Creation and characterization of TMT transplastomic lines by Southern, western blot analysis and ELISA. (a) The native chloroplast genome showing both homologous recombination sites (trnI and trnA) and the restriction enzyme sites used for Southern blot analysis. (b) The pLD-G10-γ-TMT chloroplast vector map (c) Southern blot hybridized with the flanking sequence probe showing homoplasmy. Lanes 1 to 5: T0 transplastomic lines; WT, wild-type. (d) Western blot analysis of wild-type (WT), transplastomic TMT and his-tag standard protein. Protein extracts were resolved by SDS-PAGE and transferred to nitrocellulose membrane. WT, wild-type tobacco ; SD: his-tag protein standard (~30kD GenScript Inc); lanes 1–4, his-TMT transplastomic lines- F2.3, C.1, B 3.3 and E.1. (e) ELISA quantification of his-TMT in five independent transplastomic lines-F2.3, C.1, B3.3, E.1 and H3.2.