Date of this Version
Kahlandt, E. (2018) Plant Mitochondrial DNA Damage and Repair: PvuII as a Model for Damage Induction
In contrast to the DNA contained in animal and fungal mitochondria, plant mitochondrial genomes have significantly larger amounts of DNA, but not significantly more genes. However, the genes that they do contain are very highly conserved, while the large intergenic regions contain a multitude of insertions and deletions. It is thought that this strange pattern might be the result of the way the mitochondria repair their damaged DNA; namely, by using double-strand break repair. To test this hypothesis, the endonuclease PvuII was used to create blunt-end cuts in the mitochondrial genome. By attaching the endonuclease gene to a mitochondrial targeting peptide, from the gene AOX1A, PvuII can be transported to the mitochondria. This construct was then introduced into a plasmid vector, initially pART and later pMDC7, and then transformed into plants using Agrobacterium. The plants used for this study were Arabidopsis thaliana, which have been shown to be useful in modeling plant genetics. Both vectors are inducible, meaning that upon exposure to a particular substance, the genes in the plasmid will be transcribed; pART is ethanol inducible and pMDC7 is estradiol inducible. Plants with pART were selected and bred to obtain homozygosity. Once this was achieved, studies involving inducing the PvuII activity and exposure to stress, like drought, were done. DNA was extracted and prepared for sequencing from T3 plants and is currently being examined for any interesting patterns that would show how DNA repair is regulated and carried out in mitochondria. pMDC7 is currently being transformed into agrobacterium and will be transformed into plants in the near future.