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

Authors : SHIVA HAMZEH, MOSTAFA MOTALLEBI, MOHAMMAD REZA ZAMANI

Pages : 1-11

View : 11 | Download : 7

Publication Date : 2016-12-01

Article Type : Research Paper

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 pr??omoterless β-glucuronidase insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(100%); demonstrates the effectiveness of this autoexcision system for the production of marker gene-free transgenic plants.
Keywords : Cre loxP, site specific recombination system, seed specifically regulated autoexcision system, marker free transgenic plants, BcNA1 promoter