| Summary: | Thesis (MSc)--University of Stellenbosch, 2003. === ENGLISH ABSTRACT: The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. The
DNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderived
genome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are an
attractive target for genetic engineering as high protein expression levels are readily obtained due to
the high genome copy number per organelle. The resultant proteins are contained within the plastid
organelle and the corresponding transgenes are inherited, in most crop plants, uniparentally,
preventing pollen transmission of DNA.
Plastid transformation involves the uniform modification of all the plastid genome copies, a process
facilitated by homologous recombination and the non-Mendelian segregation of plastids upon cell
division. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due to
their common genetic complement. This enables the site-specific integration of any piece of DNA
flanked by plastid targeting sequences, via homologous recombination. The attainment of
homoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectable
marker. Selective pressure favouring the propagation of the transformed genome copies, as well as the
random segregation of plastids upon cell division, make it feasible to acquire uniformity and hence
genetic stability. From this, a complete transplastomie line is obtained where all plastid genome
copies present are transgenic, having eliminated all wild-type genome copies.
The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins from
polycistronic mRNAs, allowing the introduction of entire operons in a single transformation.
Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbour
genes encoding selectable and screenable markers, as well as one or more genes of interest. Each
coding region is preceded by an appropriate translation control region to ensure efficient translation
from the polycistronic mRNA.
The function of a plastid transformation vector is to enable transfer and stable integration of foreign
genes into the chloroplast genomes of higher plants. The expression vector constructed in this
research is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from the
family, Vitaceae, is the choice species for the production of wine and therefore our target for plastid
transformation. All chloroplast derived regulatory elements and sequences included in the vector thus
originated from this species. === AFRIKAANSE OPSOMMING: Die genetiese inligting van plante word gevind in die kern, die mitochondria, en die plastiede. Die
DNA van plastiede bestaan uit veelvuldige kopieë van 'n ~ 150 kb dubbelstring, sirkulêre genoom van
prokariotiese oorsprong. Die genoomekwivalente van plastiede in hoër plante is 'n aantreklike teiken
vir genetiese manipulering, aangesien die hoë genoom kopiegetal per organel dit moontlik maak om
gereeld hoë vlakke van proteïenuitdrukking te verkry. Hierdie proteïene word tot die plastied beperk,
en die ooreenstemmende transgene word in die meeste plante sitoplasmies oorgeërf, sonder die
oordrag van DNA deur die stuifmeel.
Plastied transformasie behels die uniforme modifikasie van al die plastied genoomkopieë, 'n proses
wat deur homoloë rekombinasie en die nie-Mendeliese segregasie van plastiede tydens seldeling
gefasiliteer word. As gevolg van die gemeenskaplike genetiese komplement, vind aanhoudende interen
intra-molekulêre uitruiling van plastiedgenome plaas. Dit maak die setel-spesifieke integrasie, via
homoloë rekombinasie, van enige stuk DNA wat deur plastied teikenvolgordes begrens word,
moontlik. Vir die verkrying van homoplasmie, waar alle genome getransformeer is, word die
insluiting van 'n plastiedspesifieke selekteerbare merker benodig. Seleksiedruk wat die vermeerdering
van die getransformeerde genoomkopieë bevoordeel, en die lukrake segregasie van plastiede tydens
seldeling, maak dit moontlik om genetiese stabiliteit en uniformiteit van die genoom te verkry. Dit
kan op sy beurt tot die verkryging van 'n volledige transplastomiese lyn lei, waar alle aanwesige
plastiedgenome transgenies is, en wilde tipe genoomkopieë geëlimineer is.
Die prokariotiese aard van die chloroplas genetiese sisteem maak die uitdrukking van veelvuldige
proteïene vanaf polisistroniese mRNAs moontlik, wat die toevoeging van volledige operons in 'n
enkele transformasie toelaat. Uitdrukkingskassette in vektore bevat dus enkel regulatoriese elemente
van plastied oorsprong, gene wat kodeer vir selekteerbare en sifbare merkers, asook een of meer gene
van belang (teikengene). Voor elke koderingsstreek, is daar ook 'n toepaslike translasie beheerstreek
om doeltreffende translasie vanaf die polisistroniese mRNA te verseker.
Die funksie van 'n plastied transformasie vektor is om die oordrag en stabiele integrasie van transgene
in chloroplasgenome van hoër plante moontlik te maak. Die uitdrukkingsvektor wat in hierdie studie
gekonstrueer is, is spesifiek vir die transformasie van die druif chloroplasgenoom. Vitis vinifera L.,
van die familie Vitaceae, is die voorkeur species vir die produksie van wyn, en daarom die teiken vir
plastied transformasie. Alle chloroplast-afgeleide regulatoriese elemente en volgordes wat in hierdie
vektor ingesluit is, het huloorsprong vanaf VUis vinifera L.
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