Intein-mediated Splitting Of Cre Recombinase And Its Deletion Activity Analyse In Arabidopsis Thaliana

In the decades, plant transgenic technologies have widely applied worldwide. However, this technology exists some concerns about biosafety. The biosafety problem of genetically modified (GM) crops has been the focus of international attention. Therefore, the biosafety of transgenic technology has become a serious obstacle, resulting in restricting the application of transgenic crops. To address this problem, increasing biological technologies have been widely studied and applied to control the biosafety problems of transgenic plants may exist. Among these technologies,"Gene-deletor", which can efficiently and thoroughly remove foreign genes in transgenic plants, provides an alternative approach to solve biological safety problems of GM crops. However, the current version of Gene-deletor system can not be used in the breeding of hybrid crops due to the thorough deletion of all transgenes from GM seeds.In order to obtain an efficient "Gene-deletor" system that can be used in hybrid crops, in this study, we split the Cre recombinase into N-terminal (NCre) and C-terminal (CCre), both NCre and CCre have no recombinase activity. Then we construct a new Cre/loxp system. In this modified "Gene-deletor", NCre and CCre peptide fragments were introduced into parents for hybridization, through artificial hybridization technique, hybrid progeny obtain the two dependent parts. Under the reassembling efficiency of the protein via intein-mediated protein-splicing, NCre and CCre can recover reconstruction activity. The main results obtained are as follows:1. The select of split site. Through the analysis of the protein structure of recombinase Cre, combining previous research works, we select five splicing sites:384bp,629bp,740bp,866bp,1013bp, that mean recombinase Cre will be splited at the amino acid sequence128th,153th,190th,232th,281th. Among these five split sites, the128th amino acid is located in the functional domain of recombinase Cre, the others four amino acid sites, however, are located in the connection section between the functional domain.2. The comparison of deletor efficiency of different split site. The split Cre fragments (NCre, CCre)and intein fragments (In> Ic) were obtained by PCR. Then we connected these fragments in to the plant expression vector pCA-Loxp, which contains a pair of direction loxp site. Finally, we construct five split vector, named pCA-CRE384, pCA-CRE629, pCA-CRE740, pCA-CRE866, pCA-CRE1013, respectively. The five split vectors were transformed into Agrobacterium rhizogenes C58C1. Split Cre fragments were introduced into tobacco via leaf disc transformation for analyze the reconstruction activity of Cre. Through GUS staining of the transgenic hairy roots, we account the GUS positive ratio to compare the deletion efficiency of each splicing vector. The results show that when splited at the384bp site, there was no GUS activity detected, for all transgenic hairy roots displayed white. The GUS positive ratio of the transgenic hairy roots of the other four sites Cre629, Cre740, Cre866, Cre1013were20%,18.5%,31.3%,30.8%, respectively.3. The deletion activity analysis of split system in Arabidopsis thaliana. Accordin-g to the GUS positive ratio analyzed from transgenic tobacco hairy roots, we choosed the Cre866as the best splicing site. Through Agrobacterium tumefaciens EHA105, we transformed the vector pCA-CRE866into A. thaliana. From T2generation of screening, we obtained six lines of transgenic A. thaliana that are single copy. By analyzed the GUS staining, we found that different single copy lines also have different degree of GUS staining results. We then respectively extracted the GUS protein of these six transgenic lines, and quantitative determined the GUS protein activity by fluorescence spectrophotometer. The results showed that, when the line had the more degree of GUS staining, it would also had the higher the activity of the GUS protein.4. The deletion activity analysis of split system by the hybrid method. We introduced the plant expression vector pCA-NCre-In and pCA-Ic-CCre into Arabidopsis. After T3generation of screening, we obtained four homozygous lines of transgenic Arabidopsis that contained NCre-In, four homozygous lines of transgenic Arabidopsis that contained Ic-CCre. We used them as parents to artificial hybridization.At last; we got the hybrid progeny Arabidopsis. By GUS staining analyzed, we found that the parents only contained the single NCre or CCre can’t be dyed to blue, while hybrid progeny Arabidopsis were all dyed to different levels of blue, this declared that under the reassembling efficiency of the protein via intein-mediated protein-splicing, NCre and CCre recovered reconstruction activity.