Assessment of Chloroplast Expression Factors in Escherichia coli

Background: genetic manipulation of chloroplast in higher plants offers a number of unique prerogatives, including; undesirableof pleiotropic genome and gene silencing effects and also use as an important agronomic trait for producing essential biomaterials andindustrial enzymes. in order to manipulate chloroplast genome, specific vectors are required. these vectors can be transformed and expressedin escherichia coli due to the same evolutionary origin of bacteria and chloroplasts.objectives: the aim of the present study was to construct chloroplast vector specified for spinach and assessing the chloroplast regulatoryelements in a prokaryotic expression host, e. coli.materials and methods: flanking sequences (insl+insr) were isolated by pcr from the spinach chloroplast genome and blunt-end ligatedinto the pvuii site of puc19 vector to form an intermediate vector, pucins. then the selectable marker cassette (including aada gene, prrnpromoter and rbcl terminator) was isolated via pcr and blunt-end cloned into the unique pvuii site of pucins to make the final chloroplastvector, named pcsi.results: the constructed vector transformed to e.coli strain dh5? and several procedures such as colony pcr, digestion and sequencing wereassigned to confirm the consequence of the construct.conclusions: the appearance of bacterial colonies on the plate containing different concentrations of streptomycin indicated the strengthof resistance to streptomycin which showed the bacterial cells capability to express aada gene under the controls of chloroplast regulatoryelements.