Study Of Gene Split Technology On Controlling Transgene Flow

With the rapid development and commercialization of GMC （Genetically Modified Crop）, thepotential environmental problem caused by transgene flow from GMC to related plants has become oneof the focuses of public concern. The best strategy for promoting public acceptance of transgenicproducts is to eliminate transgene flow fundamentally. Existing physical technologies such as distanceisolation, florescence isolation or physical barrier can only reduce the frequency of transgene flow to acertain extent. So, it is important to develop new biological strategy with more satisfied efficiency oncontrolling transgene flow. In this study, the G2-aroA gene, conferring resistance to herbicideglyphosate, was used to study the transgene split technology and the reassembling efficiency of theprotein via intein-mediated protein-splicing and to explore the effect of gene split technology oncontrolling transgene flow in tobacco and rice, in the hope of establishing a technology platform tocontrol transgene flow.The main results obtained are as follows:1. Gene split technology to controlling transgene flow on tobacco::1) Seeds of T₀generation of transgenic tobacco, which harboring the integrated G2-aroA（EPSPS）,EPSPSn-In and IC-EPSPSC, respectively, were generated on kanamycin medium to analyzethe segregation ratio of KanR/KanS. It was indicated that transgenic plants N-33、C-11andG2-24were integrated with single copy of foreign gene according to the segregation ratio ofKanR/KanSand reconfirmed through southern blot and quantitative Real-time PCR study.2) Through self-polination and screening with kanamycin of transgenic tobacco plants,homozygous line of N-33and C-11were obtained. F1seeds of N-33×C-11were also obtainedby hybridization.3) The flanking sequence of inserted genes in transgenic tobacco plants N-33and C-11werecloned with Tail-PCR and it was preliminary indicated that integration of foreign genes ofEPSPSn-In and Ic-EPSPSc were occurred at different chromosomes. The KanR/KanSsegregation ratio of self-pollinated seeds from N-33×C-11was analyzed. Among3096seedlings tested,1880seedlings were KanRand the resistant proportion was60.7%.According to the basic rules of genetics, we further confirmed that the two fusion genesEPSPSn-In and Ic-EPSPSc were inserted into two non-homologous chromosomes.4) The glyphosate resistance of different types of transgenic tobacco was analyzed. It wasindicated that both N-33and C-11as well as the non-transgenic tobacco NC89weresusceptible to glyphosate, while100%seedlings from hybrid N-33×C-11harboring bothEPSPSn-In and Ic-EPSPSc showed the same resistance to glyphosate as the positive controlG2-24. RT-PCR analysis showed that each of the two gene fragments can be amplified fromhybrid N-33×C-11, but the complete gene can’t be found in hybrid. It was proved through Western blot and mass spectral analysis that the hybrid N-33×C-11plants contain thecomplete EPSPS protein. The above results showed that the two fragments of G2-aroA genecan be complemented and EPSPS activity can be recovered by intein mediated proteincomplementation, with a assembly efficiency up to100%in plant level, which can meet theneeds of practical application.5)The glyphosate resistance of the seedlings from back crossing generation of hybrid N-33×C-11and non-transgenic control NC89was analyzed. Among2812seedlings totally tested,664seedlings or23.61%of the total seedlings showed resistance to glyphosate, which fit to thehypothesis that the transgenic flow rate is25%when the two fragments were integrated intotwo non-homologous chromosomes. The results suggest that the transgene split technologycan at least reduce the gene flow frequence of75%.2. Constructing a gene split technology system for controlling transgene flow in rice1)Plant expression vectors13UG2,13UEI and13UIC, harboring the integrated G2-aroA（EPSPS）,EPSPSn-In and IC-EPSPSC, respectively, were constructed and transformed into rice byagrobacterium-mediated gene transfer and14,15,14transgenic rice plants were obtainedrespectively.2)Results from Southern-blot analysis indicated that transgenic rice plants En-1、En-3、En-12、En-19、En-31、En-33、En-34、Ec-2、Ec-5、Ec-8、G2-6、G2-7、G2-10were integrated withone copy of target gene.3)The flank sequence of transgenic rice were cloned by Tail-PCR, and chromosome localizationanalysis showed that the foreign genes in transgenic rice En-1, En-3, En-19, En-31, Ec-2,Ec-5and Ec-8were localized in chromosome2,11,1,7,4,3and9respectively.4)The quantity of foreign protein expressed in transgenic rice was indentified via Real-timequantitative PCR. The foreign protein expressing level from high to low is Ec-2, Ec-5andEc-8respectively for Ec line, while in the En line, the order is En-12, En-3, En-1, En-34,En-33and En-31.In summary, our study on tobacco has established a technological platform to control trangene flowby using gene split technology and laid the foundation for the same study in transgenic rice. In thepractical application in rice, the target gene can be split into two fragments. One fragment may beintroduced into the restore line, while another introduced into the maintainer line. There for, only thehybrids can produce complete and functional target protein, and the parents with only one fragment hasno function and will not impact on the environment. Thus it paving the way for using trangenictechnology on hybrid rice, and to maintain China’s leading position in hybrid rice research andproduction. It will has significant benefits for commercialization of transgenic rice in the future.