Analysis of effectiveness of the Cre/loxp site-specific recombinase system through subsequent generations of Arabidopsis thaliana transformants
Journal Title: Вісник Українського товариства генетиків і селекціонерів - Year 2015, Vol 13, Issue 1
Abstract
Aim. The aim of the work was to analyze the effectiveness of the Cre/loxP site-specific recombinase system through subsequent generations of Arabidopsis thaliana transformants. Methods. The increase in the number of marker-free Arabidopsis thaliana transformants was studied through several subsequent generations. Genetic transformation of plants was carried out using the Cre/loxP site-specific recombinase system under the control of 35S promoter. For this purpose two types of DNA constructs were designed that contain the marker gene gus and the recombinase gene cre flanked by loxP sites. Hygromycin resistance gene hptII in both constructs was moved out of the loxP sites, and in the case of excision event it remained in plant genome. The selection of transformants was performed on Murashige and Scoog medium with hygromycine (100 mg/l). The recombinase-mediated excision event of marker genes in genome was determined by histochemical analysis and molecular genetic analysis of three subsequent generations of transformants. Results. The number of marker-free transformants was increased with each subsequent generation for both types of DNA-constructs. Moreover, the reduction in the total amount of integrated T-DNA per genome to a minimum was identified by molecular-genetic analysis that indicates the ability of the Cre/loxP site-specific recombinase system to control the number of the integration events in plant genome. Conclusions. The developed approach is simple in usage and effective tool for obtaining marker-free transformed plants.Keуwords: selective marker genes, removal of marker genes, site-specific recombinase.
Authors and Affiliations
A. S. Sekan
Keywords
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