The construction of an expression vector for the transformation of the grape chloroplast genome

Robson, Julia (2003-12)

Thesis (MSc)--University of Stellenbosch, 2003.

Thesis

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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