The chloroplast genome of Chlamydomonas reinhardtii has been transformed with a chimeric gene consisting of the chloroplast atpA promoter and the bacterial gene for aminoglycoside adenine transferase (aadA). The atpA-aadA cassette has been placed within the chloroplast DNA EcoRI restriction enzyme fragment 14, or within the chloroplast BamH1 fragment 10. The chimeric constructs were introduced into the chloroplast by particle bombardment. Integration of the cassette into chloroplast DNA then occurred via homologous recombination of sequences flanking the cassette with their corresponding chloroplast sequences. We demonstrate that the chloroplast atpA promoter in atpA-aadA routinely recombines with its endogenous counterpart, resulting in heteroplasmic chloroplast DNA populations that may persist for many generations. The heterologous gene does not require a 3′ inverted repeat sequence for its expression. The atpA-aadA gene copy number, which is dictated here by its position in the chloroplast genome, is proportional to the steady state level of atpA-aadA mRNA. However, neither genomic position, gene copy number, or mRNA level have a significant effect on cellular resistance to spectinomycin, nor activity of the aadA gene product in vitro. These results suggest that, in the case of aadA, the limiting step for expression of this gene is at the translational or post-translational level. The atpA-aadA cassette should prove a useful model for future studies on the maintenance and expression of heterologous genes in C. reinhardtii chloroplasts.
- Chlamydomonas reinhardtii
- aminoglycoside adenine transferase
ASJC Scopus subject areas
- Aquatic Science
- Plant Science