MARKER RESCUE FROM THE NICOTIANA-TABACUM PLASTID GENOME USING A PLASTID ESCHERICHIA-COLI SHUTTLE VECTOR

We recently reported an 868-bp plastid DNA minicircle, NICE1, that for med during transformation in a transplastomic NicoTiana tabacum line. Shuttle plasmids containing NICE1 sequences were maintained extrachrom osomally in plastids and shown to undergo recombination with NICE1 seq uences on the plastid genome. To prove the general utility of the shut tle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the Co1 E1 ori for maintenance in E. coli and a spectinomcyin resistance gene (andA) for selection in both systems. In addition, pNICER1 carries a d efective kanamycin resistance gene, kan, to target the rescue of a fu nctional kanamycin resistance gene, kan, from the recipient plastid ge nome. pNICER1 was introduced into plastids where recombination could o ccur between the homologous kan/kan sequences, and subsequently rescu ed in E. coli to recover the products of recombination. Based on the e xpression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recove red at a high frequency. Efficient rescue of kan from the plastid geno me in E. coli indicates that NICE1-based plasmids are suitable for res cuing mutations from any part of the plastid genome, expanding the rep ertoire of genetic tools available for plastid biology.