System site-specific recombination to manipulate the plastid genome of higher plants.

摘要

Procedure site-specific recombination for removal of nucleic acid sequences predetermined plastid genome, said method comprising: a) providing a first nucleic acid construct, said construct comprising a promoter that is operably linked to a nucleic acid encoding a transit sequence directed to plastid which is operably linked to a nucleic acid encoding a protein having excision activity, it said construct further comprising a nucleic acid encoding a first selectable marker having nucleic acid sequences regulatory 5 'and 3' 'specific plant; b) providing a second DNA construct, said second construct comprising a nucleic acid encoding a second selectable marker and cleavage sites, optionally containing said second construct a gene of interest, said second construct further comprising sequences flanking ácidonucleico targeting plastid facilitate homologous recombination into said plastid genome; c) introducing said second DNA construct into a plant cell; d) culturing said plant cell of step c) in the presence of a selection agent and selecting those plant cells expressing proteins encoded by said second DNA construct; e) introducing said first DNA construct into plant cells from step d) in the presence of a selection agent and selecting those plant cells expressing encoded by said first construct, which when present, proteins such cleavage activity acting at those sites cleaving, cleaving thus said predetermined target sequence.