The molecular characterisation and genetic transformation of the grapevine chloroplast genome
Burger, J T
University of Stellenbosch. Department of Genetics
Laubscher, A E
Rose, B A
This project has as its ultimate goal the development of a genetic transformation system for the grapevine chloroplast genome. Towards this goal, a thorough molecular characterisation of the genome of this plant organelle is inevitable. Several shortcomings and potential hazards of nuclear transformation are addressed by plastid transformation. The most obvious of these are:
1. foreign proteins can be expressed at 100- to 1000-fold higher levels
2. insertion in the chloroplast genome is directed, i.e. accidental interference with normal gene expression is prevented
3. since chloroplast genomes of most higher plants are maternally inherited, the potential horizontal gene escape via pollen, is eliminated. In addition, we aim to establish an antibiotic marker-free plastid transformation technology.
In this multi-faceted project, we propose to develop transformation technology for the grapevine chloroplast genome, and to do a comprehensive molecular characterisation of this genome. The first goal would be to optimise a transient expression system in grapevine chloroplast by biolistic bombardment. A ‘broad-spectrum’ chloroplast transformation vector (obtained from abroad) would be used for this study. To enhance our chances for constitutive expression of transgenes in chloroplasts, a grape-specific vector will have to be developed. We plan to utilise grape chloroplast promoter and terminator elements in our vector to drive the expression of a green fluorescent protein gene and a non-antibiotic resistance gene as screenable and selectable markers, respectively. Two grapevine chloroplast-specific flanking sequences will be incorporated into the vector to facilitate the targeted insertion of this expression cassette in the chloroplast genome. The second facet of the project comprises the sequencing of the entire grapevine chloroplast genome. Chloroplast DNA will be isolated and used to construct a library of overlapping clones, which will be sequenced and compiled into the complete chloroplast sequence. Sequence information is required to isolate the various elements needed in the construction of the transformation vector.
Robson, J and Burger, J T. 2002. Construction of a grapevine-specific chloroplast transformation vector. Poster presented at the Plant, Animal and Microbe Genome X Meeting. San Diego, United States.
Robson, J and Burger, J T. 2002. Construction of a grapevine specific chloroplast transformation vector. Paper presented at the 40th Congress of the South African Society for Plant Pathology. Dikhololo, South Africa.
Robson, J and Burger, J T. 2002. Construction of a grapevine-specific chloroplast transformation vector. Paper presented at the Cape Biotechnology Congress, South Africa.
Rose, B A and Burger, J T. 2002. Molecular characterisation and partial sequencing of the grapevine chloroplast genome. Paper presented at the Cape Biotechnology Congress, South Africa.
Robson, J and Burger, J T. 2002. Construction of a grapevine-specific chloroplast transformation vector. Presentation to the Experimental Biology Group.
Rose, B A and Burger, J T. 2004. Molecular characterisation and partial sequencing of the grapevine chloroplast genome. Paper presented at the 18th Congress of the South African Genetics Society. South Africa.