Transient expression for functional gene analysis using Populus protoplasts
Transient expression for functional gene analysis using Populus protoplasts
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使用杨属原生质体进行功能基因分析的瞬时表达
DOI：
10.1007/s11240-013-0299-x
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发表时间：
2013-07-01
影响因子：
3
通讯作者：
Huang, Minren
中科院分区：
文献类型：
--
作者：
Tan, Biyue;Xu, Meng;Huang, Minren
尽管杨树基因组序列已经存在，并且遗传、基因组和转基因方法也得到了发展，但杨树的漫长寿命和其转化所需的繁琐过程阻碍了杨树基因功能的快速表征。原生质体为植物基因的高通量分析和功能表征提供了多功能且生理相关的细胞系统。这里，基于以下三个步骤开发了利用杨叶肉原生质体的高效瞬时表达系统。第一步涉及配制含有 2% 纤维素酶 C2605 和 0.5% 果胶酶 P2611 的新酶混合物，该混合物被证明能够有效大规模分离同质杨叶肉原生质体。第二步涉及优化转染条件，例如聚乙二醇浓度和质粒 DNA 量，以确保杨属原生质体的转染效率 > 80%。第三步涉及使用杨原生质体瞬时表达系统成功确定蛋白质的亚细胞定位，模拟病原体感染期间的信号事件，并制备用于蛋白质印迹和蛋白质-蛋白质相互作用测定的蛋白质提取物。这种在杨树叶肉原生质体中快速高效的瞬时基因表达系统将有助于快速鉴定杨树基因功能和阐明杨树信号通路。
Despite the availability of the Populus genome sequence and the development of genetic, genomic, and transgenic approaches for its improvement, the lengthy life span of Populus and the cumbersome process required for its transformation have impeded rapid characterization of gene functions in Populus. Protoplasts provide a versatile and physiologically relevant cell system for high-throughput analysis and functional characterization of plant genes. Here, a highly efficient transient expression system using Populus mesophyll protoplasts was developed based on the following three steps. The first step involved formulating a new enzyme cocktail containing 2 % Cellulase C2605 and 0.5 % Pectinase P2611, which was shown to enable efficient large-scale isolation of homogenous Populus mesophyll protoplasts. The second step involved optimization of transfection conditions, such as the polyethylene glycol concentration and amount of plasmid DNA to ensure a > 80 % transfection efficiency for Populus protoplasts. The third step involved using the Populus protoplast transient expression system to successfully determine the subcellular localizations of proteins, emulate signaling events during pathogen infection, and prepare protein extracts for Western blotting and protein-protein interaction assays. This rapid and highly efficient transient gene expression system in Populus mesophyll protoplasts will facilitate the rapid identification of gene functions and elucidation of signaling pathways in Populus.