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Efficient seed-specifically regulated autoexcision of marker gene (nptII) with inducible expression of interest gene in transgenic Nicotiana tabacum

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In this study, we developed the seed-specifically regulated autoexcision system based on Cre/loxP site-specific recombination for coelimination of nptII and cre genes from transgenic plants. | Turkish Journal of Biology http://journals.tubitak.gov.tr/biology/ Research Article Turk J Biol (2016) 40: 1-11 © TÜBİTAK doi:10.3906/biy-1408-32 Efficient seed-specifically regulated autoexcision of marker gene (nptII) with inducible expression of interest gene in transgenic Nicotiana tabacum Shiva HAMZEH, Mostafa MOTALLEBI*, Mohammad Reza ZAMANI National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Received: 15.08.2014 Accepted/Published Online: 24.12.2014 Final Version: 05.01.2016 Abstract: In this study, we developed the seed-specifically regulated autoexcision system based on Cre/loxP site-specific recombination for coelimination of nptII and cre genes from transgenic plants. To accomplish this, a seed-specific gene promoter, BcNA1, from Brassica napus was used to drive conditional expression of recombinase. NptII and recombinase cassettes were placed between two directly repeated loxP sites. The loxP-flanked DNA was located between the SP-DDEE synthetic pathogen inducible promoter and promoterless β-glucuronidase (Gus) gene, as a model gene of interest. Upon seed-specific expression of cre recombinase, the excision event would eliminate loxP-flanked DNA and bring the gus reporter gene under the control of the inducible promoter. This Cre/loxP system was transformed into Nicotiana tabacum via Agrobacterium-mediated transformation. The regenerated plants were obtained by selection on kanamycin medium and PCR screening. Based on seed germination assay on kanamycin-containing medium, the transgenic lines were subdivided into homogeneous and heterogeneous categories. Phenotypic and molecular analysis of T1 progeny plants indicated that completely NptII-free plants were obtained in homogeneous transgenic lines. Coelimination of NptII and recombinase cassettes were verified with PCR, sequencing, and histochemical Gus staining assay. Full excision efficiency (100%) demonstrates the effectiveness of this autoexcision system for the production