Abstract: Leaves of Penn-A4 creeping bentgrass were used as materials to establish a stable and efficient transient expression system for mesophyll protoplasts cells. The effects of different factors on the protoplast isolation of mesophyll cells were analyzed, and the optimum conditions for protoplast isolation were determined. The results showed that the leaves of 4-week-old bentgrass aseptic seedlings could be hydrolyzed in 0.6 mol·L^-1 mannitol at 28℃ for 4 h. Protoplasts with high viability could be isolated, and the extraction amount was about 6.75×10⁶ cells·g^-1. To further test whether the protoplast can be effectively used in the study of protein function, the recombinant plasmid of heat shock protein gene AsHSP26.8-GFP was constructed and introduced into mesophyll cell protoplasts under the mediation of PEG-4000 for subcellular localization. The localization of AsHSP26.8 in chloroplast was observed by laser confocal microscope. To sum up, this study established a set of relatively complete protoplast isolation and transient expression system of creeping bentgrass glume mesophyll cells, which laid a foundation for exploring the functional genomics of creeping bentgrass.

Key words: Agrostis stolonifera L., Protoplast, Transient expression, Subcellular localization

摘要： 为了建立稳定、高效的匍匐翦股颖（Agrostis stolonifera L.）叶肉细胞原生质体瞬时表达体系，本研究以匍匐翦股颖‘Penn-A4’的叶片为材料，分析不同因素对其叶肉细胞原生质体分离的影响，确定其原生质体分离的最佳条件。结果表明：苗龄为4周的翦股颖无菌苗叶片在甘露醇为0.6 mol·L^-1的酶解液中，28℃酶解4 h后，可分离得到较高活力原生质体、提取量约为6.75×10⁶个·g^-1；为进一步检测该原生质体是否可有效的应用在蛋白功能研究中，构建了热激蛋白基因AsHSP26.8-GFP重组质粒，并在聚乙二醇（PEG-4000）介导下将其导入叶肉细胞原生质体中进行了亚细胞定位；通过激光共聚焦显微镜观察到AsHSP26.8定位于叶绿体。综上所述，本研究建立了一套较为完整的匍匐翦股颖叶肉细胞原生质体分离与瞬时表达体系，为探究匍匐翦股颖的功能基因组学研究奠定了基础。

关键词: 匍匐翦股颖, 原生质体, 瞬时表达, 亚细胞定位