkomponente van die
swamselwand, waar dit 'n belangrike rol in die apikale groei van vegetatiewe hifes speel.
Chitinases (EC 3.2.1.14) is proteïene wat oorvloedig deur 'n verskeidenheid van
mikroërganismes en plante geproduseer word, waar hulle vir die hidrolise van die chitien
polimeer noodsaaklik is. Tydens die infeksie van verskeie plantspesies deur In patogeen, word
die produksie van 'n spesifieke groep proteïene, die sogenaamde patogeen-verwante (PR)
proteïene wat chitinases insluit, as deel van die plant se verdedigingsreaksie geïnduseer. Die
feit dat patogeniese swamselwande chitien bevat en dat plantchitinases tydens infeksie
geïnduseer word, het daartoe gelei dat dit bevestig is dat chitinases In integrale en kritiese deel
van die plant se natuurlike verdedigingsreaksie uitmaak. Chitinases word toenemend geteiken
in pogings om die plant se intrinsieke siekteweerstandsmeganismes te verbeter deur
transgeniese ooruitdrukking daarvan in 'n verskeidenheid van gashere.
Verskeie swamspesies, insluitend verskillende Trichodenna-spesies, is kragtige
bio-antagoniste van plantpatogeniese swamme. Die antagonistiese aksies van hierdie
biologiese beheeragente teenoor fitopatogene is gebaseer op die uitskeiding van ekstrasellulêre
hidrolitiese ensieme, soos die selwandverterende chitinase ensieme. Nietemin is biologiese
beheer nie net tot bio-antagoniste wat natuurlik voorkom beperk nie. Deur die proses van
genetiese transformasie kan ander swam- of gisspesies verbeter word om hul eie chitinases of
ander antimikrobiese substanse meer effektief te produseer, wat aanleiding sal gee tot kragtige
bio-antagoniste.
Verskeie tipes chitinases is al in die produksie van swambestande plante ingespan en
uitgebreide navorsing is gedoen op die toepassing van 'n reeks chitinases, afkomstig van 'n
verskeidenheid van mikroërganismes, as biologiese beheeragente. In teenstelling is baie min
bekend oor die antifungiese aktiwiteite van die Saccharomyces cerevisiae chitinase ensiem, wat
deur die CTS1-2 geen ge-enkodeer word. Die CTS1-2-geen is in hierdie studie gebruik vir
ooruitdrukking in beide gis- en plantuitdrukkingsisteme om die chitinase se vermoë om
swamgroei te inhibeer, te ondersoek. Die eerste oorkoepelende oogmerk van hierdie studie het hoë-vlak uitdrukking en
optimalisering van sekresie van die CTS1-2-geen in S. cerevisiae behels, met die toekomstige
doelwit om 'n rekombinante gis met verbeterde antifungiese eienskappe en met moontlike
toepassings as 'n bio-antagonis teen plantpatogeniese swamme te ontwikkel. Die hipotese was
dat hoë-vlak uitdrukking en voldoende sekresie voorvereistes vir 'n bio-antagonisras is. Omdié
rede is twee sterk promotors en termineerders by hierdie studie ingesluit en is die sekresie van
die chitinase-geen geëvalueer deur drie verskillende sekresieseine te toets. Die sekresieseine
sluit in: die wilde-tipe CTS1-2 sekresiesein, die S. cerevisiae paringsferomoon a-faktor (MFa1)
sekresiesein, en die Trichoderma reesei p-xilanase (XYN2) sekresiesein. Die fosfogliseraat
kinase 1 (PGK1) en alkohol dehidrogenase 2 (ADH2) promotors en termineerders is gebruik om
hoë-vlak uitdrukking te dryf.
Die resultate wat vanaf die analises van die rekombinante giste verkry is, het getoon dat die
PGK1 promotor-termineerder konstrukte hoë-vlak CTS1-2-uitdrukkende en
chitinase-produserende S. cerevisiae PRY488 rasse opgelewer het. Die vermoë van die
verskillende sekresieseine om die ooruitgedrukte chitinase voldoende uit te skei, is geanaliseer,
en daar is gevind dat die heteroloë sekresieseine aansienlik meer proteïene na die
ekstrasellulêre omgewing geloods het. Dit was dus duidelik dat die MFa1 en XYN2
sekresieseine beter as die wilde-tipe sekresiesein presteer het. Die antifungiese aktiwiteit van
die rekombinante chitinases wat deur hierdie konstrukte geproduseer is, is ook in in vitrotoetse
teen Botryits cinerea getoets. Die teenwoordigheid van die ensieme het gelei tot 'n aansienlike
afname in hife-ontwikkeling, veroorsaak deur ekstreme strukturele skade aan die hifepunte, die
hifeselwande, asook die vermoë van die swam om voortplanting- en oorlewingstrukture te vorm.
Die ADH2 promotor-termineerderkonstrukte het CTS1-2 transkripte vertoon, maar geen
chitinase-aktiwiteite kon in hierdie konstrukte waargeneem word nie. Die redes hiervoor is tot op
hede onbekend.
Die tweede oogmerk van hierdie studie was om die potensiaal van die gischitinase om
swaminfeksie in planta teë te werk, te ondersoek. Die CTS1-2-geen is konstitutief ooruitgedruk
in tabakplante, waarin die chitinase na beide die intra- en ekstrasellulêre omgewing geteiken is.
Resultate het getoon dat die geregenereerde transgeniese tabaklyne die transgeen stabiel
geïntegreer het, transgeenuitdrukking vertoon en dat 'n biologies aktiewe chitinase-ensiem
geproduseer is. 'n F1-generasie is aan strawwe toetse onderwerp om weerstand teen B. cinerea
te ondersoek. Beide die in vitro en in planta toetse het bevestig dat die gischitinase die plant se
verdraagsaamheid teenoor swaminfeksie verhoog het; sommige lyne het siekteweerstand van
tussen 65 en 70% getoon. Die plante wat 'n ekstrasellulêre chitinase produseer, word steeds
geëvalueer. Interessante resultate word verwag aangaande die effek van die chitinase op die
plant se oppervlak met betrekking tot siekteweerstand teen swampatogene.
Ten slotte, die gekombineerde stel resultate wat vanaf beide die gis- en
plantuitdrukkingstudies verkry is, het die sterk antifungiese effek van gischitinases bevestig. Die
gis CTS1-2 kan instrumenteel wees in die ontwikkeling van 'n nuwe generasie gisrasse met
verbeterde antifungiese eienskappe. Die ensiem kan ook 'n belangrike rol in genetiese
transformasietegnologieë, wat op verbeterde siekteweerstand gemik is, speel.
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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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Genetic improvement of growth rate in rainbow trout (Oncorhynchus mykiss)Brink, Daniel 12 1900 (has links)
Dissertation (PhD (Agric))--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: A breeding programme aimed at the genetic improvement of growth rate of rainbow trout was initiated in
1988 by the Department of Genetics, University of Stellenbosch, in collaboration with the local trout
producer's organisations. The first phase of the breeding programme included the collection, evaluation and
selection of the best available genetic material from 13 different genetic groups (nine local and four
overseas) to make up two separate base populations as odd and even year-groups. This was done to
establishment a base population with high genetic merit and variation at the onset of the breeding
programme. Statistically significant and commercially valuable genetic differences in terms of weight and
length gain were detected between the various hatchery groups.
The next two generations of the breeding program included a series of single and double crosses in order to
increase the levels of genetic variation in the base populations, and to investigate possible heterosis and
specific and general combining ability among the crosses. Significant levels of heterosis (6.7% to 9.6%) and
general combining ability was found for weight and length gain during consecutive growth stages. No
evidence was found for specific combining ability among the crosses. The crossing of selected offspring
from the original genetic groups followed by the application of intensive multi-stage selection for growth
rate within progeny groups has led to the establishment of second and third generation parental populations
with higher levels of genetic variation and improved individual genetic merit with regard to growth rate. The
exploitation of non-additive genetic variation within the base populations through crossbreeding and
heterosis during the early stages of the selection programme was delayed in favour of the utilization of
additive genetic variance through a procedure of multi-stage selection that incorporated high intensities of
selection within and between family groups.
The estimation of genetic parameters during the fourth generation on the basis of a hierarchical half-sib
family structure confirmed the presence of high levels of additive genetic variation within the respective
populations/year-groups. High heritability values in the range of 0.40 to 0.53 were recorded for body weight
and length at 150 days. Genetic correlations between the traits were also high, in the range of 0.74 to 0.82.
The cumulative realized response of 50% in body length for the EVEN year-group after six generations of
selection (8.3% per generation), and the 33% for the ODD year-group after five generations of selection
(6.6% per generation) confirms the efficiency of the multi-stage selection procedure to exploit the available
additive genetic variation for growth rate within the respective populations.
The programme is still ongoing, entering its 7th generation in 2004 and is supplying about 50-60% of
commercial material through direct supplies of broodstock, ova and fingerlings and indirect supplies via
multiplier stations (commercial hatcheries). The programme was the first of its kind in relation to
aquaculture species in the Southern African region, and has since initiated the introduction of programmes of
genetic improvement in three other indigenous species, namely tilapia (Oreochromis mossambicus), African
catfish (Clarias gariepinus) and abalone (Haliotis midae). / AFRIKAANSE OPSOMMING: ‘n Teelprogram gerig op die verbetering van groeitempo in reënboogforel is in 1988 ingestel onder toesig van
die Departement Genetika aan die Universiteit van Stellenbosch, in sameweking met die plaaslike
forelprodusenteverenigings. Die eerste fase van die teelprogram behels die versameling, evalasie en seleksie
van die beste beskikbare genetiese materiaal vanuit, 13 verskillende genetiese groepe (nege plaaslike en vier
van oorsee) om twee basispopulasies te ontwikkel in elk van die gelyke en ongelyke jaargange. Die doel
daarvan was om ’n basispopulasie met hoë genetiese meriete en variasie te ontwikkel met die aanvang van die
teelprogram gerig op genetiese verbetering, deur middel van seleksie. Statisties betekenisvolle en ekonomies
belangrike genetiese verskille in massa- en lengtetoename is aangetref, tussen die onderskeie genetiese groepe.
Die daaropvolgende twee generasies binne die teelprogram behels die uitvoering van ’n reeks enkel- en
dubbelkruisings ten einde ’n verdere toename in genetiese variasie in die basispopulasies te bewerkstellig,
sowel as om die voorkoms van heterose en algemene, sowel as spesifieke kombinerings-vermoë tussen die
kruisings te bepaal. Betekenisvolle vlakke van heterose (6.7% tot 9.6%) sowel as algemene
kombineringsvermoë, is aangetref ten opsigte van massa- en lengtetoename in opeenvolgende groeifases. Daar
kon geen aanduiding van betekenisvolle, spesifieke kombineringsvermoë gevind word nie. Die kruising van
geselekteerde nageslag vanuit die oorspronklike genetiese groepe, gevolg deur ‘n multi-fase seleksiemetode vir
groeitempo binne nageslaggroepe, het bygedra tot die ontwikkeling van ‘n tweede en derde generasie
broeipopulasie wat beskik oor hoër vlakke van genetiese variasie en verbeterde individuele meriete ten opsigte
van groeitempo. Die benutting van nie-additatiewe genetiese variasie binne die basispopulasies deur middel
van kruisteling en heterose tydens die vroee stadium van die teelprogram is uitgestel ten gunste van die
benutting van additatiewe genetiese variasie deur middel van ‘n multi-fase seleksiemetode, wat berus het op
die toepassing van hoë vlakke van seleksie-intensteit binne en tussen familiegroepe.
Die beraming van genetiese parameters tydens die vierde generasie het die voorkoms van hoe vlakke van
additatiewe variasie binne die onderskeie jaargroepe bevestig. Hoë oorerflikhede van 0.40 tot 0.53 is beraam
vir ligaamsmassa en -lengte op die ouderdom van 150 dae. Genetiese korrelasies tussen die kenmerke was ook
hoog met waardes van 0.74 tot 0.82. Die saamgestelde gerealiseerde seleksierespons van 50% vir
liggaamslengte vir die “EVEN”-jaargroep na afloop van ses generasies van seleksie (8.3% per generasie) en
die 33% van die “ODD”-jaargroep na afloop van vyf generasies van seleksie (6.6% per generasie) het die
doeltreffendheid van die multi-fase seleksiemetode bevestig ten opsigte van die benutting van die additatiewe
variasie vir groeitempo binne die onderskeie basispopulasies/jaargroepe.
Die teelprogram duur steeds voort en sal die 7de generasie in 2004 bereik. Die program voorsien nagenoeg
50-60% van die kommersiele materiaal vanuit direkte voorsiening van teelmaterial, eiers en vingerlinge asook
die indirekte voorsiening via kommersiële teelstasies. Die teelprogram was die eerste van sy soort met
betrekking tot akwakultuurspesies in Suider Afrika en het bygedra tot die implimentering van programme van
genetiese verbetering in drie inheemse spesies, naamlik die tilapia (Oreochromis mossambicus), die baber
(Clarias gariepinus) en die perlemoen (Haliotis midae).
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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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Genetic mapping of adult plant stripe rust resistance in the wheat cultivar KariegaRamburan, Viresh Premraj 04 1900 (has links)
Thesis (PhD (Agric)) -- Stellenbosch University, 2003. / ENGLISH ABSTRACT: Stripe (yellow) rust of wheat, caused by Puccinia striiformis f.sp. tritici, was first detected as a
single introduction into South Africa in 1996. Two additional pathotypes have since been
identified. Control of the disease may be achieved by use of genetic adult plant resistance
(APR) as is present in the local cultivar 'Kariega'. The aim of this project was to understand the
genetic basis of the APR in 'Kariega' to facilitate breeding of new varieties with genetic
resistance to stripe rust.
A partial linkage map of a 'Kariega X Avocet S' doubled haploid population covering all 21
wheat chromosomes was generated using 208 DNA markers, viz, 62 SSR, 133 AFLP, 3 RGA
and 10 SRAP markers, and 4 alternative loci. The different marker techniques detected varying
polymorphism, viz, overall SSR: 46%, AFLP: 7%, SRAP: 6% and RGA: 9%, and the markers
produced low levels of missing data (4%) and segregation distortion (5%). A significant feature
of the linkage map was the low polymorphism found in the D genome, viz, 19% of all mapped
DNA markers, 11% of all AFLP markers and 30% of the total genome map distance. A region
exhibiting significant segregation distortion was mapped to chromosome 4A and a seedling
resistance gene for stem rust (Puccinia graminis f.sp . tritici), Sr26, mapped to chromosome 6A
close to three SSR markers. The leaf tip necrosis gene, Ltn, which was also segregating in the
population, mapped to chromosome 7D. Protocols for SRAP and RGA were optimised, and
SRAP marker use in wheat genetic linkage studies is reported for the first time.
The linkage map was used together with growth chamber and replicated field disease scores for
QTL mapping. Chromosomes showing statistically significant QTL effects were then targeted
with supplementary SSR markers for higher resolution mapping. The quality of disease
resistance phenotypic data was confirmed by correlation analysis between the different scorers
for reaction type (0.799±0.023) and for transformed percentage leaf area infected
(0.942±0.007).
Major QTL were consistently identified on chromosome 7D (explaining some 25-48% of the
variation) and on chromosome 2B (21-46%) using transformed percentage leaf area infected and transformed reaction type scores (early and final) with interval mapping and modified
interval mapping techniques. Both chromosomal regions have previously been identified in
other studies and the 7D QTL is thought likely to be the previously mapped APR gene Yr 18.
Minor QTL were identified on chromosomes lA and 4A with the QTL on 4A being more
prominent at the early field scoring for both score types. A QTL evidently originating from
'Avocet S' was detected under growth chamber conditions but was not detected in the field,
suggesting genotype-environment interaction and highlighting the need for modifications of
growth chamber conditions to better simulate conditions in the field.
The genetic basis of the APR to stripe rust exhibited by 'Kariega' was established by mapping
of QTL controlling this trait. The linkage map constructed will be a valuable resource for
future genetic studies and provides a facility for mapping other polymorphic traits in the
parents of this population with a considerable saving in costs. / AFRIKAANSE OPSOMMING: Streep of geelroes van koring word veroorsaak deur Puccinia striiformis f. sp tritici, en is die
eerste keer in 1996 in Suid-Afrika na introduksie van 'n enkele patotipe waargeneem. Twee
verdere patotipes is sedertdien in Suid-Afrika gei"dentifiseer. Beheer van die siekte word veral
moontlik gemaak deur die gebruik van genetiese volwasseplantweerstand soos gei"dentifiseer in
die plaaslike kultivar 'Kariega'. Die doel van hierdie studie was om die genetiese grondslag van
die streeproesweerstand te ontrafel ten einde die teling van nuwe bestande kultivars moontlik te
maak.
'n Verdubbelde haplo1ede populasie uit die kruising 'Kariega X Avocet S' is aangewend om 'n
gedeeltelike koppelingskaart vir die volle stel van 21 koring chromosome saam te stel. Die kaart
het uit 208 DNA merkers, nl., 62 SSR, 133 AFLP, 3 RGA, 10 SRAP merkers en 4 ander lokusse
bestaan. Totale polimorfisme wat deur die verskillende merkersisteme opgespoor is, was as volg:
SSR: 46%, RGA: 9%, AFLP: 7% en SRAP: 6%. Die mate van ontbrekende data was gering
(4%) asook die mate van segregasie distorsie (5%) van 'n enkele geval wat op chromosoom 4A
gekarteer is. 'n Prominente kenmerk van die koppelingskaart is die relatiewe gebrek aan
polimorfiese merkers op die D-genoom, nl., slegs 19% van alle DNA merkers en 11% van alle
AFLP merkers wat slegs 30% van die totale genoom kaartafstand bestaan het. Die stamroes
(Puccinia graminis f. sp. tritici) saailingweerstandsgeen, Sr26, karteer op chromosoom 6A naby
drie SSR merkers. Die geen vir blaartipnekrose, Ltn, karteer op chromosoom 7D. Protokolle vir
SRAP en RGA merkers is ge-optimiseer en gebruik van SRAP merkers in koppelings-analise
word vir die eerste keer in koring gerapporteer.
Die koppelingskaart is in kombinasie met groeikamerdata en gerepliseerde veldproefdata gebruik
om die gene (QTL) vir volwasseplant streeproesweerstand te karteer. Chromosome met statisties
betekenisvolle QTL is met aanvullende SSR merkers geteiken om die resolusie van kartering
verder te verhoog. Die kwaliteit van fenotipiese data, soos in die proewe aangeteken, is bevestig
deur korrelasies te bereken tussen lesings geneem deur onafhanklike plantpataloe (0.799 ± 0.023
vir reaksietipe en 0.942 ± 0.007 vir getransformeerde persentasie blaaroppervlakte besmet).
Hoofeffek QTL vir die twee maatstawwe van weerstand is deur middel van die metodes van
interval QTL kartering en gemodifiseerde interval QTL kartering konsekwent op chromosome
7D (25-48% van variasie verklaar) en 2B (21-46% van variasie verklaar) ge"identifiseer. In
vorige studies is aangetoon dat beide chromosome 7D en 2B QTL vir volwasseplant
streeproesweerstand dra. Die 7D QTL is waarskynlik die weerstandsgeen, Yr 18. QTL met klein
effekte op weerstand is op chromosome lA en 4A ge"identifiseer. Die effek van laasgenoemde
geen was meer prominent in die velddata in die vroee datum van weerstandsbeoordeling. Een
QTL, afkomstig van 'Avocet S', is slegs onder groeikamertoestande identifiseerbaar. Dit dui op
moontlike genotipe-omgewing wisselwerking en beklemtoon die noodsaaklikheid om
aanpassings te maak in groeikamertoestande vir beter simulasie van veldproeftoestande.
Die genetiese grondslag van volwasseplantweerstand teen streeproes in die kultivar 'Kariega' is
deur QTL kartering bepaal. Die 'Kariega X Avocet S' koppelingskaart kan as 'n waardevolle basis
dien vir toekomstige genetiese ontledings van ander polimorfiese kenmerke in die populasie.
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Mannoprotein production and wine haze reduction by wine yeast strainsNdlovu, Thulile 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Wine protein haze formation is a major challenge for wine makers, and several wine clarifying agents such as bentonite are used in the industry to protect wine from this occurrence. However, clarifying agents may have an undesirable impact on wine quality. Yeast mannoproteins have been shown to possess haze-protective properties, while also positively impacting on the sensorial properties of the product. However, while such mannoproteins are released into the wine during the wine making process, the amounts are low and therefore of limited oenological significance. However, and although commercial wine yeast strains display significant genotypic and phenotypic diversity, no broader assessment of haze protective activity and of mannoproteins release by different wine yeast strains has been undertaken.
In this study, several yeast strains were screened for their impact on wine haze formation in Chardonnay must and in a grape juice model system. The data show that strains of the species Saccharomyces paradoxus possess better haze protective properties than the common Saccharomyces cerevisiae wine yeast strains. Differences in the nature of the proteins released by these two species were investigated, and indicated that several mannoproteins were released at significantly higher levels by S. paradoxus, and that some of these proteins might indeed contribute to the haze-protective activity. A further exploration of yeast cell wall properties indicated that the cell walls of haze-protective S. paradoxus strains contained higher levels of chitin than non-haze protective strains. Grape chitinases are likely to be primarily responsible for wine haze formation, and the data clearly demonstrate that these enzymes are able to bind to the yeast cell walls, and that strains with higher amounts of chitin in the cell wall will bind more chitinases. This finding suggests that the haze-protective nature of the strains is at least in part linked to the chitin levels of the strains.
Furthermore, the impact of some genetic modifications in two wine strains (namely S. cerevisiae VIN13 and S. paradoxus RO88) suggests that several proteins contribute to wine haze protection. However, none of the mannoprotein-encoding flocculation genes, FLO1, FLO5, and FLO11 showed any impact on this property.
Further studies are required to assess the full impact of the S. paradoxus strains on haze protection. In particular, the possible use of such strains as starter cultures or the use of S. paradoxus yeast hulls as clarifying agent needs to be further explored. / AFRIKAANSE OPSOMMING: Wyn proteïen-waas vorming is 'n groot uitdaging vir wynmakers en verskeie wyn verhelderings agente soos bentoniet word in die wynbedryf gebruik om wyn te beskerm teen die vorming van waas. Hierdie verheldering agente het egter 'n ongewenste impak op wynkwaliteit. Gis mannoproteïene is uitgewys as proteïene met moontlike waas-beskermende eienskappe wat ook 'n positiewe uitwerking op die sensoriese eienskappe van die produk het. Al word hierdie mannoproteïene egter vrygestel in die wyn tydens die wynmaak proses, is die hoeveelhede oor die algemeen te laag om van wynkundige belang te wees. Verder, ten spyte van die beduidende genotipiese en fenotipiese diversiteit van kommersiële wyngisrasse is daar nog geen breër assessering van die waas beskermende aktiwiteit van mannoproteïene, vrygestel deur verskillende rasse, tot dusver onderneem nie.
In hierdie studie is verskeie gisrasse gekeur vir hul impak op wyn waas-vorming in Chardonnay mos en ook in 'n model druiwesap. Die data wys dat rasse van die spesie Saccharomyces paradoxus besit beter waas beskermende eienskappe as die algemene Saccharomyces cerevisiae wyngisrasse. Verskille in die aard van die proteïene wat vrygestel is deur hierdie twee spesies is ondersoek, en dit is aangedui aangedui dat verskeie mannoproteins vrygestel aan aansienlik hoër vlakke deur S. Paradoxus. Dit is ook aangedui dat sommige van hierdie proteïene wel bydra tot die waas-beskermende aktiwiteit.
'n Verdere verkenning van gis selwand eienskappe het aangedui dat die selwande van waas-beskermende rasse van S. paradoxus hoër vlakke chitien as nie-waas beskermende stamme bevat. Druiwe chitinases is waarskynlik hoofsaaklik verantwoordelik vir wyn waas vorming, en die data toon duidelik dat hierdie ensieme in staat is om te bind aan die gis selwande, en dat die stamme met hoër vlakke chitien in die selwand meer chitinases sal bind. Hierdie bevinding dui daarop dat die waas-beskermende aard van die stamme ten minste gedeeltelik gekoppel is aan die chitien vlakke van die stamme. Die impak van sekere genetiese modifikasies in twee verskillende gisrasse, naamlik die S. cerevisiae ras VIN13 en die S. paradoxus ras RO88, dui verder daarop dat verskeie proteïene dra by tot die beskerming teen wyn waas. Geeneen van die mannoprotein-koderende flokkulasie gene, FLO1, FLO5 en FLO11 het egter 'n impak op hierdie eienskap nie.
Verdere studies is nodig om die volle impak van die S. paradoxus rasse op waas beskerming te assesseer. In die besonder, die moontlike gebruik van sulke rasse as 'n inkolasie kultuur of die gebruik van S. paradoxus gis doppe as verheldering agent moet verder ondersoek word.
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