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
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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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53763 |
| Date | 03 1900 |
| Creators | Rose, B. A. (Beverley Ann) |
| Contributors | Burger, J. T., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 126 p. : ill. |
| Rights | Stellenbosch University |
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