A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice
A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice
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DOI：
10.1111/j.1364-3703.2006.00346.x
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发表时间：
2006-09-01
影响因子：
4.9
通讯作者：
Wang, Guo-Liang
中科院分区：
文献类型：
--
作者：
Chen, Songbiao;Tao, Lizen;Wang, Guo-Liang
基于叶肉细胞或培养细胞来源的原生质体的瞬时检测系统已经在多种植物中得到开发，并已成为快速基因功能分析和生化操作的有力工具。然而，由于难以从叶片或悬浮培养细胞中大规模分离出可存活的水稻原生质体，该系统尚未在水稻上广泛应用。在这里，我们描述了一种显著改进的方法，从幼树和成熟植物的茎和鞘组织中分离出大量的原生质体。在茎和鞘来源的原生质体中，观察到多种结构的高水平共表达和外源和内源基因的有效抑制。建立了基于瞬时绿色荧光蛋白和荧光素酶的水稻防御相关基因表达分析报告系统，为筛选和鉴定水稻防御信号途径相关基因奠定了基础。此外，还建立了一个基于原生质体的双分子荧光互补(BIFC)系统，用于检测活体水稻细胞中蛋白质与蛋白质的相互作用。通过GUS和细胞死亡相关蛋白SPIN1的同源二聚化，在转基因原生质体中观察到了两个分裂的YFP片段的YFP互补。结合遗传学、基因组学和蛋白质组学方法，建立的多功能原生质体瞬时检测系统将有助于对水稻防御相关基因的大规模功能分析。
The transient assay system based on mesophyll or cultured cell-derived protoplasts has been exploited in several plant species and has become a powerful tool for rapid gene functional analysis and biochemical manipulations. However, the system has not been widely used in rice owing to the difficulties in large-scale isolation of viable rice protoplasts from leaves or suspension-cultured cells. Here, we describe a significantly improved method to isolate a large number of protoplasts from stem and sheath tissues of both young and mature plants. High-level coexpression of multiple constructs and efficient suppression of exogenous and endogenous genes were observed in the stem- and sheath-derived protoplasts. A transient green fluorescent protein and luciferase-based reporter system for defence-related genes expression analysis has been established, which is useful for screening and characterizing genes involved in rice defence signalling pathways. Furthermore, a protoplast-based bimolecular fluorescence complementation (BiFC) system for the detection of protein-protein interactions in living rice cells was developed. The YFP complementation of two split-YFP halves mediated by homodimerization of the GUS and SPIN1, a cell-death related protein, was observed in transfected protoplasts. In combination with genetic, genomic and proteomic approaches, the established versatile protoplast transient assay system will facilitate large-scale functional analysis of defence-related genes in rice.