Chen Jiang, Yi Qiang, Song Qiaoheng, Gu Yong, Zhang Junjie, Hu Yufeng, Liu Hanmei, Liu Yinghong, Yu Guowu, Huang Yubi
Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Maize Research Institute, Sichuan Agricultural University, 211 Huiming Road, Chengdu, 611130, China.
Plant Cell Rep. 2015 Jul;34(7):1239-51. doi: 10.1007/s00299-015-1783-z. Epub 2015 Mar 20.
Conditions for the isolation and transfection of maize nucellus protoplasts were established. We demonstrated its utilization for protein expression, localization, protein-protein interaction, and the investigation of PCD-related processes. Plant protoplasts are an important and versatile cell system that is widely used in the analysis of gene characterization and diverse signaling pathways. Programmed cell death (PCD) occurs throughout the life of plants from embryogenesis to fertilization. The maize nucellus undergoes typical PCD during development of the embryo sac. The nucellus protoplast shows potential for use in research of PCD-related processes. No studies have reported previously the isolation and transfection of nucellus protoplasts. In this study, conditions for the isolation and transfection of maize nucellus protoplasts were established. The maize protoplast system can be used for protein expression, localization, and protein-protein interaction. We applied this system to investigate PCD-related processes. Quantitative real-time PCR analysis revealed that transient expression of MADS29 in the maize nucellus protoplast increases Cys-protease gene transcript level. In addition, β-glucuronidase and luciferase activity assays showed that MADS29 could enhance the promoter activities of the Cys-protease gene. Thus, we demonstrated the potential of a highly efficient maize nucellus protoplast system for transient gene expression and investigation of PCD-related processes.
建立了玉米珠心原生质体的分离和转染条件。我们展示了其在蛋白质表达、定位、蛋白质-蛋白质相互作用以及程序性细胞死亡（PCD）相关过程研究中的应用。植物原生质体是一种重要且通用的细胞系统，广泛用于基因特性分析和多种信号通路研究。程序性细胞死亡在植物从胚胎发生到受精的整个生命周期中都会发生。玉米珠心在胚囊发育过程中经历典型的程序性细胞死亡。珠心原生质体在程序性细胞死亡相关过程的研究中显示出应用潜力。此前尚无关于珠心原生质体分离和转染的研究报道。在本研究中，建立了玉米珠心原生质体的分离和转染条件。玉米原生质体系统可用于蛋白质表达、定位以及蛋白质-蛋白质相互作用研究。我们应用该系统研究程序性细胞死亡相关过程。定量实时PCR分析表明，MADS29在玉米珠心原生质体中的瞬时表达增加了半胱氨酸蛋白酶基因转录水平。此外，β-葡萄糖醛酸酶和荧光素酶活性测定表明，MADS29可增强半胱氨酸蛋白酶基因的启动子活性。因此，我们证明了高效玉米珠心原生质体系统在瞬时基因表达和程序性细胞死亡相关过程研究中的潜力。
