Designing and construction of specific plasmid constructs for targeted plastome transformation

Integration of foreign genes into plastid genomes occur through homologous recombination between flanking sequences of gene in plastid vectors. In this study, by analysis of plastid genomes using bioinformatics databases, we succeed to select a region of plastome as alien gene targeting sequence including the appropriate length for homologous recombination and integration in plastome, specific plastid origin of replication, targeting gene to inverted repeat region of plastid genome and unique restriction enzymes recognition sites in the center of flanking region sequences for alien gene integration. Since similarity (%) of the flanking region sequences would increase transformation efficiency of plastome dramatically, therefore, a pair of primers was designed to isolate the plastome flanking regions. These primers were100% similar to all plant plastomes, but the length and sequence of their amplified fragments were variable in different plant plastomes. Flanking region sequence from tobacco, cotton, corn, lettuce, tomato, carrot, and even the canola and lemon plastomes that still their plastome sequences are not available in Gene Bank were isolated and cloned. The accuracy of cloning, present and direction of flanking regions fragment from different plant plastomes were confirmed by enzymatic digestion analysis using HindIII and BamHI. Then different types of chloroplastid vectors using different regulation elements were constructed. The regulation elements that could be used for efficient expression of any alien genes in different plastids were including plastid constitutive or inducing promoters and terminators, suitable untraslated regions, Ribosome binding sites of plastid, suitable restriction enzyme recognition sites for cloning and expression of desired gene in single or polycistronic status, reporter genes, antibiotic or non-antibiotic markers and sequences for removing of marker gene. These recombinant chloroplast plasmids including pFNGi and pFNG can be used as universal and specific vectors for chloroplast transformation. Thus, in this study we succeeded to design the high efficient and specific plastid vectors, not only for plants which their plastomes were completely sequenced, but also for other plant species that there is not any sequence of their plastomes availaable in databanks.