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A genetic strategy to reduce sulfite reductase activity in Saccharomyces cerevisiae / by Catherine M. Sutherland.Sutherland, Catherine M. (Catherine Maree). January 2000 (has links)
Erratum pages attached to back page. / Bibliography : leaves 125-147. / 147, [41] leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study was undertaken to derive a strategy to reduce the potential of S. cerevisiae to produce hydrogen sulfide under oenologial conditions by altering the levels of active sulfite reductase in the cell. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2000
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Gel electrophoresis for determining isozyme differences in 'superior seedless' grapesSchwennesen, Jean Clarke January 1981 (has links)
No description available.
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The characterisation and partial sequencing of the grapevine chloroplast genomeRose, B. A. (Beverley Ann) 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: A number of proteins essential for the survival of a plant are encoded by the chloroplast
genome. The characterization and sequencing of a number of algal and plant chloroplast
genomes has facilitated researchers understanding of cellular functions and metabolism.
Chloroplast DNA (cpDNA) has also been used to determine inter- and intraspecies
evolutionary relationships and this organelle offers an alternative means of expressing foreign
genes. Although a number of species' chloroplast genomes have been characterized and
sequenced, no previous attempts of this kind have been made for a chloroplast genome of the
family Vitaceae.
In this study, attempts were made to characterize and partially sequence the chloroplast
genome of Vilis vinifera. Chloroplast DNA was isolated from the Sultana and Sugra 1
cultivars and digested with restriction enzymes that produced cpDNA fragments of a suitable
size for cloning. The fragments were shotgun-cloned into a plasmid vector and white colonies
were screened by means of PCR and colony blotting. Three EcoRI-digested clones and one
PstI-digested clone were obtained in this manner. Walking outwards from a previously
sequenced grapevine rrn 16 gene region by means of PCR also allowed us to sequence a
further -3310 bp region of the Sultana chloroplast genome.
BAC clones containing V. vinifera cv L. Cabernet Sauvignon cpDNA inserts became
available later in the project. It was decided to use these clones for further library
construction instead of isolated cpDNA. The 5' and 3' end sequences of seven of the 24 BAC
clones were obtained. These were compared to sequences found in the NCBI database to find
-
homologous chloroplast regions and determine the size of each BAC insert. One clone
appeared to contain the entire grapevine chloroplast genome, apart from a 500 bp region.
This clone was selected for further analysis. The BAC clone DNA was isolated and
restriction-digested fragments were shotgun-cloned into a plasmid vector. White colonies
were screened by isolating the plasmid DNA and digesting it with appropriate restriction
enzy~es. The 5' and 3' ends of putative positive clones were sequenced and mapped onto the
Atropa belladonna chloroplast genome.
A total of 15 clones were obtained in this project. Five of these contain cpDNA isolated from
grapevine leaves and 10 contain fragments sub-cloned from the BAC clone. The biggest problem encountered with both methods used for library construction was genomic DNA
contamination. Genomic DNA either originated from the plant nuclear genome or from the
bacterial host cells in which the BAC clones were maintained. Many of the clones screened
contained genomic DNA, and these could only be identified and removed once the clones had
been sequenced. Even when a commercial kit was used for BAC clone isolation, 31% of the
clones screened contained genomic DNA. This kit was specifically designed for the isolation
of genomic DNA-free large constructs.
The clones obtained from the two strategies provided a good representation of the grapevine
chloroplast genome. The only region not represented was the Small Single Copy (SSC)
region. Approximately 40% of the grapevine chloroplast genome was covered by these
clones. This provides a basis for further genome characterization, physical mapping and
sequencing of the grapevine chloroplast genome. / AFRIKAANSE OPSOMMING: Die chloroplasgenoom kodeer VIr 'n hele aantal proteïene wat essensieel is VIr die
voortbestaan van 'n plant. Die karakterisering en volgorde bepaling van 'n aantal alg en plant
chloroplasgenome het dit. vir navorsers moontlik gemaak om sellulêre funksies en
metabolisme van plante te ontrafel. Chloroplas DNA (cpDNA) is ook gebruik om intra- en
interspecies evolusionêre verwantskappe vas te stel. Dié organel verskaf ook 'n alternatiewe
manier vir die uitdrukking van transgene. Alhoewel die chloroplasgenome van 'n hele aantal
species al gekarakteriseer is en die DNA volgorde daarvan bepaal is, is daar nog geen
navorsing van bogenoemde aard op die chloroplasgenoom van die Vitaceae familie gedoen
rue.
In hierdie studie is beoog om die chloroplasgenoom van Vitis vinifera te karakteriseer en
gedeeltelike volgordebepaling daarvan te doen. Chloroplas DNA is geïsoleer vanaf Sultana
en Sugra 1 kultivars en restriksie-ensiem vertering is gedoen met ensieme wat cpDNA
fragmente, met geskikte grootte vir klonering, produseer. Dié fragmente is in 'n
plasmiedvektor gekloneer met die haelgeweer-metode en wit kolonies is gesif deur middel
van PKR en die kolonieklad metode. Op hierdie manier is drie EcoRI-verteerde klone en een
PstI-verteerde kloon verkry. Deur uitwaarts te loop, deur middel van PKR, vanaf 'n druif
rrnl6 geenstreek, waarvan die volgorde voorafbepaal is, was dit vir ons moontlik om ook die
volgorde te bepaal van 'n verdere ~3310 bp streek van die Sultana chloroplasgenoom.
BAC klone wat V. vinifera cv L. Cabernet Sauvignon cpDNA fragmente bevat, het later in die
projek beskikbaar geraak. Daar is besluit om hierdie klone, i.p.v. die geïsoleerde cpDNA, te
gebruik vir verdere biblioteek konstruksie. Die 5' en 3' entpuntvolgordes van sewe uit die 24
BAC ~lone is verkry. Hierdie volgordes is vergelyk met volgordes in die NCB Idatabasis om
homoloë chloroplas streke te identifiseer, en die grootte van elke BAC fragment te bepaal.
Die het geblyk dat die hele druif chloroplasgenoom in een van die klone vervat is, behalwe vir
'n 500 bp streek. Die BAC-kloon DNA is geïsoleer en die restriksie-verteerde fragmente is in
'n plasmiedvektor gekloon d.m.V. die haelgeweer-metode. Wit kolonies is gesif deur die
isolering van plasmied DNA en die vertering daarvan met geskikte restriksie-ensieme. Die
volgorde van die 5' en 3' entpunte van skynbare positiewe klone is bepaal en gekarteer op die
Atropa belladonna chloroplasgenoom. In hierdie studie is 'n totaal van 15 klone verkry. Vyf hiervan bevat cpDNA wat vanaf
druifblare geïsoleer is, en 10 bevat fragmente wat vanaf die BAC-klone gesubkloneer is.
Genorniese DNA kontaminasie was die grootste probleem wat ondervind is tydens beide
metodes wat gebruik is vir biblioteek konstruksie. Genomiese DNA was afkomstig vanaf óf
die plant nukleêre genoom óf die bakteriële gasheerselle waarin die BAC-klone gehou is.
Baie van die klone wat gesif is, het genomiese DNA bevat, en dit kon eers geïdentifiseer en
verwyder word nadat die volgorde van die klone bepaal is. Selfs al is 'n kommersiële produk
vir BAC-kloon isolasie gebruik, het 31% van die gesifde klone steeds genomiese DNA bevat.
Dié kommersiële produk is spesifiek vir die isolasie van groot konstrukte, wat genomiese
DNA vry is, ontwerp.
Die klone wat deur die twee strategeë verkry is, het 'n goeie verteenwoordiging van die druif
chloroplasgenoom gegee. Die enigste streek wat die verteenwoordig is nie, was die Klein
Enkelkopie (SSC) streek. Ongeveer 40% van die druif chloroplasgenoom is deur hierdie
klone gedek. Dit verskaf 'n basis vir verdere genoomkarakterisering, fisiese kartering en
volgordebepaling van die druif chloroplasgenoom.
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Studies on the biology and genetic variation of phomopsis on grapevineScheper, Reiny W. A. (Reiny Wendelke Anna) January 2001 (has links) (PDF)
Bibliography: leaves 212-227.
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Studies on the biology and genetic variation of phomopsis on grapevine / Reiny W. A. Scheper.Scheper, Reiny W. A. (Reiny Wendelke Anna) January 2001 (has links)
Bibliography: leaves 212-227. / viii, 227 leaves, [19] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2001
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Differential gene expression during berry ripening in Vitis vinifera (cv Chardonnay) : isolation of specific sequences through subtractive cloningOlivier, Abraham Jacobus 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Grapevine is worldwide an agronomically important crop. Traditionally selective
breeding has been used to improve existing cultivars. In the last ten years, however,
the advent of biotechnology has shortened these breeding programmes by producing
transgenic grapevine. Because this new technology is aimed at the possible genetic
manipulation of the ripening process in grape berries, it is important to elucidate all
the mechanisms that may be involved in ripening. The aim of the present study was
the identification of genes that play an important role during the ripening process in
grape berries. This was achieved by investigation of putative differentially expressed
genes in ripening Chardonnay berries isolated through subtractive hybridisation. Two
subtraction libraries, representing early and late ripening stages were constructed.
Four of the ten genes analysed exhibited expression during berry ripening. One of the
four genes was expressed in a tissue and stage specific manner. Further
characterisation of eight of the DNA and protein sequences revealed that the putative
translation products of these clones had homologues that are involved in amongst
others cell wall structure in other species. These included UDP-glucose
dehydrogenase, which is involved in the synthesis of hemicellulose precursors. The
remaining seven clones encoded putative stress response proteins. These included
two heat shock proteins, a vacuolar pyrophosphatase and a protein involved in cell
division. It is suggested that specific grape mRNAs accumulate in response to
stresses such as the storage of high concentrations of sugars and rapid cell expansion.
These processes occur rapidly during the ripening of berries. Accumulation of specific
mRNAs can be attributed to part of the normal ripening developmental programme. / AFRIKAANSE OPSOMMING: Druiwe is wêreldwyd 'n belangrike landbougewas en kultivars word tradisioneel deur
middel van tydsame selektiewe teling verbeter. Die tyd wat hieraan bestee word, kan
verkort word deur die implementering van biotegnologie en die produksie van
transgeniese duiwe. Omdat hierdie nuwe tegnologie op die moontlike genetiese
manipulering van die rypwordingsproses in druiwe gemik is, is dit belangrik dat alle
meganismes betrokke by rypwording ondersoek en verstaan word. Die doel van
hierdie studie was om gene wat moontlik tydens die rypwordingsproses in druiwe 'n
rol kan speel, te identifiseer. Hierdie doel is bereik deurdat differensieel uitgedrukte
gene uit die kultivar Chardonnay geïsoleer is met behulp van verrykingsbiblioteke
vanuit jong en volwasse druiwekorrels. Vier van die tien gene wat geanaliseer is,
word uitgedruk tydens die rypwordingsproses. Verder het een van die vier gene
weefsel- en rypwordingstadium- spesifisiteit getoon. Volledige karakterisering van
agt van die DNA- en proteïenvolgordes het aangedui dat die proteïenprodukte van
hierdie gene homoloog is aan volgordes wat onder andere by selwandstruktuur
betrokke is. Dit sluit UDP-glukose dehidrogenase in, wat betrokke is by die sintese
van hemi-sellulose boustene. Die ander sewe gene kodeer vir moontlike
spanningsproteïene. Twee hitteskokproteïene, 'n vakuolêre pirofosfatase en 'n
proteïen wat betrokke is by selverdeling is geïdentifiseer. Daar word voorgestel dat
druiwe mRNA versamel in reaksie op spanningsituasies soos die berging van hoë
konsentrasies suikers en selvergroting. Hierdie prosesse vind baie vinnig plaas tydens
rypwording. Versameling van spesifieke mRNAs kan toegeskryf word as 'n normale
deel van die rypwordingsproses.
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Isolation and characterisation of carotenoid biosynthetic genes from Vitis viniferaTaylor, Kerry Lyn 03 1900 (has links)
Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. / Plants are constantly exposed to adverse environmental conditions including variations in
light intensity and the availability of water resources. These abiotic factors are expected to
worsen as the changing global climate places additional daily and seasonal demands on
plant growth and productivity. As plants are incapable of avoiding stress they have
developed a number of mechanisms to manage and adapt to the unfavourable conditions.
Carotenoids represent one of these mechanisms; with the xanthophylls (oxygenated
carotenes) playing an essential role in photoprotection following exposure to excess light
energy. They are also precursors to the plant hormone abscisic acid (ABA) which plays a
known role in stomatal regulation and thus drought tolerance. Carotenoids have been
identified as potential targets for genetic manipulation to meet the existing nutritional
demands (particularly vitamin A) and to enable plants to survive the climatic variations
predicted. Thorough investigations into the regulation and functioning of each carotenoid
biosynthetic gene in vivo as well as the roles of their encoded proteins are prerequisite.
Within the Grapevine Biotechnology Programme, a number of isoprenoid biosynthetic genes
have been isolated from Vitis vinifera L. cv. Pinotage. From this vast resource two genes
were chosen; namely a lycopene b-cyclase (b-LCY) and 9-cis epoxycarotenoid dioxygenase
(NCED) for detailed in planta analyses to address knowledge gaps in our current
understanding of carotenoid biosynthesis in general, its regulation and the roles of the two
target genes in these processes. Currently, the role of b-LCY within the chloroplasts is not
well known. Although the relationship between NCED overexpression, ABA levels, reduced
stomatal conductance and increased tolerance to water stress has been well-established,
comprehensive physiological analysis of the resulting mutants during conditions of both
water availability and shortage is not well documented. To assess their in planta role,
functional copies of both genes were isolated from Vitis vinifera (cv. Pinotage), characterised
and independently transformed into the genome of the model plant, Arabidopsis thaliana, in
the sense orientation under a constitutive promoter.
In order to investigate these pertinent scientific questions and thus to evaluate the
physiological role of each gene in vivo, a number of technologies were developed and/or
adopted. These included a high-performance liquid chromatography method for profiling the
major plant pigments in leaf tissue, a combination vapour phase extraction and electron
impact-gas chromatography/mass spectrometry method for the phytohormone profiling as
well as various physiological analyses including the use of chlorophyll a fluorescence to
assess the photosynthetic and non-photochemical quenching (NPQ) capacities of the plants.
Overexpression of grapevine b-LCY (Vvb-LCY) decreased lutein levels due to preferential
partitioning of lycopene into the b-branch. This decrease was not met by an increase in
either b-carotene or the xanthophyll cycle pigments implying that Vvb-LCY is not able to
regulate the flow of carbon through the pathway and provides additional evidence to the
fluidity of this pathway whereby pigment levels are continually balanced. The decreased
lutein levels observed under low light (LL) did not compromise the plants’ ability to induce
and maintain NPQ over a wide actinic light range. Vvb-LCY transgenics also had lower neoxanthin levels (and specifically the cis-isomer) under both LL and following exposure to
high light (HL), which could be correlated to an increase in malondialdehyde. Although not
corroborated, a novel and unexpected finding was an essential role for neoxanthin, and
potentially lutein, in preventing or at least reducing lipid peroxidation under HL stress. The
lower neoxanthin amounts may be due to silencing of the Arabidopsis b-LCY by the
Vvb-LCY, as the former may function as a NSY paralog as NSY is not encoded for in the
Arabidopsis genome. Clearly, this study has confirmed that Vvb-LCY partitions the carbon
flux between the a- and b-branches, however, the catalytic action of this enzyme is
dependent on the amount of substrate available and is thus not a regulatory step directing
the flux within the pathway. Enzyme kinetic and detailed transcriptional analyses would
confirm the above findings.
Overexpression of grapevine NCED1 (VvNCED1) increased ABA concentrations, delayed
seed germination and resulted in a slight to severe reduction in the overall plant growth rate.
NCED cleaves the 9-cis xanthophylls regulating ABA synthesis. However, contrary to
expectations, constitutive levels of this regulatory enzyme did not deplete the total and
individual chlorophylls and carotenoids in well-watered plants. Instead the VvNCED1
transgenics simply exhibited a lower chloroplastic pigment complement with no concomitant
effects on their photosynthetic capacity. Of particular interest, well-watered plants
overexpressing the VvNCED1 gene had an increased NPQ capacity of which the thermal
energy dissipation component (qE) was the most significant. It has been speculated that this
NPQ is associated with the phenotype conferred by VvNCED1 overexpression and occurs
independently of the xanthophyll cycle, and specifically zeaxanthin. This study confirmed
that VvNCED1 functions during drought tolerance via ABA regulation of stomatal
conductance. A detailed study was done to understand the plants’ response during water
deficit. Typically, decreases in total and individual carotenoids and the maximum efficiency
of photochemistry (Fv/Fm) as well as the relative water and soil moisture content were
recorded. No changes were recorded in salicylic acid (SA) levels, while indole acetic acid
(IAA) was positively correlated to ABA or vice versa. In contrast, the physiology of VvNCED1
overexpressing lines was largely unaffected, indicating that a reduced stomatal conductance
protects the plants against water stress.
This study has resulted in the isolation and characterisation of a carotenoid biosynthetic gene
(b-LCY) and an abscisic acid synthesising gene (NCED). Significant advancements in our
existing knowledge of the in planta role of both genes have been achieved. We have also
reaffirmed that strict regulatory control and fluidity exists within the carotenoid biosynthetic
pathway whereby individual pigment levels are constantly brought back into balance despite
constitutive expression of one of the pathway gene members. These analyses provide
valuable baseline information about individual genes which can be extended upon with other
omic technologies in order to comprehend the full complexity involved in carotenogenesis.
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The isolation and characterisation of a developmentally-regulated gene from Vitis vinifera L. berriesBurger, Anita L. 12 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2004. / 152 Leaves printed single pages, preliminary pages i-xiv and 129 numberd pages. Includes bibliography. List of abbreviations. / ENGLISH ABSTRACT: Despite increased focus on ripening-related gene transcription in grapevine, and the large number of
ripening-related cDNAs identified from grapes in recent years, the molecular basis of processes
involved in grape berry ripening is still poorly understood. Moreover, little is known about the
mechanisms involved in the ripening-related regulation of fruit-specific genes, since the isolation
and characterisation of no ripening-related, fruit-specific promoter elements has been reported to
date. This study was aimed at the isolation and characterisation of a fruit-specific, ripeningregulated
gene from Vitis vinifera L.
In the first phase of the work, gene transcription in ripening berries of Cabernet Sauvignon (a good
quality wine cultivar) and Clairette blanche (a poor quality wine cultivar) were studied by Amplified
Fragment Length Polymorphism analysis of complementary DNA (cDNA-AFLP analysis). Total
RNA from immature (14-weeks post flowering, wpf) and mature (18-wpf) berries was used for the
analysis. A total of 1 276 cDNA fragments were visualised, of which 175 appeared to be ripening
related. Average pairwise difference of the fragments amplified from immature and mature
Clairette and Cabernet berries, suggested that ripening-related gene transcription in these two
phenotypically different cultivars is remarkably similar. Nevertheless, it was shown that seventy
percent of the 175 ripening-related cDNA fragments were cultivar-specific. It was suggested that
these differences should be targeted to identify genes related to the phenotypical differences
between the two cultivars, but also to identify genes possibly involved berry quality. Moreover, the
analysis illustrated the usefulness of cDNA-AFLPs for the analysis of ripening-related gene
transcription during grape berry ripening.
In the second phase of the work, one of the ripening-related cDNAs identified by the cDNA-AFLP
analysis, was selected for further characterisation. This work highlighted the limitation placed on
the isolation of a single specific sequence from a cDNA-AFLP gel, indicating the presence of
multiple ripening-related genes in a single band excised from a cDNA-AFLP gel. Steps to
overcome this limitation of cDNA-AFLP analysis to identify and clone a specific ripening-related
gene, were implemented. In short, the band corresponding to the particular ripening-related cDNA
was band was excised from the cDNA-AFLP polyacrylamide gel and re-amplified. Northern blot
analysis using the re-amplified, uncloned product confirmed the ripening-related transcription
demonstrated by cDNA-AFLP analysis. The re-amplified, uncloned product was then cloned.
Sequence analysis of two randomly selected candidate clones revealed two distinctly different
sequences, of which neither hybridised to messenger RNA from ripening grape berries. Furtheranalysis revealed an additional five cDNAs with terminal sequences corresponding to the selective
nucleotides of the primers used for selective amplification, in the re-amplified, uncloned product.
Of these, only two were abundantly expressed in ripening grape berries, accounting for the ripeningrelated
transcription visualised by cDNA-AFLP analysis. All seven cDNAs identified from the
particular excised band were shown to be ripening-regulated during berry development, although
most were characterised by low levels of transcription during berry ripening. One of the clones,
based on the relative high levels of the transcript and the initiation of gene transcription at the onset
of véraison (10- to 12-wpf), was identified for isolation and characterisation of the full length
coding sequence.
In the third phase of the work, it was shown that this cloned sequence corresponded to a gene
encoding a proline-rich protein (PRP) associated with ripening in Merlot and Chardonnay (mrip1,
Merlot ripening-induced protein 1). It was shown that the gene is specifically transcribed in the fruit
tissue, seed and bunchstems of grapes, from 10-wpf (véraison) to the final stages of berry ripening.
The results showed that mrip1 encodes a distinct member of the plant PRP family. Most obvious is
the central region of mrip1, which is comprised of eight consecutive repeats of 19 amino acid
residues each. In comparison with other grapevine PRPs, mrip1 revealed single amino acid
differences and deletion of one of the 19 amino acid residues repeats, all in the central region of
mrip1. In situ hybridisation studies showed that accumulation of the mrip1 transcript in the ripening
berry is limited to the mesocarp and exocarp cells of the ripening grape berry. No transcript with
high sequences similarity to mrip1 could be detected in ripening strawberry or tomato fruit. Based
on the properties and proposed function of PRPs, and the results obtained in this study, potential
applications for the use of this gene in the control of cell wall architecture in fruits, were proposed.
Furthermore, as manipulation of fruit properties in grape berries would be most important in the
later stages of ripening, mrip1 was proposed an ideal candidate gene for the isolation of a fruit- and
late-ripening-specific promoter to achieve transgene transcription in genetically modified grapevine.
The final phase of the work was dedicated to the isolation and characterisation of the mrip1
promoter element. A 5.5 kb sequence corresponding to the mrip1 5’ untranslated (UTR) flanking
region was isolated and characterised by sequence analysis. In the 2.8 kb sequence directly
upstream of the mrip1 transcription initiation site, several putative cis-acting regulatory elements
were identified. These include a spectrum of hormone-, light-, phytochrome-, sugar-and stressresponsive
elements, as well as elements implicated in tissue-specific transcription. Analysis of the
sequence further upstream (3.6 – 5.5 kb) of the mrip1 transcription initiation site (TIS), revealed the
presence of another proline-rich protein directly upstream of mrip1. Sequence identity of this
sequence (mprp2) to the mrip1 coding sequence was 88%. This information provided the first insight into the chromosomal organisation of grapevine PRPs. For functional analysis of the mrip1
promoter element, the 2.2 kb sequence directly upstream of the mrip1 TIS, was translationally fused
to the sgfpS65T reporter gene. Functionality of the mrip1:sgfpS65T fusion was verified by transient
expression in green pepper pericarp tissue, before introduction into tobacco by Agrobacteriummediated
transformation. In transgenic tobacco, transcription of the mrip1:sgfpS65T fusion was
developmentally-regulated and specific to the ovary and nectary-tissue of the developing flower.
Whilst low in immature flowers, the green fluorescent protein (GFP) rapidly accumulated to the
high level of expression visualised in the flower in full-bloom, followed by a decrease in the final
stages of ovary development. These observations suggested that the 2.2 kb mrip1 promoter is
functional and that this promoter region harbours cis-elements necessary for tissue- and
developmental-specific regulation of GFP accumulation. It furthermore suggested that the
transcriptional activation of mrip1 is mediated by developmental signals present in both grapevine
berries and tobacco flowers. Results presented, suggest that the use of tobacco as heterologous
system for the analysis of ripening-related promoters, can be more generally applied. Evidently,
characterisation of the mrip1 promoter region contributes towards a better understanding of the
regulatory mechanisms involved in non-climacteric fruit ripening, and forms a basis for future
experiments defining the cis-acting elements necessary for tissue- and cell-specific gene regulation
in fruit, more specifically in grapevine. Moreover, the mrip1 promoter is an ideal candidate for the
ripening-related, tissue-specific regulation of transgene transcription in genetically modified
grapevine. / AFRIKAANSE OPSOMMING: Ten spyte van toenemende fokus op rypwordings-verwante geentranskripsie in druiwe, en die groot
aantal rypwordings-verwante komplimentere DNA (cDNA) fragmente wat gedurende die laaste paar
jaar in druiwe geïdentifiseer is, word die molekulêre basis van prosesse betrokke by die rypwording
van die druif, steeds swak begryp. Nog te meer, is baie min bekend oor die meganismes betrokke in
the rypwordings-verwante regulering van vrugspesifieke gene, aangesien die isolering en
karakterisering van nie een rypwordings-verwante, vrugspesifieke promoter tot dusver gerapporteer
is nie. Die doel van hierdie studie was die isolering en karakterisering van ‘n vrugspesifieke,
rypwordings-verwante geen uit druiwe (Vitis vinifera L).
In die eerste fase van die werk, is geentranskripsie in rypwordende druiwekorrels van Cabernet
Sauvignon (‘n goeie kwaliteit wyn kultivar) en Clairette blanche (‘n swak kwaliteit wyn kultivar)
bestudeer deur middel van cDNA-AFLP vingerafdrukke. Totale RNA van onvolwasse (14-weke na
blom vorming) en volwasse (18-weke na blom vorming) druiwekorrels was gebruik vir die analise.
‘n Totaal van 1 276 cDNA fragmente is gevisualiseer, waarvan 175 as rypwordings-verwant
voorgekom het. Gemiddelde paarsgewyse verskille van die fragmente wat vanaf onvolwasse en
volwasse Clairette en Cabernet druiwekorrels geamplifiseer is, het aangedui dat rypwordingverwante
geentranskripsie in die twee kultivars, wat fenotipies baie van mekaar verskil,
merkwaardig soortgelyk is. Nieteenstaande, is daar gewys dat sewentig persent van die 175
rypwordings-verwante cDNA fragmente, kultivar-spesifiek is. Daar is voorgestel dat hierdie
spesifieke cDNAs verder geanaliseer word om gene betrokke by die fenotipiese verskille tussen die
twee kultivars te identifiseer; maar ook om gene te identifiseer wat moontlik by die kwaliteit van die
druiwekorrel betrokke is. Voorts, het die analise die bruikbaarheid van die cDNA-AFLP tegniek vir
die karakterisering van rypwordings-verwante geentranskripsie in rypwordende druiwekorrels,
geïllustreer.
In die tweede fase van die werk, is een van die rypwordings-verwante cDNAs wat met die cDNAAFLP
analise geïdentifiseer is, geselekteer vir verdere karakterisering. ‘n Aantal rypwordingsverwante
cDNAs is in die enkele band wat uit die cDNA-AFLP gel gesny is, geïdentfiseer. Dit het
die beperking wat geplaas word op die isolering van ‘n enkel, spesifieke cDNA uit die cDNA-AFLP
gel, beklemtoon. Stappe om hierdie beperking te oorkom, en ‘n spesifieke rypwordings-verwante
cDNA te identfiseer en te kloneer, is beskryf. In kort, die band oorstemmend met die spesifieke
rypwordings-verwante cDNA, is uit die cDNA-AFLP poli-akrielamied gel gesny en gereamplifiseer.
Noordelike klad analise waarin die ge-reamplifiseerde, ongekloneerde produk aspeiler gebruik is, het die rypwordings-verwante transkripsie soos deur cDNA-AFLP analise
aangedui, bevestig. Die ge-reamplifiseerde, ongekloneerde produk is daarna gekloneer. Nukleotied
volgorde bepaling van twee ewekansig geselekteerde kandidaat klone, het twee duidelik
verskillende cDNAs aangetoon, waarvan nie een enige hibridisering met boodskapper RNA van
rypwordende druiwekorrels getoon het nie. Verder analise het die teenwoordigheid van ‘n verder
vyf cDNAs met terminale nukleotied volgordes ooreenstemmend met die selektiewe nukleotiede
van die voorlopers wat gebruik is vir selektiewe amplifisering, aangetoon. Van hierdie, het slegs
twee hoë vlakke van geentranskripsie in rypwordende druiwekorrels getoon; heel moontlik
verteenwoordigend van die rypwordings-verwante geentranskripsie wat met die cDNA-AFLP
analise gevisualiseer is. Die studie het gewys dat al sewe cDNAs rypwordings-verwant is, alhoewel
die meeste van hierdie cDNAs baie lae vlakke van geentranskripsie tydens duiwekorrel rypwording
getoon het. Gebaseer op relatief hoë vlakke van die transkrip, en die inisiering van geen transkripsie
met die aanvang van vrugrypwording (véraison, 10- tot 12-weke na blomvorming), is een van die
cDNAs geselekteer vir isolering en karakterisering van die vollengte koderings volgorde.
In die derde fase van die werk, is dit aangetoon dat hierdie cDNA ooreenstem met ‘n geen wat vir ‘n
proline-ryke proteïen (PRP), geassosieerd met vrugrypwording in Merlot en Chardonnay, kodeer.
Hierdie geen is genoem Merlot rypwording-geïnduseerde proteïen 1 (mrip1). Die studie het verder
aangetoon dat hierdie geen spesifiek in die weefsel van druiwekorrels, saad and stammetjies van die
druiwetros getranskribeer word, vanaf 10-weke na blomvorming (véraison) tot 16-weke na
blomvorming. Resultate het aangetoon dat mrip1 vir ‘n unieke lid van die plant PRP familie kodeer.
Mees opvallend, is die sentrale gedeelte van mrip1, wat uit agt opeenvolgende herhalings van
negentien aminosure elk bestaan. In vergelyking met ander druif PRPs, toon mrip1 enkel aminosuur
verskille en ‘n delesie van een van die negentien aminosuur herhalings, alles in die sentrale gedeelte
van mrip1. In situ hibridisering het getoon dat akkumulering van die mrip1 transkrip net in selle van
die mesocarp en eksokarp van die rypwordende druif plaasvind. Geen transkip met hoë nukleotied
gelyksoortigheid aan mrip1 kon in rypwordende aarbeie of tamatie vrugte aangetoon word nie.
Gebaseer op die eienskappe en funksie van PRPs soos voorgestel in die literatuur, en die bevindinge
van hierdie studie, is potensiële toepassings vir die gebruik van die geen in die beheer van selwand
argitektuur in vrugte, voorgestel. Verder, aangesien die manipulering van vrugkwaliteit in die druif
veral belangrik is vanaf die aanvang van vrugrypwording (véraison), is daar voorgestel dat mrip1 ‘n
ideale kandidaat is vir die isolering van ‘n vrugspesifieke en rypwording-verwante promoter vir
gebruik in geneties gemodifiseerde druiwe.
Die laaste fase van die studie was gewy aan die isolering en karakterisering van die mrip1 promotor
element. ‘n 5.5 kb fragment ooreenstemmend met die mrip1 5’ ongetransleerde area is geisoleer en gekarakteriseer deur middel van nukleotied volgorde bepaling. In die 2.8 kb area direk stroomop
van die mrip1 transkripsie inisiasie punt (TIS), is verskeie moontlike cis-beherende regulatoriese
elemente geïdentifiseer. Hierdie sluit in ‘n spektrum van hormoon-, lig-, fitochroom-, suiker- en
stress-reagerende elemente, asook elemente geïmpliseer in weefselspesifieke geentranskripsie.
Analise van die area verder stroomop (3.6 – 5.5 kb) van die mrip1 TIS, het die teenwoordigheid van
‘n ander PRP direk stroomop van mrip1 getoon. Nukleotied gelyksoortigheid van hierdie geen
(MPRP2) aan die mrip1 koderingsgebied was slegs 88%. Hierdie inligting verskaf die eerste insig
in die chromosomale organisasie van druif PRPs. Vir funksionele analise van die mrip1 promotor
element, is die 2.2 kb area direk stroomop van die mrip1 TIS transkripsioneel verenig met die
sgfpS65T merker geen. Funksionaliteit van die mrip1: sgfpS65T fusie is bevestig deur middel van
kortstondige (transient) geenuitdrukking in die perikarp van groenrissie, voordat dit ingevoer is in
tabak met Agrobacterium-bemiddelde genetiese transformasie. In transgeniese tabak was
transkripsie van die mrip1:sgfpS65T fusie ontwikkelingsstadium-gereguleerd, en spesifiek in die
ovarium en heuningsakkie (nektarium) van die ontwikkelende blomme. Terwyl die vlak van
geenuitdrukking laag was in die jong blomme, het GFP baie vinnig akkumuleer tot die hoë vlakke
wat in die blomme in volle-blom gevisualiseer is. Daarna het dit weer vinnig afgeneem tydens die
finale stadiums van ovarium ontwikkeling. Hierdie waarnemings dui daarop dat die 2.2 kb mrip1
promotor element funksioneel is en dit al die nodige cis-beherende regulatoriese element bevat wat
nodig is vir weefsel- en ontwikkelingsstadium-spesifieke regulering van GFP akkumulering. Dit dui
verder daarop dat transkripsionele aktivering van mrip1 beheer word deur ontwikkelingsstadium
seine teenwoordig in beide die druif en tabakblomme. Hierdie resultate stel voor dat tabak meer
algemeen gebruik kan word as heteroloë sisteem vir die analise van rypwording-verwante
promotors. Duidelik dra die karakterisering van die mrip1 promoter element by tot ‘n beter begrip
van die regulatoriese meganismes betrokke by die rypwordingsproses van nie-klimateriese vrugte,
en vorm die basis vir toekomstige eksperimente waarin die cis-beherende regulatoriese elemente vir
vrug- en sel-spesifieke geen regulering, meer spesifiek die druif, bepaal sal word. Meer nog, is die
mrip1 promotor ‘n ideale kandidaat vir weefsel-spefieke en rypwording-verwante regulering van
transkripsie van die transgeen in geneties gemodifiseerde druiwe.
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9 |
Molecular analyses of candidate carotenoid biosynthetic genes in Vitis vinifera L.Young, Philip Richard, 1973- 03 1900 (has links)
Thesis (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Plants cannot avoid stress and must therefore be capable of rapidly responding to
extreme environmental changes. An inability to control and regulate the
photosynthetic process during stress conditions will lead to the formation of highly
reactive oxygen species that concomitantly causes photo-oxidative damage to the
pigments and proteins of the photosynthetic apparatus. Since light is the primary
source of energy for the photosynthetic process, it is clear that plants are
continuously required to balance the light energy absorbed for the photochemical
reactions against photoprotection in a dynamic way in order to survive. Carotenoids
are precursors of abscisic acid, but more importantly structural components of the
photosynthetic apparatus. During photosynthesis carotenoids function as accessory
light-harvesting pigments, and also fulfil a photoprotective function by quenching the
reactive molecules formed during conditions that saturate the photosynthetic process.
Due to the importance of carotenoids to plant fitness and human health (as
Vitamin A precursors) this study has attempted to isolate and characterise genes that
are directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In total
eleven full-Iength- and eight partial genes have been isolated, cloned and
sequenced. These genes can be grouped into the following pathways: (i) the 1-
deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP)
pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) the
mevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii)
the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as a
degradation product of carotenoids); and general isoprenoid biosynthetic pathways
(as precursors of carotenoids).
The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXP
synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPP
isomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoene
synthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH),
zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyl
diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) have
been isolated from cDNA. In addition, the full-length genomic copy and putative
promoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated from
genomic DNA by the construction and screening of sub-genomic libraries.
Alignments of the genomic copies of these genes to the corresponding cDNA
sequences have provided useful information regarding the genomic organisation of
these genes, including the intron-exon junction sites in V. vinifera. The copy number
of the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genome
have been determined. DXS, PSY, BCH and lEP are single copy genes, whereas
LBCY and NCED have two and three copies, respectively.
The transcriptional activity of the putative promoters of six of the isolated genes
(i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reporter
gene assay. None of the putative promoters tested showed any transcriptional
activity of the reporter gene. The transcription of these genes, has however been
shown using northern blot analysis and/or RT-PCR. Preliminary expression profiles
for PSY, LBCY, BCH, and lEP were determined in different plant organs and the
expression of these genes was generally higher in photosynthetically active tissues.
The expression of these genes following different treatments (abscisic acid, NaCI and
wounding) was also assayed. The functionality of five of the isolated full-length genes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colour
complementation assay.
In silica analysis of the predicted protein sequences of all eleven isolated
genes revealed that they are conserved and share a high degree of homology to the
corresponding proteins in other plant species. The sequences were further analysed
for conserved domains in the protein sequences, and these proteins typically
demonstrated similar domain profiles to homologues in other species (plant, bacteria
and algae). The predicted protein sequences were further analysed for transit
peptides, the presence of which would provide evidence for the sub-cellular
localisation of the mature peptides. Since these genes are involved in biosynthetic
pathways that are active in discrete organelles, the sub-cellular localisation of most of
these proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH and
ZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathway
genes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonate
pathway gene, HMGS, was localised to both the cytosol and the mitochondria, and
the general isoprenoid precursor genes, FPS and GGPS, were localised to the
cytosol and the chloroplast, respectively. All these results are in agreement with the
localisation of the respective pathways.
In order to increase our understanding of carotenoid biosynthesis and functions
in plants, we constitutively overexpressed one of the isolated genes (BCH) in the
model plant, Nicotiana tabacum. Plants expressing the BCH gene in the sense
orientation maintained a healthy photosynthetic rate under stress conditions that
typically caused photoinhibition and photodamage in the untransformed control
plants. This result was inferred using chlorophyll fluorescence and confirmed using
CO2 assimilation rates and stomatal conductance. Chlorophyll fluorescence
measurements indicated that the photo protective non-photochemical quenching
ability of the BCH-expressing plants increased, enabling the plants to maintain
photosynthesis under conditions that elicited a stress response in the untransformed
control plants. An integral photosynthetic protein component, the D1 protein, was
specifically protected by the additional zeaxanthin in the BCH sense plants. Plants
expressing an antisense BCH proved the converse, i.e. lower levels of BCH resulted
in decreased zeaxanthin levels and made the transgenic plants more susceptible to
high-light induced stress. These results have shown the crucial role of carotenoids
(specifically the xanthophylls) in the photoprotective mechanism in plants. The
increased photoprotection provided by the BCH expressing plants suggests that the
scenario in plants is not optimal and can be improved. Any improvement in the
photoprotective ability of a plant will affect both the fitness and productivity of the
plant as a whole and will therefore find application in a number of crop plants on a
global scale. This study has resulted in the successful isolation and characterisation
of genes involved in the direct, or indirect, carotenoid biosynthetic pathways. The
further study and manipulation of these genes in model plants will provide useful
insights into the physiological role of specific carotenoids in photosynthesis and in
plants as a whole. / AFRIKAANSE OPSOMMING: Plante het nie die vermoë om stres te ontwyk nie en moet dus vinnig op veranderinge
in hulomgewingstoestande kan reageer. Indien hulle nie die fotosinteseproses kan
kontroleer en reguleer tydens streskondisies nie, sal dit tot die vorming van hoogs
reaktiewe suurstofspesies lei, wat beide die pigmente en proteiene van die
fotosintetiese apparaat sal beskadig. Lig is die primêre energiebron vir fotosintese
en daarom is dit noodsaaklik dat plante deurgaans 'n dinamiese balans tussen
fotosintese en fotobeskerming moet handhaaf. Karotenoiëde is voorlopers vir die
vorming van absisiensuur, maar meer belangrik vir die plant, ook integrale
komponente van die fotosintetiese apparaat. Tydens fotosintese word karotenoiëde
vir die opneem van lig benodig, terwyl dit ook die fotosintetiese apparaat beskerm
wanneer lig 'n versadigingspunt bereik vir fotosintese.
Weens die belang van karotenoiëde vir plant- en menslike gesondheid (as
Vitamiene A voorlopers), het hierdie studie beoog om gene te isoleer en
karakteriseer wat direk of indirek 'n rol in karoteenbiosintese in Vitis vinifera speel.
Elf vollengte- en agt gedeeltelike gene is geïsoleer, gekloneer, en gekarakteriseer.
Hierdie gene kan in die volgende biosintetiese paaie gegroepeer word: (i) die 1-
deoksi-D-xilulose 5-fosfaat (DOXP)/2-C-metiel-D-eritritol-4-fosfaat (MEP) pad (d.w.s.
die plastiediese isopenteniel difosfaat biosintetiese pad); (ii) die mevalonaat pad
(d.w.s. the sitosoliese/mitokondriale IPP biosintetiese pad); (iii) die karotenoiëd
biosintetiese pad; (iv) die absisiensuur biosintetiese pad (as 'n afbraak produk van
karotenoiëde) en die algemene isoprenoïed bisintetiese paaie (as voorlopers van
karotenoiëde ).
Die vollengte gene (d.w.s. vanaf die geskatte ATG tot die STOP kodon) van
DOXP-sintase (DXS), 4-hidroksi-3-metielbut-2-eniel difosfaatreduktase (lytB), IPPisomerase
(IPI), 3-hidroksi-3-metielglutariel koensiem A sintase (HMGS), fitoeën
sintase (PSY), likopeen p-siklase (LBCY), p-karoteen hidroksilase (BCH), zeaxantien
oksidase (ZEP), 9-cis-epoksi karotenoiëd dioksigenase (NCED), farnesiel difosfaat
sintase (FPS)en geranielgeraniel difosfaat sintase (GGPS) is met behulp van. RTPKR
vanaf eDNA geïsoleer. Die vollengte genomiese kopieë en die verwagte
promotors van die DXS, PSY, LBCY, BCH, NCED and ZEP gene is ook geïsoleer
d.m.v. die opstel en sifting van subgenomiese biblioteke. Vergelykende analises van
die genoom- en eDNA kopieë het insiggewende data oor die genomiese rangskikking
van die gene, insluitende die intron-ekson setels in V. vinifera gelewer. Die
kopiegetalle van DXS, PSY, LBCY, BCH, NCED en ZEP is bepaal. DXS, PSY, BCH
en ZEP is in die Vitis-genoom as enkel kopieë teenwoordig, terwyl LBCYen NCED
twee en drie kopieë, repektiewelik, beslaan.
Die transkipsionele aktiwiteit van die verwagte promotors van ses van die
geïsoleerde gene (naamlik DXS, PSY, LBCY, BCH, ZEP en NCED) is d.m.v. 'n
tydelike verklikkergeentoets ondersoek. Geeneen van die promotors het die
transkripsie van die verklikkergeen bemiddel nie. Die transkripsie van die gene is
egter wel bewys deur van northernhibridisasies en/of RT-PKR gebruik te maak. Die
promotors van hierdie gene kan dus as transkipsioneel aktief beskou word.
Voorlopige uitdrukkingsprofiele van PSY, LBCY, BCH, en ZEP is in verskillende
plantorgane bepaal; die profiele was deurgaans hoër in fotosinteties aktiewe
weefsels. Die uitdrukkingsprofiele van die gene is verder ook in reaksie op
verskillende induktiewe behandelings (absisiensuur, NaCI en beskadiging) bepaal. Vyf van die vollengte gene (IPI, GGPS, PSY, LBCYen BCH) is funksioneel bewys in
'n bakteriese funksionele kleurkomplementasiesisteem.
In silico analises van die afgeleide proteïene van al elf geïsoleerde gene het 'n
hoë vlak van homologie met ooreenstemende proteiene van ander plantspesies
getoon. Gekonserveerde domeine is ook in die proteïensekwense van die
geïsoleerde gene teenwoordig. Hierdie proteïene het deurgaans dieselfde
domeinprofiele vertoontoon as homoloë in ander spesies (bakterieë, alge en plante).
Die sub-sellulêre teikening van die gene kon voorspel word deur die seinpeptiede in
die proteiensekwense te eien. Aangesien hierdie gene betrokke is by biosintetiese
paaie wat in diskrete kompartemente plaasvind; is die sub-selluiêre lokalisering van
hierdie proteïene voorspelbaar. Die karotenoïed biosintetiese gene (PSY, LBCY,
BCH en ZEP), die absisiensuur biosintetiese geen, NCED, sowel as die DOXP/MEP
pad se gene (DXS, lytB en IPI) kom almal in die chloroplast voor. Die
mevalonaatpadgeen, HMGS, word na beide die sitosol en die mitokondria geteiken,
terwyl die algemene isoprenoïed voorlopergene, FPS en GGPS, onderskeidelik na
die sitosol en die chloroplast geteiken word. Die verkreë voorspellings stem met die
lokalisering van die biosintetiese paaie in die selooreen.
Om ons kennis rakende karotenoïed biosintese en veral hulle funksie(s) in
plante te verbreed, het ons een van die geïsoleerde gene, BCH, in die model plant,
Nicotiana tabacum, konstitutief ooruitgedruk. Plante wat die BCH geen in die "sense"
orientasie uitgedruk het, kon normale fotosintetiese aktiwiteit handhaaf onder
kondisies wat foto-inhibisie en foto-osidatiewe skade in die ongetransformeerde
kontrole plante veroorsaak het. Hierdie resultaat is met chlorofil fluoresensie
analises aangetoon terwyl dit met CO2 assimilasie- en huidmondjie
geleidingseksperimente bevestig is. Chlorofil fluoresensie metings het aangetoon
dat die beskermingsvermoë van die transgeniese plante verhoog is, en dit dan die
plante in staat stelom fotosintetese te handhaaf onder streskondisies van hoë lig.
Proteïen analises het aangetoon dat 'n integrale fotosintetiese proteien, die 01
proteïen, word veral deur die verhoogde zeaxantien vlakke in die BCH transgeniese
plante beskerm. Plante wat verminderde zeaxantien vlakke gehad het, weens die
konstitutiewe ooruitdrukking van die BCH geen in die anti-"sense" orientasie, het die
teenoorgestelde bewys. Met ander woorde. laer BCH vlakke (en dus laer zeaxantien
vlakke) het tot plante wat meer vatbaar was vir hoë lig geïnduseerde stress gelei.
Hierdie resultate het die essensiële beskermende rol wat karotenoiede tydens
fotosintese speel, uitgelig. Die vermoë om hierdie beskermende meganisme te
manipuleer in transgenies plante het aangetoon dat die sisteem in plante, alhoewel
effektief, nie optimaal is nie. Enige verbetering in 'n plant se inherente vermoë om
streskondisies te weerstaan sal die plant se algemene gesondheid en dus
produktiwiteit beïnvloed. As sulks sal hierdie in meeste gewasspesies toepassing
vind. Hierdie studie beskryf die isolering en karakterisering van gene wat direk, of
indirek, by karotenoïedbiosintese betrokke is. Verdere studies, en veral die
manipulering van hierdie gene in model plante, sal die fisiologiese rol van spesifieke
karotenoïeede in fotosintese, en die plant as 'n geheel, ontrafel.
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10 |
The construction of plant expression vectors for the introduction of leafroll disease resistance in grapevineVan Straten, Celene Debra 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Grapevine leafroll is one of the most damaging viral diseases that affect many
viticultural regions of the world. Numerous reports over the last few years
have associated closterovirus-like particles with leafroll disease. To date,
eight serologically distinct closteroviruses have been isolated from leafroll
infected vines, of which grapevine leafroll associated closterovirus-3
(GLRaV-3) is the best characterized.
Virus resistance in transgenic plants based on the expression of a virusderived
gene is known as pathogen-derived resistance. The viral coat protein
(CP) gene, which expresses a structural protein responsible for coating the
virus particles, was used in the first demonstration of virus-derived resistance.
Coat protein-mediated resistance is currently the most feasible and most
widely used method to obtain virus resistance in crop plants.
The CP gene of a South African isolate of GLRaV-3 infected grapevine was
isolated, cloned and sequenced. Double stranded RNA (dsRNA) was
extracted from GLRaV-3 infected material and a high molecular weight band,
of -18 kb was identified from infected vines. The dsRNA was used as a
template in a reverse transcription PCR together with GLRaV-3 CP gene
specific primers for the amplification of the GLRaV-3 CP gene (975 bp). The
GLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Clones
hosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-)
orientations respectively were obtained. The sequence obtained from these
two clones showed 99.26 % similarity to the only other GLRaV-3 CP
nucleotide sequence available. The GLRaV-3 CP gene was excised from
pLR3CP+ and pLR3CP- and subcloned into a plant expression vector,
pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense
(pCamBLR3CP-) orientations respectively, therefore enabling sense and
antisense gene expression in transgenic plants. The GLRaV-3 CP gene was
also subcloned from pCamBLR3CP+ into another plant expression vector,
pCAMBIA 2301 in the sense orientation and designated as pCVSLR3CP+.
These three constructs were given to Dr. M. Vivier (Institute for Wine
Biotechnology, Stellenbosch) for grapevine transformation experiments. Two
of these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacterium
tumefaciens-mediated transformation. Plants were selected for their ability to
withstand the herbicide, Basta. This resistance is due to the presence of a
plant selectable marker gene on each of these constructs, known as the bar
gene. PCR with GLRaV-3 CP gene specific primers showed no amplification
of the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ and
pCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene as
hybridization probe showed no signal for these plants, thus confirming the
PCR results. PCR with bar gene specific primers showed no amplification of
the bar gene in the plants infected with pCAMBIA 3301. The plants
transformed with pCamBLR3CP+ and pCamBLR3CP- were also screened for
the presence of the bar gene. Three of the eight plants tested showed
amplification of the -560 bp bar gene. This result suggests that these plants
were transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3
CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected.
This project provides preliminary work for the subsequent transformation of
grapevine with the GLRaV-3 CP gene, in an attempt to impart virus
resistance. / AFRIKAANSE OPSOMMING: Wingerd rolblaar is een van die mees beskadigende virale siektes wat baie
wingerd areas in die wêreld aantas. In Aantal verslae oor die afgelope jare
het closterovirus partikels met wingerd rolblaar geassosieer. Tot hede, is agt
serologiese onderskeibare closterovirusse geïsoleer vanuit geaffekteerde
wingerde, waarvan wingerd rolblaar geassosieerde closterovirus-3 (GLRaV-3)
die beste gekarakteriseerd is.
Virus bestandheid in transgeniese plante gebaseer op die uitdrukking van
gene afkomstig vanaf virusse, staan bekend as patogeen-afgeleide
weerstand. Die virale kapsule protein (CP) geen vervaardig In strukturele
protein wat verantwoordelik is vir die bedekking van die virus partikel. Dié
geen was gebruik in die eerste demonstrasie van patogeen-afgeleide
weerstand. Kapsuul protein-bemiddelde weerstand is tans die mees praktiese
en algemene gebruikte metode om virus weerstand in plant gewasse te
verkry. Die CP geen van In Suid Afrikaanse isolaat van GLRaV-3
geïnfekteerde wingerde is geïsoleer, gekloneer en die volgorde is bepaal.
Dubbelstring RNA (dsRNA) was uit GLRaV-3 geïnfekteerde materiaal
geëkstraheer en In hoë molekulêre gewig band van -18 kb is geïdentifiseer.
Die dsRNA is gebruik as In templaat vir In omgekeerde transkripsie PKR
saam met GLRaV-3 CP geen spesifieke inleiers vir die amplifikasie van die
GLRaV-3 CP geen (975 bp). Die GLRaV-3 CP geen is gekloneer in die
pGem®-T Easy vektor. Klone met die CP geen in die sin (pLR3CP+) en
teensin (pLR3CP-) oriëntasies respektiewelik is verkry. Die volgorde wat
verkry is vanuit hierdie twee klone dui op In 99.26 % ooreenstemming met die
enigste ander GLRaV-3 CP geen volgorde wat beskikbaar is. Die GLRaV-3
CP geen is uit pLR3CP+ en pLR3CP- gesny en is gesubkloneer in In plant
ekspressie vektor, pCAMBIA 3301 in die sin (pCamBLR3CP+) en teensin
(pCamBLR3CP-) oriëntasies respektiewelik, wat die sin en teensin geen
ekspressie in transgeniese plante in staat stel. Die GLRaV-3 CP geen was
ook gesubkloneer vanaf pCamBLR3CP+ in In ander plant ekspressie vektor,
pCAMBIA 2301 in die sin orientasie en is as pCVSLR3CP+ benoem. Hierdie
drie konstruksies is aan Dr. M. Vivier (Instituut vir Wyn Biotegnologie,
Stellenbosch) gegee vir wingerd transformasie eksperimente. Twee van hierdie konstruksies, pCamBLR3CP+ en pCamBLR3CP- asook pCAMBIA
3301 is gebruik om Nicotiana tabacum deur middel van Agrobacterium
tumefaciens-bemiddelde transformasie te transformeer. Plante is geselekteer
vir hul vermoë om die onkruiddoder, Basta, te weerstaan. Die
teenwoordigheid van die plant selekteerbare merker geen, bar, op elke
konstruksie lui tot dié weerstand. Die plante wat getransformeer is met
pCamBLR3CP+ en pCamBLR3CP- is deur PKR saam met die GLRaV-3 CP
geen spesifieke inleiers getoets, en geen amplifikasie van die GLRaV-3 CP
geen is getoon nie. Southern blot analise met die GLRaV-3 CP geen as
hibridisasie peiler het geen sein gewys vir hierdie plante nie, wat die PKR
resultate bevestig. Die plante wat getransformeer is met pCAMBIA 3301 is
deur PKR saam met die bar geen spesifieke inleiers getoets, en geen
amplifikasie van die bar geen is getoon nie. Die plante wat getransformeer is
met pCamBLR3CP+ en pCamBLR3CP- is ook getoets vir die
teenwoordigheid vir die bar geen. Drie van die agt plante wat getoets is, het
amplifikasie van die -560 bp bar geen getoon. Hierdie onverwagte resultate
stel voor dat dié plante met pCAMBIA 3301 (vektor sonder die geligeerde
GLRaV-3 CP geen) en nie met pCamBLR3CP+ en pCamBLR3CPgetransformeer
is nie. Hierdie projek verskaf voorlopige werk vir die
daaropvolgende transformasie van wingerd met die GLRaV-3 CP geen in 'n
poging om virus bestandheid te verskaf.
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