Effect Of Several NLSs On Deletion Efficiency Of Site-specific Recombinase

To date,more than 80 million hectares of transgenic crops have been grown worldwide. The potentially negative environmental impact of transgenic plants has been of concern to both the public and the scientific communities.However,none of the existing technologies to address this problem is broadly applicable for field crops.Although site-specific recombination systems,such as Cre/loxP of the phage P1,R/RS of Zygosaccharomyces rouxii and FLP/FRT of Saccharmyces cerevisiae,have been used for removing short DNA sequences such as antibiotic marker genes or spacer sequences from plant genome,their excision efficiencies are generally low in higher plants.In our previous study,we report a novel "GM-gene-deletor" system that is exceptionally efficient for excising all foreign genes from pollen and seeds of GM plants.To improve the deletion efficiency of "GM-gene-deletor" system,we investigated effects of several nuclear localization signal(NLS) sequences,which have been demonstrated to function in higher plants,on deletion efficiency of site-specific recombinase.Firstly,four NLSs were artificially synthesized and fused to GUS::GFP gene and introduced into tobacco plants via Agrobacterium-mediated method.The NLS with the highest efficiency of nuclear transfer was obtained and combined with site-specific recombinase Cre. After transformation,we further determined the effects of the NLS sequence on improvement of the nuclear transfer Cre recombinase and on elimination efficiency of "GM-gene-deletor" system. 1.Comparison of mediated-nuclear import efficiency of NLS sequencesFour kinds of classical nuclear localization signals(NLS),named as SV40NLS,O₂NLS,NLSC and rpl25NLS,were artificially synthesized and fused with GFP::GUS reporter gene.Confocal study revealed that green fluorescence protein(GFP) was localized in the nucleus of epithelial cells of transgenic tobacco plants containing pBI-SV40NLS-GFP.GUS,while GFP was detected throughout the whole cell in these transgenic plants containing other NLSs.As a negative control,no GFP signal was observed in wild-type plants.Quantitative assays of GUS activity in the nuclei from individual lines of transgenic tobacco plants harboring GFP:GUS,SV40NLS-GFP:GUS,O₂NLS-GFP:GUS,NLSC-GFP:GUS and rpl25NLS-GFP:GUS,respectively,showed that the highest GUS activity(90-380 nmol/mg/min) was detected in transgenic SV40NLS-GFP:GUS events,which level was 2-to 4-fold higher than that of transgenic plants transformed with GFP:GUS,rp125NLS-GFP:GUS,O₂NLS-GFP:GUS, NLSC-GFP:GUS.These results indicated that SV40 NLS was the most effective to mediate nuclear import in tobacco plants.2.Effect of NLS sequence on deletion efficiency of site-specific recombinaseTo investigate effect of SV40NLS on deletion efficiency of Cre recombinase,two expression vectors,pBI-Cre and pBI-SV40NLS-Cre in which SV40NLS was fused to the N-terminus of Cre gene, were introduced into transgenic tobacco containming vector pLF-GN with loxPFRT-35S-GUS:NPTII-loxPFRT fragment,respectively,via Agrobacterium rhizogenes-mediated transformation.Deletion efficiencies of the transgenes in transgenic tobacco plants were examined by GUS histochemical staining of the transgenic hairy roots.The results revealed that addition of an N-terminal NLS did not increase deletion efficiency of Cre recombinase in transgenic tobacco,but promoted the localization of the Cre protein into the nucleus,resulting in a faster recombination reaction.