Nopo Luis, Woffenden Bonnie J, Reed Deborah G, Buswell Scott, Zhang Chenming, Medina-Bolivar Fabricio
Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA.
Methods Mol Biol. 2012;824:501-26. doi: 10.1007/978-1-61779-433-9_27.
In order to identify a promoter system for high-level expression of transgenes in hairy roots, we characterized the chimeric super-promoter fused to the translational enhancer from tobacco etch virus (TEV). Transgenic tobacco plants and hairy roots were generated with the super-promoter:TEV sequence and a modified green fluorescence protein (mGFP5) as a reporter gene. To exploit the utility of hairy root cultures as a secretion-based expression system, the signal peptide of patatin was fused to mGFP5 to direct its secretion into the culture medium. Levels of mGFP5 RNA were on average sixfold higher in hairy roots than leaves. Likewise, GFP protein levels per gram of fresh weight were at least tenfold higher in hairy roots than leaves. Furthermore, more than 10% of the recombinant protein produced in the hairy root culture system was found in the medium. Immunoblotting with anti-GFP antibodies showed two products of 27.1 and 29.9 kDa in all leaf and hairy root tissue extracts, whereas a single 27.1-kDa product was detected in the medium. Inducibility of the promoter was studied with mature leaves and 14-day (midlog phase) hairy roots. A twofold increase in mRNA levels was found immediately after wounding in both mature leaves and hairy roots, with a corresponding increase in mGFP5 protein after 24 h. Our studies demonstrate the utility of the super-promoter:TEV system for high-level expression of recombinant proteins in hairy root bioreactors.
为了鉴定用于毛状根中转基因高水平表达的启动子系统，我们对与烟草蚀纹病毒（TEV）翻译增强子融合的嵌合超级启动子进行了表征。利用超级启动子：TEV序列和修饰的绿色荧光蛋白（mGFP5）作为报告基因，构建了转基因烟草植株和毛状根。为了利用毛状根培养物作为基于分泌的表达系统的效用，将马铃薯块茎蛋白的信号肽与mGFP5融合，以将其分泌到培养基中。毛状根中mGFP5 RNA的水平平均比叶片高六倍。同样，每克鲜重的GFP蛋白水平在毛状根中比叶片至少高十倍。此外，在毛状根培养系统中产生的重组蛋白有超过10%存在于培养基中。用抗GFP抗体进行免疫印迹分析显示，在所有叶片和毛状根组织提取物中均有27.1 kDa和29.9 kDa两种产物，而在培养基中检测到单一的27.1 kDa产物。用成熟叶片和14天龄（对数中期）的毛状根研究了启动子的诱导性。在成熟叶片和毛状根受伤后立即发现mRNA水平增加了两倍，24小时后mGFP5蛋白相应增加。我们的研究证明了超级启动子：TEV系统在毛状根生物反应器中用于重组蛋白高水平表达的效用。
