Pre-screening plastid transgene expression cassettes in Escherichia coli may be unreliable as a predictor of expression levels in chloroplast-transformed plants

Abstract

A di-cistronic expression cassette ( Hb) encoding the α- and β-subunits of human adult hemoglobin, under the transcriptional control of a phage T7 promoter, was introduced into the tobacco plastid genome. The resulting chloroplast-transformed line, Hb1, was crossed with a nuclear-transformed line, PR-T7A, expressing a salicylic acid-inducible plastid-targeted T7 RNA polymerase in order to activate Hb transcription. Even in the absence of induction, Hb transcripts were expressed constitutively in Hb1xPR-T7A progeny plants. Treatment of leaves with salicylic acid resulted in an additional five-fold increase in Hb transcript levels. However, despite the very high-level of Hb transcript accumulation in Hb1xPR-T7A plants and the fact that the Hb expression cassette directed the synthesis of hemoglobin in Escherichia coli, recombinant hemoglobin did not accumulate at levels detectable by immunoblot analysis in chloroplast-transformed plants. Furthermore, Hb transcripts present in total leaf RNA isolated from Hb1xPR-T7A plants directed hemoglobin synthesis in an E. coli-derived in vitro translation system thus excluding the possibility that Hb mRNA might have been rendered untranslatable by the plastid RNA editing machinery.