Transient expression for functional gene analysis using Populus protoplasts
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- 作者：Biyue Tan (1) (2)
Meng Xu (1) (2)
Ying Chen (1) (2)
Minren Huang (1) (2) - 关键词：Mesophyll cells ; Enzyme cocktail ; PEG ; mediated transfection ; Western blotting ; Protein interaction
- 刊名：Plant Cell, Tissue and Organ Culture
- 出版年：2013
- 出版时间：July 2013
- 年：2013
- 卷：114
- 期：1
- 页码：11-18
- 全文大小：606KB
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Meng Xu (1) (2)
Ying Chen (1) (2)
Minren Huang (1) (2)
1. Jiangsu Key Laboratory for Poplar Germplasm Enhancement and Variety Improvement, Nanjing Forestry University, Nanjing, 210037, China
2. Key Laboratory of Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, 201137, China - ISSN：1573-5044
文摘
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.