Yu Guohui, Cheng Qiang, Xie Zheni, Xu Bin, Huang Bingru, Zhao Bingyu
College of Agro-grassland Science, Nanjing Agricultural University, Nanjing, 210095 People's Republic of China.
Jiangsu Key Laboratory for Poplar Germplasm Enhancement and Variety Improvement, Nanjing Forestry University, Nanjing, 210037 People's Republic of China.
Plant Methods. 2017 Jun 5;13:46. doi: 10.1186/s13007-017-0196-0. eCollection 2017.
Perennial ryegrass ( L.) is an important temperate grass used for turf and forage purposes. With the increasing accumulation of genomic and transcriptomic data of perennial ryegrass, an efficient protoplast and transient gene expression protocol is highly desirable for in vivo gene functional studies in its homologous system.
In this report, a highly efficient protoplast isolation (5.6 × 10 protoplasts per gram of leaf material) and transient expression (plasmid transformation efficiency at 55.2%) was developed and the detailed protocol presented. Using this protocol, the subcellular locations of two ryegrass proteins were visualized in chloroplasts and nuclei, respectively, and protein-protein interaction between two chlorophyll catabolic enzymes (LpNOL and LpNYC1) was recorded in its homologous system for the first time.
This efficient protoplast isolation and transformation protocol is sufficient for studies on protein subcellular localization and protein-protein interaction, and shall be suitable for many other molecular biology applications where the mesophyll protoplast system is desirable in perennial ryegrass.
多年生黑麦草是一种重要的温带草种，用于草坪和饲料用途。随着多年生黑麦草基因组和转录组数据的不断积累，一种高效的原生质体和瞬时基因表达方案对于其同源系统中的体内基因功能研究非常必要。
在本报告中，开发了一种高效的原生质体分离方法（每克叶片材料可获得5.6×10个原生质体）和瞬时表达方法（质粒转化效率为55.2%），并给出了详细方案。使用该方案，分别在叶绿体和细胞核中观察到了两种黑麦草蛋白的亚细胞定位，首次在其同源系统中记录到了两种叶绿素分解代谢酶（LpNOL和LpNYC1）之间的蛋白质-蛋白质相互作用。
这种高效的原生质体分离和转化方案足以用于蛋白质亚细胞定位和蛋白质-蛋白质相互作用的研究，并且适用于多年生黑麦草中需要叶肉原生质体系统的许多其他分子生物学应用。
