Construction and Characterization of a Chloroplast Targeting Plasmid DNA Vector in Nicotiana tabacum

A chimeric plasmid DNA vector designated as pMS65LBgus was constructed with the aim of targeting chloroplasts in Nicotiana tabacum. The Plasmid was designed to contain β-glucuronidase (gusA) gene under a modified chloroplast 16S rRNA gene promotor and a psbA gene terminator sequence. Adjacent to gusA gene, plasmid vector was designed to contain a selectable marker aminoglycoside 3’ adynyle-transferase (aadA) gene under atpA gene promotor and rbcL gene terminator sequences. The entire cassette with two genes was flanked by ~1.0 kb fragment on each side, spreading through 3’rps12 gene and orf131 of the tobacco chloroplast genome. Both gusA and aadA genes orient in opposite directions and are expressed in Escherichia coli. The entire cassette with aadA and gusA genes along with their expression signals can be excised by Nde I restriction endonuclease digestion. Multiple cloning sites at both ends of the gusA coding sequence enable a desired passenger gene to be replaced with gusA. PCR amplification, Southern hybridization, restriction mapping and dot blot analysis of the vector revealed a high degree of homology of the 2 kb tobacco chloroplast targeting fragment in the vector with various plant species, suggesting that the targeting vector may also be useful in other plants.