A protocol for the study of plant transformation was created to understand the role of catalase, a plant enzyme, in plant defense systems. Our lab has isolated two catalase clones (pBKAM and pBSAL) from the bean plant Phaseolus vulgaris. pBKAM and pBSAL were mapped using endonuclease digestion analysis and Northern blot analysis was used to determine the orientation of each insert in the vector. Using an immunological detection system, the orientation of pBKAM was determined. Transcription of pBKAM from the T3 promoter results in “sense” RNA (produces the coding mRNA), whereas transcription from the T7 promoter results in “antisense” (non-coding) RNA. Results for pBSAL were inconclusive.
With this information, we can insert our catalase cDNA clones into plant cells using a natural vector system with Agrobacterium tumefaciens. The natural vector system we are using consists of the binary vector pAS2023. Catalase inserts from pBKAM and pBSAL have been subcloned into the binary vector and the orientation of the clones was determined using endonuclease digestion analysis of the vector pAS2023. Using this vector allows us to add additional copies of the catalase gene into the plant cells in both the “sense” and “antisense” orientation.
If plant defense is regulated transcriptionally by the catalase gene, adding unregulated sense copies of this gene will result in a failure to induce cell necrosis following cell perception of a pathogen. Addition of extra antisense copies of the gene will result in a failure to relieve the initial oxidative burst following a pathogenic challenge. Using these results, we will be able to continue to investigate the exact role of catalase in plant defense.
Amanda Gerber, ’01 Cedar Rapids, IA
Sovady Phe, ’02 Brooklyn Park, MN
Sponsor: Craig Tepper