SITE-SPECIFIC INTEGRATION OF AGROBACTERIUM T-DNA IN ARABIDOPSIS-THALIANA MEDIATED BY CRE RECOMBINASE

In this study Agrobacterium tumefaciens transferred DNA (T-DNA) was ta rgeted to a chromosomally introduced lox site in Arabidopsis thaliana by employing the Cre recombinase system, To this end, Arabidopsis targ et lines were constructed which harboured an active chimeric promoter- lox-cre gene stably integrated in the plant genome. A T-DNA vector wit h a promoterless lox-neomycin phosphotransferase (nptII) fusion was ta rgeted to this genomic lox site with an efficiency of 1.2-2.3% of the number of random events. Cre-catalyzed site-specific recombination res ulted in restoration of nptII expression by translational fusion of th e lox-nptII sequence in the integration vector with the transcription and translation initiation sequences present at the target site, allow ing selective enrichment on medium containing kanamycin, Simultaneousl y, the coding sequence of the Cre recombinase was disconnected from th ese same transcription and translation initiation signals by displacem ent, aimed at preventing the efficient reversible excision reaction, O f the site-specific recombinants, 89% were the result of precise integ ration. Furthermore, similar to 50% of these integrants were single co py transformants, based on PCR analysis. Agrobacterium T-DNA, which is transferred to plant cells as a single-stranded linear DNA structure, is in principle incompatible with Cre-mediated integration. Neverthel ess, the results presented here clearly demonstrate the feasibility of the Agrobacterium-mediated transformation system, which is generally used for transformation of plants, to obtain site-specific integration .