| The Cre/loxP site-specific recombination system, derived from bacteriophage P1, has been widely used for the DNA recombination research on prokaryote and eukaryote both in vitro and in vivo. The high specificity and efficiency of the system make it play an important role in many regions, such as cloning tools, gene trapping, chromosome engineering, removal of specific genes or targeted integration. In order to develop the use of Cre/loxP system, a visible in vivo assay to evaluate the recombination efficiency was established. We also designed a new transformation vector, which can directly monitor the removal of the selection marker gene from transgenic plants. Furthermore, a novel strategy was tested, in which the Cre recombinase was transiently expressed from a modified virus vector, to remove the selection marker effectively.In the first part of our work, a simple and visible assay is designed, in which two incompatible plasmids, separately carrying the Cre gene and loxP-flanked GFP gene, were co-transferred into E.coli. The cre gene was inserted into a kanamycin-resistant bacterial expression vector, designated pET30a-Cre. The GFP gene (gfp), flanked by directly repeated loxP sites, was cloned into an ampicillin-resistant expression vector to generate pET23b-loxGFP. E.coli BL21(DE3) was cotransformed with pET30a-Cre and pET23b-loxGFP, and cultured in simultaneous presence of ampicillin and kanamycin. Under UV illumination, the recombination events can be detected easily. The fidelity of recombination was verified by SDS-PAGE analysis and restriction analysis followed by DNA sequencing.In the second part of our work, The GFP gene was also used to trace the fate of the selectable marker gene in transgenic plants. Green fluorescent protein can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in monitoring whether the selectable marker gene was removed from transgenic plants or not. For that purpose, the pGNG binary vector was constructed which contains one pair of directly orientated loxP sites. The GFP gene (gfp) linked to the expression cassette NosP-nptII-NosT was cloned between the two loxP sites. The plasmid also contains the CaMV 35S promoter which placed before the first loxP site and used to drive GFP expression. The β-glucuronidase gene(gus) of Escherichia coli, without promoter, was cloned behind the second loxP site, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of loxP-flanked genes, herein gfp and nptII, from the plant genome and brought the gus gene to be adjacent to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation. At last, a modified TMV-based 30B vector, which can transiently expressed Cre rocombinase, was tested to remove the selectable marker gene from transgenic plants. |
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