Analysis of plastid recombination and fluorescent protein expression in transplastomic plants

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Abstract

The first aim of this thesis was to exploit homologous recombination between two 1.4kb imperfect rbcL direct repeats (2.4% base divergence) to generate novel plastid alleles in planta. The rbcL gene encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Multiple recombination events were observed between the two rbcL repeats producing eight novel rbcL alleles, including mosaic sequences. Direct repeat recombination excised aadA and dao marker genes at high frequency allowing the isolation of marker-free plants in the T0 generation. High marker excision rates explained regeneration of plastid transformants on both D-alanine (marker-containing shoots) and D- valine (marker-free shoots) and the leaf-lamina-loss phenotype observed on spectinomycin. The second aim of this thesis was to assemble a vector comprised of imperfect direct repeats (1.4% base divergence) of the cyan fluorescent gene (cfp) and the yellow fluorescent gene (yfp). Both coding regions were fused to the whirly DNA binding protein. The cfp and yfp genes were made using overlap PCR on a codon-optimised gfp template. A change from cyan to yellow fluorescence would allow recombination to be visualized in plastids. The whirly binding domain would localize the fluorescence to nucleoids, potentially the nucleoids from which they were transcribed if transcription and translation were coupled. The vectors were partly assembled but due to a lack of CFP fluorescence and the apparent toxicity of the whirly fusion protein the project was not completed. The final aim of this thesis was to express, for the first time, the green-to-red photoconvertible Dendra2 protein, in plastids to track protein trafficking within and between plastids. The Dendra2 protein accumulated to high amounts in transplastomic plants and was visible as a distinct band when total leaf protein was fractionated by SDS- polyacrylamide- gel electrophoresis. Wild type and transplastomic Dendra2 seedlings were grown on 0.2 μM norflurazon to reduce red chlorophyll autofluorescence. Using a Leica SP5 inverted confocal microscope, Dendra2 was photoconverted with 405 nm UV at 20- 30% laser strength and an exposure time of 20-30 seconds. Photoconversion resulted in a change from green to red fluorescence, which was stable over the time observed. Photoconverted red stromules in individual plastids rapidly changed to yellow showing mixing of their contents with the green fluorescence in the stroma of the same plastids. Photoconverted whole red plastids remained red over several minutes despite apparent collisions with other more abundant green fluorescent plastids indicating limited or no mixing of the stromal proteins between plastids.

Keyword(s)

Dendra2; Photoconvertible fluorescent protein; Plant biotechnology; Recombination; Rubisco modification; Site-directed mutagenesis

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