A highly efficient maize nucellus protoplast system for transient gene expression and studying programmed cell death-related processes
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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.
KeywordsProtoplasts Maize nucellus PCD Transient expression Cys-protease
This work was supported by the National Natural Science Foundation of China (No: 91435114), the National Key Basic Research Program of China (No: 2014CB138200), and the Research Fund for Excellent Doctoral Program of Sichuan Agriculture University. We acknowledge Dr. Huang Yanyan (Rice Research Institute, Sichuan Agricultural University, Sichuan) for the kind help of Confocal laser scanning microscopy, and Liang Yueyang (Rice Research Institute, Sichuan Agricultural University, Sichuan) and Zhang (School of Life Sciences, Sun Yat-sen University, Guangzhou) for their support vectors for protein location.
Conflict of interest
All these authors declare that they have no conflict of interest.
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