Construction Of A Novel Cre/Loxp System Based On Splitting Of Cre Recombinase And Its Deletion Activity In Transgenic Tobacco

After thirty years of development, plant transgenic technology has been widely used in modern agricultural production and practice since its birth in the1980s. This technology plays a very important role in improving crop yields and quality and reducing the use of herbicides, pesticides and other chemicals.Transgenic technology brings us much economic benefits. At the same time, in the global energy crisis increasingly serious circumstances, the research and application of transgenic technology in biological energy have become increasingly prominent. Genetically modified (GM) products are closely related to our life. However, the biosafety concerns brought by transgenic technology have not been eliminated. People always debate on the topic "Whether genetically modified product is safe". This severely restricts the further popularization and application of transgenic technology. Therefore, how to let people benefits a lot and do not worry about the dangers of transgenic technology, the problem seemed to be solved as soon as possible.To address the concerns of biosafety,’Gene deletor’system as a effective technology has been developed in a previous study. This system can remove foreign genes accurately and efficiently, but it can not be used in plant sexual reproduction and hybrid crops. This greatly limits the popularization and application of the system. In order to overcome the above shortcomings, we split the Cre recombinase into N-terminal (NCre) and the C-terminal (CCre) and rebuilt a new Cre/loxp system.Through vitro and vivo experiments, we proved that the split Cre/loxp system had a high reconstruction activity. It is very important for further modification of’Gene deletor’. The specific results are as follows:1. Prokaryotic expression and activity detection of split protein in vitro The prokaryotic expression vectors were constructed.They contained the N terminal (NCre) and the C terminal (CCre) of the split Cre recombinase,and the whole-length Cre recombinase. Results of activity detection showed that the the N terminal (NCre) had no activity, while the C terminal had the activity. At the same time, when the split protein N terminal (NCre) and C terminal (CCre) were together (NCre+CCre), it regained activity.2. Expression vectors construction The split Cre fragments NCre and CCre and the full length Cre were amplified and cloned into the mediate vector pCXSN. And then the correct fragments were connected into the vector pCA-LoxP, which contains two direction site LoxP. Screened the all of the recombinant plasmid, and then both of the plant expression vectors were transformed into Agrobacterium rhizogenes C58C1.3. Recombination effect analysis of Cre protein in vivoThe possive transgenic hairy roots were detected by PCR. GUS staining of possive transgenic hairy roots further showed that the single split Cre (NCre or CCre) had no restructuring activity because the GUS staining of these ransgenic hairy roots displayed white. While when both NCre and CCre were in the same cell, the restructuring activity would be recovered because the GUS staining of these ransgenic hairy roots displayed blue. This result was same as the whole Cre recombinase. At last, sequencing results further proved that the split Cre—NCre and CCre could regain activity when they were both in the cell and delete all foreign genes between two LoxP recognition sites.4. Analysis of GUS positive ratio statisticsTransgenic hairy roots were analyzed through GUS staining for the deletion efficiency of each vector. GUS-positive hairy roots were accounted to calculate the deletion efficiency and it was repeated three times. It was showed that the deletion efficiency in the transgenic hair roots containing the whole Cre recombinase was about to49%, while the Cre recombinase plus the nuclear localization signal (NLS) had a higher efficiency (60%). We detected no recombination activity in the split Cre N-terminal (NCre) and C-terminal (CCre). While these split fragments including the N-and C-terminal domains were constructed into a plant binary vector and transformed into tobacco to gain transgenic hairy roots, we found that the recombination activity of Cre recombinase was recovered and deletion efficiency was up to46%and67%.