Shcherbak N. Investigation of lox-dependent expression of transferred genes in transgenic plants

The thesis is devoted to the investigation of lox-mediated expression in transgenic plants provided by the vectors containing sequence of lox site from the Cre/lox recombination system of bacteriophage P1 instead of genes promoter. Wild and mutated types of lox sites were tested for their effect upon bar and gus genes expression in transgenic plants of Nicotiana africana, Nicotiana tabacum and Lactuca sativa. An effective and reproducible method for Agrobacterium-mediated genetic transformation of N. africana has been developed using roots of aseptic plants as explants. Transgenic N. africana plants transformed with vectors carrying lox site between the right border of T-DNA and the promoterless bar gene (RB-lox-bar) unexpectedly were found to be resistant to phosphinothricin (PPT). Such unpredictable result led us to carry out an investigation of lox-mediated expression by plant genetic transformation with the set of vectors based on lox site disposition adjacent to promoterless bar and gus gene sequences. Most of plasmid vectors held nptII gene under control of nos promoter as well. In the experiments with the vectors containing nptII gene transgenic plants were selected according to their growth capacity on the medium with kanamycin and then were tested on the selective medium containing PPT. The presence of bar gene in selected tobacco plants was confirmed by PCR analysis. Herbicide resistance/sensitivity studies of transformants harboring RB-lox-bar sequence reproducibly resulted in approximately 80% of PPT-resistant transgenic plants. Neither the localization of nos terminator between lox site and the RB nor the replacement of the mutated loxA site with a wild tipe loxP site had an effect on bar gene expression. The frequency of PPT-resistant transgenic plants of Nicotiana africana and Nicotiana tabacum obtained using vectors with general constitution RB-lox-bar did not differ from each other at >95% confides using Students T-test. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. In contrast with previous data, disposition -lox-bar- internally in the T-DNA thoroughly changed the result of PPT tests. Transformation experiments with such vectors resulted without obtaining any PPT-resistant transgenic plants. Another strategy of experiments with direct selection on the regeneration medium with PPT immediately after co-cultivation also was used for these constructs. The invariable result was shown in the numerous transformation experiments: no PPT-resistant transgenic plants were obtained after co-cultivation with the Agrobacterium strain containing the appropriate vector. Experiments with vector containing one lox site placed near left border (LB) upstream of bar gene demonstrated possibilities of lox-depended expression also. Based on these data we suggest that lox-mediated expression occurred only when lox site was associated with T-DNA borders. According to the results of biolistic transformation experiments only 3% of obtained kanamycin resistant plants were tolerant to PPT. The result of direct transformation led us to conclude that lox-mediated expression of bar gene depended on the method of DNA delivery and occurred mainly in plants obtained via Agrobacterium-mediated transformation. Expression of bar gene from the vectors containing lox site near RB allowed recovery of PPT-resistant transgenic plants of such important crop as Lactuca sativa. The results of herbicide Basta application to transgenic plants in greenhouse have entirely agreed with in vitro test results for all plant species mentioned in this report. Self-fertilization seeds (R1) from the primary transformants were harvested and germinated on the medium containing 5mg/l PPT. The inheritance of PPT-resistant phenotype for all transgenic lettuce seedlings and several transgenic tobacco lines as the 3:1 segregation of the selectable marker gene in progeny of tobacco transgenic plants was observed. We have analyzed reporter gus gene lox-mediated expression from the vectors that were similarly designed: promoterless gus gene together with the lox site adjacent to the RB (RB-lox-gus). Constructed vectors were shown to provide ?-glucuronidase activity in transient expression assays using an Agrobacterium-based leaf infiltration of Nicotiana benthamiana. Proposed lox-depended expression system was also tested for gus gene activity in transgenic tobacco plants. These experiments in addition showed the effect of independent 35S promoter sequence on lox-mediated expression of gus gene. In the absence of 35S promoter sequence gus gene was active in 1.5% transgenic plants and it's expression level was low. With 35S promoter sequence in the same T-DNA, the frequency of transgenic plants exhibiting ?-glucuronidase activity has increased to 10% and this vector could drive three fold higher level of gus gene expression.