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

[摘要] ENGLISH ABSTRACT:The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. TheDNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderivedgenome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are anattractive target for genetic engineering as high protein expression levels are readily obtained due tothe high genome copy number per organelle. The resultant proteins are contained within the plastidorganelle 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 processfacilitated by homologous recombination and the non-Mendelian segregation of plastids upon celldivision. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due totheir common genetic complement. This enables the site-specific integration of any piece of DNAflanked by plastid targeting sequences, via homologous recombination. The attainment ofhomoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectablemarker. Selective pressure favouring the propagation of the transformed genome copies, as well as therandom segregation of plastids upon cell division, make it feasible to acquire uniformity and hencegenetic stability. From this, a complete transplastomie line is obtained where all plastid genomecopies present are transgenic, having eliminated all wild-type genome copies.The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins frompolycistronic mRNAs, allowing the introduction of entire operons in a single transformation.Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbourgenes encoding selectable and screenable markers, as well as one or more genes of interest. Eachcoding region is preceded by an appropriate translation control region to ensure efficient translationfrom the polycistronic mRNA.The function of a plastid transformation vector is to enable transfer and stable integration of foreigngenes into the chloroplast genomes of higher plants. The expression vector constructed in thisresearch is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from thefamily, Vitaceae, is the choice species for the production of wine and therefore our target for plastidtransformation. All chloroplast derived regulatory elements and sequences included in the vector thusoriginated from this species.

[发布机构] Stellenbosch University