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

Turkish Journal of Biology

SHIVA HAMZEH¹ 

关键词: Cre/loxP;    site-specific recombination system;    seed-specifically regulated autoexcision system;    marker-free transgenic plants;    BcNA1 promoter;   

学科分类：生物科学（综合）

来源: Scientific and Technical Research Council of Turkey - TUBITAK

【 摘 要 】

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 (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 of marker gene-free transgenic plants.

【 授权许可】

Unknown