Tobacco Plastid Transformation Vector Construction

A tobacco plastid transformation vector was constructed based on the iVEC technology (Figure 1A). The vector mediates integration of the expression cassettes into the intergenic spacer region between the trnfM and trnG genes of the tobacco plastid genome. For vector assembly, we first amplified five DNA fragments which have pairwise homologous end sequences by Pfu DNA polymerase using a standard PCR protocol (95°C 3 min; 95°C 30 s, 55°C 30 s, 72°C 3 min, 30 cycles; 72°C 7 min). The two DNA fragments providing the flanking regions for homologous recombination, LHRR (containing psaB, rps14 and trnfM, 1,940 bp) and RHRR (containing psbZ and trnG, 723 bp), were amplified from tobacco genomic DNA by PCR using primer pairs LHRR-F/LHRR-R and RHRR-F/RHRR-R, respectively. The primer sequences are listed in Table 1. Two additional DNA fragments were generated by PCR amplification with primer pairs aadA-F/aadA-R and gfp-F/gfp-R using aadA and gfp expression cassettes as templates that had been chemically synthesized (GeneCreate, China). The selectable marker gene aadA is driven by the Chlamydomonas reinhardtii psbA promoter (CrPpsbA), and followed by the 3′UTR from the C. reinhardtii rbcL gene (CrTrbcL). The cassette (1,647 bp) is flanked by two loxP sites to facilitate marker gene excision by Cre-mediated site-specific recombination (Corneille et al., 2001 (link); Zhou et al., 2008 (link)). The reporter gene gfp is controlled by the tobacco plastid rRNA operon promoter combined with the 5′UTR from gene10 of bacteriophage T7 (NtPrrn:T7g10) (Kuroda and Maliga, 2001 (link)), and the 3′UTR from the E. coli ribosomal RNA operon rrnB (TrrnB). NcoI and XbaI restriction sites were introduced at the 5′ and 3′ end, respectively, of the gfp gene (1,282 bp). The fifth DNA fragment (SK, 2,916 bp) was the vector backbone fragment from pBluescript II SK (+) without the multiple cloning site (MCS) amplified using primers pBS-F/pBS-R (Table 1). The five DNA fragments comprising the four DNA insert pieces (LHRR, 133 ng; RHRR, 50 ng; aadA expression cassette, 113 ng; gfp expression cassette, 88 ng) and the linear vector backbone fragment (100 ng) were mixed in a stoichiometric ratio of 2:2:2:2:1, and then co-transformed into E. coli (XL10-Gold, Agilent technologies) chemically competent cells (>2 × 10⁸ cfu/μg assayed on pUC19). Positive clones were identified by selection for both ampicillin and spectinomycin resistance and further confirmed by their green fluorescence and by DNA sequencing.