Overexpression of 5-enolpyruvylshikimate-3-phosphate synthase gene (CP4EPSPS) under the control of leafy promoter enhances glyphosate tolerance of budlet | Leafy启动子控制下5-烯醇式丙酮酰-莽草酸-3-磷酸合酶基因(CP4EPSPS)的表达增强芽对草甘膦的抗性

Chino. 抗草甘膦基因在转基因植物体内持续高效表达，不但增加植物代谢压力，有的甚至改变植物形态造成植物的生长发育畸形。为了减少转基因植株的代谢负担和能源浪费，从拟南芥菜(Arabidopsis thaliana)基因组中克隆了Leafy组织特异性启动子替代CaMV35S启动子，用其驱动改造后加二磷酸核酮糖羧化酶(rubisco)小亚基引导肽的5-烯醇丙酮酰-莽草酸-3-磷酸合酶(CP4EPSPS)基因，同时调控报告基因gus的编码区构建植物表达载体p3300-Leafy-gus和p3300-Leafy-CP4EPSPS，嵌合基因经农杆菌(Agrobacterium)介导转化烟草。稳定表达后经GUS组织染色分析表明，Leafy驱动的gus表达仅局限在植物茎尖和幼叶部分，转基因植株成熟的叶片、茎部和根系均未能检测到GUS活性。草甘膦试验分析表明，Leafy驱动的CP4EPSPS的转基因植株幼芽部位有草甘膦抗性。结果表明，Leafy启动子驱动CP4EPSPS表达增强植株芽端对草甘膦的抗性。

Inglés. Continuous and efficient expression of glyphosate-resistance gene in transgenic plants increases plant metabolism pressure, even causes abnormality of plant growth. To reduce the burden of plant metabolism and to prevent energy waste, we have used a Leafy promoter from genome of Arabidopsis thaliana instead of CaMV35S promoter to govern the expression of a modified 5-enolpyruvylshikimate-3-phosphate synthase gene (CP4EPSPS) with addition of the transit peptide-coding sequence of rubisco small ribosome subunit. Meanwhile, the Leafy promoter was used to regulate gus, a reporter gene. Two plant expression vectors, p3300-Leafy-gus and p3300-Leafy-CP4EPSPS were constructed and used to transform tobacco W38 through Agrobacterium-mediated procedure. Under these conditions, stable expression of the CP4EPSPS and gus genes was only observed in stem apex and extremely young apical leaves of transgenic tobaccos. The gus activity was not detectable in mature leaves, stems or roots. It is demonstrated that the budlet of transgenic tobaccos shows tolerance to herbicide glyphosate.