The application of gene splitting technique for controlling transgene flow in rice

Controlling transgene flow in China is important, as this country is part of the center of origin of rice. A gene-splitting technique based on intein-mediated trans-splicing represents a new strategy for controlling transgene flow via biological measures. In this study, the G2-aroA gene which provides glyphosate tolerance was split into an N-terminal and a C-terminal region, which were then fused to intein N and intein C of the Ssp DnaE intein, ultimately forming EPSPSn:In and Ic:EPSPSc fusion genes, respectively. These fusion genes were subsequently transformed into the rice cultivar Zhonghua 11 via the Agrobacterium-mediated method. The two split gene fragments were then introduced into the same rice genome by genetic crossings. Glyphosate tolerance analysis revealed that the functional target protein was reconstituted by Ssp DnaE intein-mediated trans-splicing and that the resultant hybrid rice was glyphosate tolerant. The reassembly efficiency of the split gene fragments ranged from 67 to 91% at the molecular level, and 100% of the hybrid F1 progeny were glyphosate tolerant. Transgene flow experiments showed that when the split gene fragments are inserted into homologous chromosomes, the gene-splitting technique can completely avoid the escape of the target trait to the environment. This report is the first on the reassembly efficiency and effectiveness of transgene flow containment via gene splitting in rice. This study provides not only a new biological strategy for controlling rice transgene flow but also a new method for cultivating hybrid transgenic rice.