疏水蛋白HFBI融合标签能显著提高外源基因在植物中的表达水平

摘要

Objective To explore the mechanisms by which HFBI fusions increase recombinant fusion protein accumulation in plants. Methods The HFBI sequence from Trichoderma reesei was synthesized and two plant expression vectors for expression of green fluorescence protein (GFP) and GFP-HFBI were constructed. The vectors were inoculated in Nicotiana benthamiana plants through agroinfiltration, and the expression levels and mRNA accumulation levels of GFP in Nicotiana leaves were examined by Western blotting, ELISA and RT-PCR. Results The HFBI fusion tag significantly enhanced the accumulation of GFP in the leaves of N. benthamiana without causing toxic effects. Endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of spherical protein particles in the plant cells. Conclusion HFBI fusions can increase the accumulation of its fusion partner in plants by forming stable protein particles, which probably shields the target protein from endogenous protease-induced degadation. HFBI fusion technology provides an alternative to improving recombinant protein expression in plants from agroinfection-compatible expression vectors.%目的：针对植物生物反应器中外源基因表达水平普遍较低的问题，探索和评价丝状真菌瑞氏木霉（Trichoderma reesei）编码的小分子疏水蛋白（Hydrophobin, HFBI）作为融合标签在植物生物反应器中提高系统表达量的应用潜力；分析其提高外源基因表达水平的可能机制。方法以绿色荧光蛋白（GFP）为报道基因，采用基因体外合成技术和亚克隆技术构建GFP和GFP-HFBI融合植物表达载体。利用农杆菌渗滤技术接种植物本明烟（Nicotiana benthamiana）。通过GFP荧光观察，荧光显微镜镜检， Western blot，ELISA和RT-PCR等实验手段测定报告基因GFP在植物中的表达情况，探明HFBI融合标签在植物中表达外源基因的作用效果和特点并分析其可能的作用机制。结果 HFBI融合标签对植物细胞无明显的细胞毒性；GFP-HFBI融合蛋白在植物中的积累水平显著高于对照；GFP-HFBI融合蛋白在细胞内形成致密的蛋白质颗粒。结论 HFBI融合标签能够显著提高外源基因在植物中的积累水平。推测形成的蛋白质颗粒隔绝了细胞内源性蛋白酶对目的表达产物的降解，进而提高了外源基因产物在细胞中的积累。