Fig. 1.
Introduction of cyanobacterial genes for DNA methylation enzymes into the tobacco plastid genome. a Map of the plastid genome (ptDNA) region to which the transgenes were targeted. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Restrictions sites used for transgene insertion (SpeI) and RFLP analyses (BamHI) are indicated. b Map of the plastid transformation vectors pDam and pDcm constructed in this study. The vectors are based on the previously constructed plasmids pRB94 (Ruf et al. 2001) and pRB96 (Wurbs et al. 2007). The chimeric selectable marker gene aadA is driven by the ribosomal RNA operon promoter (Prrn; Svab and Maliga 1993). The DNA methyltransferase genes (dam and dcm) are transcribed from the atpI promoter (PatpI; Miyagi et al. 1998; Wurbs et al. 2007). The 3′ UTR of the aadA cassette is from the psbA gene (TpsbA; Svab and Maliga, 1993), the 3′ UTR of the methylase transgenes from the rps16 gene (Wurbs et al. 2007). c Southern blot analysis to confirm transgene integration by homologous recombination. DNA samples from the wild-type (Nt-Wt), three Nt-dam lines and three Nt-dcm plants (representing two independently generated transplastomic lines, Nt-dcm3 and Nt-dcm9) were digested with BamHI and hybridized to a psaB-derived probe. The size difference between the hybridizing bands in the wild-type and the transplastomic lines corresponds to the combined size of the two transgene cassettes (aadA + methylase gene). The faint wild-type-like band represents promiscuous chloroplast DNA in the nuclear genome as demonstrated earlier (Wurbs et al. 2007). M: DNA size marker (band sizes given in kb). d Seed assays to confirm homoplasmy of the transplastomic lines. Exemplarily, seed assays for one Nt-dam and one Nt-dcm plant are shown. As a control, the wild-type on medium with (+Spec) and without spectinomycin (–Spec) is also shown. Resistance to spectinomycin and lack of phenotypic segregation in the T1 generation demonstrate homoplasmy of the Nt-dam and Nt-dcm lines