Chloroplast transformation vectors and integration of transgenes into the chloroplast genome. (A) Tobacco chloroplast expression vectors. Homologous chloroplast genome flanking sequences comprising 16S (16S rRNA), isoleucine tRNA (trnI), alanine tRNA (trnA) gene sequences. In both vectors, a glycine-proline-glycine-proline hinge and furin cleavage site (RRKR) is included between CTB and the FVIII domain sequence. The restriction site of AflIII and the sizes of Southern blot fragments are indicated. (B) Southern blot, tobacco CTB-HC, wild-type (WT) (untransformed), 1-3 transplastomic lines. Tobacco total genomic DNA was digested with AflIII and probed with 0.81 kb trnI/trnA flanking region fragment. (C) Southern blot, tobacco CTB-C2, WT (untransformed WT), 1-4 transplastomic lines. 3′ UTR, 3′ UTR of tobacco psbA gene; 5′ UTR, promoter and 5′ UTR of tobacco psbA gene; aadA, aminoglycoside 3′-adenylyltransferase gene to confer spectinomycin resistance; Nt, Nicotiana tabacum. Prrn, ribosomal RNA operon promoter with GGAG ribosome binding site; WT, untransformed WT.