Department of Plant Pathology, 201F Plant Science Building, University of Kentucky, 1405 Veterans Drive, Lexington, KY 40546-0312, USA.
Plant Methods. 2011 Jun 13;7:16. doi: 10.1186/1746-4811-7-16.
Common bean (Phaseolus vulgaris L.) is a crop of economic and nutritious importance in many parts of the world. The lack of genomic resources have impeded the advancement of common bean genomics and thereby crop improvement. Although concerted efforts from the "Phaseomics" consortium have resulted in the development of several genomic resources, functional studies have continued to lag due to the recalcitrance of this crop for genetic transformation.
Here we describe the use of a bean pod mottle virus (BPMV)-based vector for silencing of endogenous genes in common bean as well as for protein expression. This BPMV-based vector was originally developed for use in soybean. It has been successfully employed for both protein expression and gene silencing in this species. We tested this vector for applications in common bean by targeting common bean genes encoding nodulin 22 and stearoyl-acyl carrier protein desaturase for silencing. Our results indicate that the BPMV vector can indeed be employed for reverse genetics studies of diverse biological processes in common bean. We also used the BPMV-based vector for expressing the green fluorescent protein (GFP) in common bean and demonstrate stable GFP expression in all common bean tissues where BPMV was detected.
The availability of this vector is an important advance for the common bean research community not only because it provides a rapid means for functional studies in common bean, but also because it does so without generating genetically modified plants. Here we describe the detailed methodology and provide essential guidelines for the use of this vector for both gene silencing and protein expression in common bean. The entire VIGS procedure can be completed in 4-5 weeks.
普通菜豆（Phaseolus vulgaris L.）是世界许多地区具有经济和营养重要性的作物。由于缺乏基因组资源，普通菜豆的基因组学进展受到阻碍，从而影响了作物改良。尽管“Phaseomics”联盟的协同努力已经开发了几种基因组资源，但由于该作物遗传转化的顽固性，功能研究仍然滞后。
在这里，我们描述了使用豆斑驳病毒（BPMV）为基础的载体在普通菜豆中进行内源基因沉默和蛋白质表达。这种基于 BPMV 的载体最初是为在大豆中使用而开发的。它已成功地用于该物种的蛋白质表达和基因沉默。我们通过靶向普通菜豆基因编码的豆球蛋白 22 和硬脂酰-酰基载体蛋白去饱和酶来测试该载体在普通菜豆中的应用，以进行沉默。我们的结果表明，BPMV 载体确实可以用于普通菜豆中各种生物学过程的反向遗传学研究。我们还使用基于 BPMV 的载体在普通菜豆中表达绿色荧光蛋白（GFP），并证明在所有检测到 BPMV 的普通菜豆组织中均稳定表达 GFP。
该载体的可用性是普通菜豆研究界的一个重要进展，不仅因为它为普通菜豆的功能研究提供了一种快速手段，而且因为它不会产生转基因植物。在这里，我们描述了详细的方法，并为该载体在普通菜豆中的基因沉默和蛋白质表达提供了必要的指导。整个 VIGS 过程可以在 4-5 周内完成。
