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Application of Arabidopsis tissue-specific CRUC promoter in the Cre/loxP self-excision strategy for generation of marker-free oilseed rape: potential advantages and drawbacks

Boszorádová, Eva, Libantová, Jana, Matušíková, Ildikó, Moravčíková, Jana
Acta physiologiae plantarum 2014 v.36 no.6 pp. 1399-1409
Arabidopsis, Brassica napus var. napus, Nicotiana tabacum, cultivars, excision, reporter genes, tobacco, transfer DNA
The aim of the present study was to explore the usability of Arabidopsis cruciferin C (CRUC) promoter in the Cre/loxP self-excision strategy with the minimal rate of an ectopic expression of the cre recombinase. For this, a plant transformation vector containing the gus reporter gene driven by the light-sensitive Lhca3.St1 instead of double dCaMV 35S promoter and one pair loxP sites flanking the cre and the nptII genes was prepared. Agrobacterium-mediated transformations of three economically important oilseed rape (Brassica napus L.) cultivars Campino, Hunter and Topas as well as tobacco (Nicotiana tabacum L.) as a reference system were performed. The integration of the T-DNA into genome of all Brassica cultivars was accompanied by DNA deletions/rearrangements on the both T-DNA ends. The disruption of the Cre/loxP recombination system in oilseed rape was observed. On the contrary, no such events were detected in tobacco. The applied strategy did restrict an ectopic CRUC activity to some extent and a set of transgenic tobacco T₀ plants without premature excision event was obtained. Our data showed that a choice of the promoter can limit undesired activation of the Cre/loxP cassette.