Kachroo Aardra, Ghabrial Said
Department of Plant Pathology, University of Kentucky, Lexington, KY, USA.
Methods Mol Biol. 2012;894:287-97. doi: 10.1007/978-1-61779-882-5_19.
Understanding the biology of a system requires the association of gene functions to phenotypes and vice versa. Although a large number of resources and genomic tools are available for studies in the crop plant soybean, investigations related to gene function have been lacking. This is largely due to the fact that rapid functional genomics tools have been unavailable for application in soybean. This constraint was recently alleviated when a novel bean pod mottle virus (BPMV)-based system for virus-induced gene silencing in soybean was developed. This methodology exploits the plant's ability to silence virus-encoded sequences as a defense mechanism. Plants infected with recombinant viruses carrying sequences that are identical/near-identical to the plant's own genes suppress the expression of the target endogenous gene(s). Phenotypes associated with suppression of target sequence enable the assignment of functions to specific genes. The strategy involves inserting short sequences of target soybean genes into the viral genome to generate recombinant vectors. Soybean plants are then inoculated with the recombinant vectors, assessed for BPMV propagation and silencing of the target genes. Plants silenced for specific targets can then be analyzed for various different traits depending upon the known/predicted functions of the target genes. Using this strategy, we have identified genes participating in basal, resistance gene-mediated, and systemic immunity in soybean.
了解一个系统的生物学特性需要将基因功能与表型联系起来,反之亦然。尽管有大量资源和基因组工具可用于农作物大豆的研究,但与基因功能相关的研究却一直缺乏。这主要是因为快速的功能基因组学工具无法应用于大豆。当一种基于新型豆荚斑驳病毒(BPMV)的大豆病毒诱导基因沉默系统被开发出来后,这一限制最近得到了缓解。这种方法利用了植物将病毒编码序列沉默作为一种防御机制的能力。感染了携带与植物自身基因相同/近乎相同序列的重组病毒的植物会抑制目标内源基因的表达。与目标序列抑制相关的表型能够将功能赋予特定基因。该策略包括将目标大豆基因的短序列插入病毒基因组以产生重组载体。然后用重组载体接种大豆植株,评估BPMV的传播情况以及目标基因的沉默情况。根据目标基因的已知/预测功能,然后可以对特定目标沉默的植株进行各种不同性状的分析。利用这种策略,我们已经鉴定出了参与大豆基础免疫、抗性基因介导的免疫和系统免疫的基因。