Department of Crop Science, North Carolina State University, Raleigh, NC 27695, USA.
Plant Methods. 2012 Aug 1;8(1):27. doi: 10.1186/1746-4811-8-27.
We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns.
The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively.
These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.
我们先前开发了一种来自二分体伴随病毒棉花皱叶病毒（CLCrV）的棉花病毒诱导基因沉默（VIGS）载体。最初的 CLCrV VIGS 载体是为基因枪的弹道式传递而设计的。这个前提条件限制了该系统的使用范围，只能在有基因枪设备的实验室中使用。在这里，我们描述了该系统通过农杆菌（Agrobacterium tumefaciens）传递的适应性改造。我们还描述了两种低成本粒子流入枪的构建。
生物弹道式的 CLCrV 载体被转移到两个农杆菌二元质粒中。将二元质粒的农杆菌接种到棉花中，导致沉默和 GFP 表达与生物弹道式载体相当。两个自制的低成本基因枪分别成功地用 CLCrV VIGS 载体或番茄金黄花叶病毒（TGMV）VIGS 载体接种棉花（G. hirsutum）和 N. benthamiana。
这些创新扩展了基于 CLCrV 的 VIGS 在分析棉花中基因功能的多功能性。这两个低成本基因枪通过提供一种有价值的替代昂贵的商用基因枪的方法，使 VIGS 实验在研究和教学实验室中都能负担得起。
