| Plant transgenic technology was widely used in crops.It can increase the yields of crops,improve the quality of agricultural products and strengthen the resistance of crops to adversity.However,it also brought about a series of biosafety issues,due to the proliferation of foreign genes to environment.These biological safety issues have attracted much attention and concern,therefore,more and more related biotechnology was adopted to solve these problems.Because it's simple and efficient,the site-specific recombinase systems,especially Cre/loxP system,have been widely used to delete exogenous genes in genetically modified plants and animals.However,the system itself has obvious faults that it can't be applied to hybrid crops.According to previous studies,the intact protein can be split into two complementary parts,when co-expressed,split pieces were possible to rebuilt the structure and restore the activity of the protein before.Here,we split the Cre recombinase into two complementary pieces,the N-terminal and the C-terminal at the first 866 bp site.We have successfully constructed two plant expression vectors,and carried artificial hybridization.If the flower-specific promoter was employed to control the expression of the split fragments,after hybridization,the exogenous functional genes will still be retained in vegetative growth stage,and in the reproductive growth stage,recombinant enzyme will remove the exogenous genes,making the seeds and fruits free from foreign genes.So genetically modified crops producing non-GM products will come true factually.This system promoted the application of split-Cre system in the agricultural field.The main results are as follows:1.Analysis of the recombination efficiency of split-Cre system mediated by intein.The Cre gene was split into N-terminal(0-866 bp)and C-terminal(867-1202 bp)at the first 866 bp site.Based on the pCA-loxP vector,we constructed two plant expression vectors.One vector was constructed by intein-mediated protein split technique,named pNCre-CCre;the other was by splitting Cre protein directly,named pN-C.The two vectors were then transformed into Agrobacterium rhizogenes C58C1 and infected wild type tobacco.We found that the GUS staining positive rate of vector pNCre-CCre(31.3%)was significantly higher than that of pN-C vector.It declared that intein-mediated protein split technique could greatly improve the recombination efficiency of split-Cre fragments.2.Analysis of the recombination efficiency of split-Cre protein in progenies of Arabidopsis thaliana after hybridization.The N-terminal of split-Cre protein was fused in frame with the N-terminal of intein,and the C-terminal of intein was fused with the C-terminal of split-Cre protein.Based on the vector pCA-loxP,we constructed two plant expression vectors,named pCA-NCre-In and pCA-Ic-CCre,then transformed them into wild-type Arabidopsis thaliana.We did artificial hybridization using homozygous Arabidopsis.By GUS staining and detection of GUS enzymatic activity,we found that the split-Cre protein system could efficiently remove the foreign genes in progenies,efficiency up to 96.736%.3.Establishment of a modified gene deletion system.In order to improve the Cre/loxP system,we put all the exogenous genes into two loxP sites with the same direction,that is to say,the left border and right border of T-DNA were flanking the two loxP site.So,when Cre recombinase recognized the two loxP sites,it would delete all of the exogenous genes.After transformation into wild-type Arabidopsis thaliana,we did artificial hybridization using homozygous plants.Through detection of GUS and GFP reporter gene,as well as amplification of target genes in hybrid progenies,we found that,in most cases,the deletion was partial,not complete,namely,some cells or tissues generated entirely remove,but,not every plant cell has undergone deletion.However,complete deletion had happened separately in N-terminal or C-terminal.Therefore,this study will improve the application of recombinase system Cre/loxP in hybrid crops. |
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