抗草甘膦基因(EPSPS)植物双价表达载体的构建

针对目前报道的抗草甘膦转基因作物中,强组成型启动子驱动抗草甘膦基因在转基因植物的所有部位和所有发育阶段都表达,增加植株代谢负担,对植物的产量可能引起负效应的这一问题,利用Ag2这种草甘膦胁迫诱导启动子来驱动epsps基因,只在草甘膦喷施后高效表达对草甘膦的抗性.另外,同时利用具有组织特异性的启动子RuBP,使epsps基因在植物草甘膦农药田间喷施主要部位叶片中高效表达,应可以进一步增强转基因植物的抗草甘膦能力,但又不致过多地增加转基因植物的代谢负担,以利培育高抗草甘膦且农艺性状优良的转基因植物.实验分别以pBI121-E-M-Bt(Kanar)和pBI121-CP4E(Kanar)质粒为基础,通过载体pUC19及对EcoRⅠ位点的接头改造,构建了由RuBP启动子和新发现的诱导型启动子Ag2共同调控epsps基因的植物表达载体pGBI-Ag2EM-RuBPEM和pGBI-Ag2CP4E-RuBPCP4E,选择标记为卡那霉素,通过冻融法将重组质粒导入根癌农杆菌LBA4404,并通过农杆菌介导的浸花法转化拟南芥,获得了T0代种子,为利用epsps基因改良植物对草甘膦的抗性奠定了物质基础.

Structure of Two plant Expression Vectors of Resistance to Glyphosate Gene

By the present reports,there are sciences of strong promoter driving resistance gene expression in the glyphosate genetically modified crops.It made resistance glyphosate gene be highly expressed in almost all positions of plant and all developmental stages.It increases the burden of the metabolism of the plant and the plant output is also likely to cause negative effects.In this paper,the use of glyphosate stressinduced promoter Ag2 drive the epsps gene to make plant highly express resistance to glyphosate only after spraying glyphosate.In addition,the tissue-specific promoter RuBP enable epsps genes highly express,when the glyphosate pesticide was sprayed over the main positions of plant leaves which should be able to increase the resistance to glyphosate of transgenic plants,but not to increase the metabolism burden of transgenic plant.It can help cultivate transgenic plants with strong resistance to glyphosate and high-quality agronomic traits. Based on plasmid of pB1121-E-M-Bt(Kanar)and pB1121-CP4E(Kanar),so pUC19 and EcoR locus were reformed through the site joint of the transformation of intermediate vector.The plant expression vector pCBI-Ag2EM-RuBPEM and pGBI-Ag2CP4E-RuBPCP4E of epsps gene controlled by RuBP promoter and the newly discovered indacible promoter Ag2,and the selectable marker is Kanamycin.The transformation of Arabidopsis thaliana was carried out by floral dip method and Agrobacterium-mediated.The T0 generation seed was obtained which laid a material foundation for improving the resistance of plants to glyphosate by anti-epsps gene.