Submitted to: In Vitro Biology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: April 1, 2005
Publication Date: June 30, 2005
Citation: Ruhlman, T.A., Rajasekaran, K., Cary, J.W. 2005. Plastids transformed: Expressing microbial resistance genes from the chloroplast genome. In Vitro Biology Meeting, June 2005, Baltimore, MD. 41:39-A. Technical Abstract: While genetic improvement of susceptible crop species may enhance resistance to microbial pathogens and facilitate reduced pesticide load, the possibility for transmission of novel genes to wild relatives has hampered acceptance of GM crops in some markets. Chloroplast transformation presents an attractive alternative to nuclear transformation and offers the potential to ameliorate environmental concerns. Most agronomically important species exhibit maternal inheritance of organellar genomes eliminating the threat of transgene escape through pollen and gene silencing is absent. The border sequences in our vector, pLD-CtV, direct single copy insertion by homologous recombination in the inverted repeat region of the tobacco plastome. Each transformant contains up to 20,000 copies of the transgene in a leaf cell, proteins can accumulate to high levels (up to 50 percent of total soluble protein) and are retained within the chloroplast envelope protecting them from degradation by host cytoplasmic proteases. Two antimicrobial gene constructs, a) a bacterial chloroperoxidase (cpo-p) and b) D4E1, a synthetic, lytic peptide, were ligated into pLD.CtV for transformation experiments. Transformants were identified by selection on regeneration media with 500 mg L**-1 spectinomycin, resistance to which was conferred by the aminoglycoside-3'-adenyltransferase (aadA) gene in the plasmid vector. PCR and Southern analysis has confirmed integration in the plastome. Western analysis confirms the presence of the CPO-P in higher abundance in chloroplast transformants than nuclear. Although integration and expression was achieved in these experiments, concomitant increase in the level of protein accumulation was not observed. Northern analysis of primary transformants for cpo-p showed 15 fold higher transcript abundance than nuclear transformants; yet this was not reflected in western blot, enzyme or bioassay. In an effort to enhance the rate of translation a new construct was developed replacing the single ribosome binding site between aadA and cpo-p with the tobacco full length psbA 5' UTR.