Gene Split Approach For Controlling Transgene Flow

The possible impacts of transgene flow on environment and human health is one of the safety issues of GMC (gene modified crops) concerned by the scientific community and public. One of the best strategy for promoting public acceptance of transgenic products is to protect the environment and food safety through eliminating transgene flow fundamentally. Exploring new technology of biological containment to prevent form transgene flow, has become a hot topic internationally. It is not only a new generation of technology in basic research, but also is of great significance for further promoting and facilitating the commercializaton of transgenic crops. In this study, the epsps gene, conferring resistance to herbicide glyphosate, was used to study the gene split approach and the reassembling efficiency of the protein via intein-mediated protein-splicing, thus to confirm the effect of biological containment through gene split methodology. The main results obtained are as follows:1) Plant expression vectors pG₂, pEPSPSn-In and pIC-EPSPSC, harboring the integrated G₂-aroA(EPSPS), EPSPSn-In and IC-EPSPSC, respectively, were constructed and used for transformation of tobacco. Transgenic tobacco plants containing with G₂-aroA (intact EPSPS gene), and EPSPSn-In or IC-EPSPSC gene fragment were obtained and confirmed by PCR analysis.2) The analyse of glyphosate resistance of transgenic tobacoo integrated G₂-aroA show that T₁ generation of transgenic tobacco seeds can germinate and grow on the medium with 676.3 ppm glyphosate, while the seeds of NC89 died on the medium with 33.8 ppm glyphosate; transgenic tobaccos plants can tolerance 4000 ppm glyphosate in the field, while non-transgenic tobaccos died on 1500 ppm.3) Transgenic tobacco plants integrated with EPSPSn-In or IC-EPSPSC fragment were cross-pollinated and hybrid seeds integrated with the two fragments were obtained. Glyphosate-resistance of different type of transgenic tobacoo was investigated to confirm the reassembling efficiency after the gene split. Hybrid seeds integrated with the two fragments could germinate and grow on the medium with 84.5 ppm glyphosate, while seeds integrated with one of the each fragment died on the medium containing 84.5 ppm glyphosate. RT-PCR test show that glyphosate resistance of hybrid is due to protein reassembling of two fragments by intein-mediated protein-splicing.4) It was estimated that the reassembling efficiency of the split fragments could be 100 % via selecting their offspring, considering the segregation ratio of different type of transgenic tobacoo on the medium with 100 mg/L Kan.