Abstract: The environmental risk caused by gene flow from herbicide-tolerant rice to normal rice is focused by the public and has become a key factor affecting the use of herbicide-tolerant rice in rice production. Gene splitting technique can effectively resolve the problem of the environmental risk and be applied on the development of herbicide-tolerant rice. In this study, the herbicide-tolerant gene G2-aroA was separated into N-terminal (EPSPSn) and C-terminal (EPSPSc) and were fused with Ssp DnaE intein, N-terminal (Intein-N) and C-terminal (Intein-C), to form fusion genes EPSPSn-In and Ic-EPSPSc, respectively. EPSPSn-In and Ic-EPSPSc were separately transferred into Zhonghua11 by Agrobacterium-mediated method. Southern hybridization demonstrated that the transgenic lines En-12 and Ec-22 were integrated with a single copy of insertion. The flanking sequence analysis demonstrated that the foreign genes in the transgenic lines En-12 and Ec-22 were respectively inserted into chromosomes 2 and 6. The homozygous lines of En-12 and Ec-22 were screened using the optimized four-primer method, and the hybrid En×Ec containing both EPSPSn-In and Ic-EPSPSc was obtained through sexual hybridization. Glyphosate resistance analysis revealed that transgenic rice containing only one gene fragment did not possess glyphosate tolerance, while transgenic rice En×Ec containing both gene fragments showed glyphosate tolerance. The glyphosate tolerance of the hybrid En×Ec was slightly weaker than that of the transgenic lines containing whole G2-aroA, but could meet the requirement in rice production. The results of this study provide a scientific basis for the use of gene splitting technology to develop safe transgenic glyphosate-tolerant rice, and also a new technology platform for genetically engineering safe transgenic hybrid rice.

Key words: glyphosate-tolerance')">glyphosate-tolerance, transgenic rice, gene splitting