Transformation of Tobacco cpDNA with Fusion E7GGG/GUS Gene and Homologous Recombination Mediated Elimination of the Marker Gene

ABSTRACT

A transformation vector harboring the marker gene aadA conferring spectinomycin resistance and the fusion E7GGG/gus gene of interest was employed for the transformation of tobacco cpDNA via biolistics. The gene of interest consisted of the oncogenic E7GGG gene from human papillomavirus strain 16 fused with the reporter gus gene. Both transgenes were equipped with the same promoter and termination sequences arranged as direct repeats. Biolistics with circular vector yielded 20 shoots rooted in spectinomycin containing medium; with linear vector no rooted plants were obtained. After 4 months of in vitro selection the plants were burst into flower in a greenhouse and pollinated with non-transgenic plant pollen. Seeds were harvested individually from each plant capsule and planted onto selection medium. Rare white seedlings were recovered by transfer onto medium without spectinomycin and self-pollinated. GUS activity of 15 seedlings from each self-pollinated plant was measured and 26 plants with higher activity (and one without any GUS activity) were PCR analyzed for the presence of both the marker gene and the gene of interest. Finally, 8 plants were analyzed in detail by Southern hybridization, RT-PCR, and Western blot. We identified 6 plants harboring the E7GGG/gus gene only, where the marker gene was eliminated by homologous recombination; one plant with both transgenes; and one with the aadA gene only. RT-PCR showed the presence of the respective mRNAs in all 8 plants analyzed but Western blot proved that only the GUS part of the fusion E7GGG/GUS protein was present in all plants harboring the fusion gene. We discuss why E7GGG protein could not be detected.

Keywords:

• E7GGG oncogene
• chloroplast transformation
• homologous recombination
• marker-free plant
• gus reporter gene