抗草甘膦基因植物双价表达载体的构建-生物化学与分子生物学专业毕业论文.docxVIP

摘 摘 要 本文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A92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基冈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。 因此，本文分别以pBll21 E M Bt(Kana‘)和pBll21．CP4E(Kana‘)质粒为基础，通过载体pUCl9 及对EcoR I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A醇共同调控epsps 基因的植物表达载体pGBI-A92EM-RuBPEM和pGBI-A92CP4E·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 To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。 关键词：草甘膦，EPSPS，A92，RuBP，拟南芥 AbstractIn Abstract In the present reports，there composed of strong promoter driving resistance gene expression in the glyphosate genetically modified crops．It made resistance glyphosate gene highly expressed in almost all plant and all developmental stages．It increase the burden the metabolism of the plant and the plant’S ompm is also likely to negative effects．In this paper,the of glyphosate stress-induced promoter A92 drive the epsps gene made plant high express of resistance to glyphosate only after spraying glyphosate．In addition，the tissue—specific promoter RuBP enable epsps genes high expression in plant leaves which is the field of glyphosate pesticide spraying major part．Use the RuBP and inducible promoter A92 driving epsps gene at the same time should be able to enhance the anti-transgenic plants ability to glyphosate further and bring the metabolism burden of transgenic plants not too much．It Can made transgenic plants with strong resistance to glyphosate and high-quality agronomic traits． So that the plant expression vector pBI 1 2 1—·E·-M··Bt(Kand)and pBl l 2 1·-CP4E(Kand)are based the pBl]2 1-E(mutations)一M-Bt(Kana)and pBl l 2 1-epsps(Kana)plasmid respectively．They constructed throggh an intermediate vector pUC 1 9 and the EcoR I sites joint transformation．Their anti-epsps gene (mutation and unmutation)common reg}tlated by the newly discovered inducible promoter A92 and the tissue—specific promoter RuBP and the sel