Sekan A. Using the site-specific recombination system Cre/loxP for the one-step obtaining of marker-free Arabidopsis thaliana transformants.

Object of study: the efficiency of the site-specific system Cre/loxP in the genome of transformed lines A.thaliana within the vector DNA-constructions through three transgene generations. Purpose of the study: one-step obtaining of A.thaliana transformants, using a new approach with site-specific recombination system Cre/loxP, which aims to complete excision of marker genes with gene cre, without involving tissue-specific or inducible promoters.Developed and designed two types of DNA-constructions with site-specific recombination system Cre/loxP (Cre/loxP1 and Cre/loxP2) to determine the more successful design for the obtaining of A.thaliana transgenic plants, free from loxP-bounded T-DNA sequences. Genetically modified plant lines A. thaliana (generation T0) obtained in one-step after agrobacterium-transformation by floral dip method with using of the created DNA-constructions. Effective system of selection for transgenic plant lines with site-specific recombinase Cre/loxP in their genome developed and obtained two subsequent generations of transgenic lines (T1 and T2 generation). Histochemical and molecular-genetic analysis of each generation transformants is carried out with aim to identify excision events of the loxP-limited T-DNA sequences with marker genes. Identified marker-free transformants of first generation owing to using the developed aproach with the site-specific recombination system Cre/loxP : 10% of plants transformed by DNA-construction Cre/loxP1, and 12% - by Cre/loxP2. It is shown that the number of marker-free transformed plant lines with one or another DNA-construction increases on 5-7% for each further generation. It is determined that the efficiency of the developed approach with using of the site-specific recombination system Cre/loxP for obtaining the genetically modified marker-free A.thaliana plants, for vector construction Cre/loxP1 is 53% and for the Cre/loxP2 - 43 %. Statistical analysis of the efficiency of using both studied DNA-constructions is obtained and it is shown that the changing of gene location on vector cassettes rises to different genetic events and resulting to the obtaining of transgenic plants with different types of integrated T-DNA. Statistical classification model is developed with aim to highlight the critical stages of experimental studies on efficiency in using of the designed DNA-constructions, that allows to predict the effectiveness of use this DNA-constructions on other plant objects with the ability to predictability for further results on 60%