利用基因拆分技术培育耐草甘膦转基因水稻的研究

耐除草剂转基因水稻基因飘流可能产生的环境安全问题是人们关注的焦点之一,并已成为耐除草剂转基因水稻能否在我国生产上发挥效益的限制因素。基因拆分技术能够有效地控制转基因目标性状飘流,为培育耐除草剂转基因水稻提供新的途径和思路。本研究将耐除草剂基因G2-aroA拆分成N端(EPSPSn,1～295aa)和C端(EPSPSc,296～435aa),分别与SspDnaE蛋白内含肽的N端(Intein-N)和C端(Intein-C)连接形成融合基因EPSPSn-In与Ic-EPSPSc,并分别通过农杆菌介导法转入受体材料中花11。Southern杂交证明转基因水稻En-12和Ec-22中外源基因为单拷贝插入。侧翼序列分析证明转基因水稻En-12和Ec-22中外源基因分别插入第2和第6染色体。利用四引物法筛选出转基因水稻En-12和Ec-22的纯合系,并通过有性杂交获得同时含有EPSPSn-In与Ic-EPSPSc的转基因水稻En×Ec。草甘膦抗性分析发现,单独含有1个基因片段的转基因水稻En-12和Ec-22不具有耐受草甘膦特性,而同时含有2个基因片段的转基因水稻En×Ec具有耐受草甘膦的特性,说明拆分后的2个蛋白片段在intein的介导下重新组装成完整有功能蛋白,并赋予转基因水稻耐受草甘膦的特性。转基因水稻En×Ec与含有完整G2-aroA转基因水稻G2-6相比,其耐受草甘膦的能力有所下降,但能够满足生产需求。本研究结果为利用基因拆分技术培育转基因耐草甘膦水稻提供了科学依据,同时也为利用基因工程手段培育转基因杂交稻提供了新的技术平台。

Cultivation of herbicide tolerant transgenic rice by gene spliting technique

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.