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. 2010 Mar 22;107(14):6204–6209. doi: 10.1073/pnas.0914423107

Fig. 3.

Fig. 3.

Generation of plastid transformants with vectors carrying gfp gene constructs under the control of riboswitch elements. (A) Physical map of the targeting region in the plastid genome. (B) Structure of plastid transformation vectors of the pAV series harboring riboswitch-containing GFP expression cassettes. Relevant restriction sites are marked. The transgenes are targeted to the intergenic region between the trnfM and trnG genes (37). The GFP expression cassette consists of the ribosomal RNA operon promoter (Prrn) fused to the riboswitch (RS) element (see Fig. 1 and Fig. S1) and the 3′UTR from the plastid rps16 gene (Trps16). The expected sizes of DNA fragments in restriction fragment length polymorphism analyses with the enzyme BglII are indicated. The location of the RFLP probe is shown as a black bar. The selectable marker gene aadA is driven by a chimeric ribosomal RNA operon promoter (Prrn) and fused to the 3′UTR from the plastid psbA gene [(TpsbA (38)]. (C) RFLP analysis of transplastomic tobacco lines. Total cellular DNA was digested with BglII and hybridized to a radiolabeled probe detecting the region of the plastid genome that flanks the transgene insertion site. Fragment sizes for the wild-type and the transplastomic lines are indicated. Absence of a hybridization signal for the wild-type genome indicates homoplasmy of the transplastomic lines. Line Nt-pAV4-10A shows the hybridization signal for the wild-type genome suggesting that this line represents a spontaneous antibiotic-resistant mutant. The transplastomic lines harbor the following riboswitches: Nt-pAV1: gly-RS; Nt-pAV2: ade-RS; Nt-pAV3: tpp-RS; Nt-pAV4: s.gly-RS; Nt-pAV5: s.ade-RS; Nt-pAV6: s.theo-RS.