The characterisation and partial sequencing of the grapevine chloroplast genome

Rose, B. A. (Beverley Ann) (2004-03)

Thesis (MSc)--University of Stellenbosch, 2004.

Thesis

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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