Expression Of Bacterial RpoD Gene In Higher Plant Of Nicotiana Tabacum

Evidence has shown that Chloroplasts were derived from previously free-living cyanobacteria. Chloroplasts have retained many prokaryotic features including transcription and translation mechanisms. Transcription in plant plastids is mediated by at least two distinct transcription systems, one of which is primarily plastid-encoded plastid RNA polymerase (PEP) and one of which is exclusively nuclear-encoded plastid RNA polymerase (NEP). The PEP polymerase is a multi-subunit enzyme complex that depends on sigma like factors (SLFs) to recognize and initiate at eubacterial-type plastid promoters. Amino acid sequence comparison between plastid SLFs and bacterial σ-70 showed that four highly conserved domains are located at C-terminal, the functional region. Molecular assays also revealed that chloroplast psbA promoter can be recognized by bacterial RNA polymerase holoenzyme including σ-70 factor in vitro and the green fluorescent protein gene (gfp) driven by bacterial trc promoter can express in chloroplasts of transplastomic tobacco plants in vivo. We therefore cloned and transformed bacterial E.coli rpoD gene, the principal sigma factor (σ-70) in bacteria into the tobacco, Nicotiana tabacum, nuclear genome. Bacterial σ-70 factor was fused to a petunia plastid targeting leading peptide, therefore the protein of bacterial sigma 70 goes into plastids. Southern hybridization showed that rpoD genes were inserted into different plant nuclear sites with different copy numbers in individual lines. Expression levels of the gene revealed by Northern blotting indicated that multi copy insertions would result in relatively low gene expression compared with that of single copy insertion. Further assays on the...