Abstract
The red fluorescent protein mCherry is widely used as a tagged protein in intracellular protein positioning and dynamic tracing due to its stable characteristics in color and monomer molecule. In this study, mCherry was used as a tag to explore the cell wall-directed binding of the carbohydrate-binding module (CBM), in order to intuitively demonstrate that the fusion protein anchored on the cell wall. Two constitutive expression vectors harboring CBM-mCherry fusion gene were constructed for rice transformation. The results of fluorescent signal detection showed that both the ubiquitin promoter and the CaMV35S promoter could drive the expression of mCherry fusion gene in seeds, leaves, and roots. The results of subcellular localization by different methods, such as cytological observation, immunofluorescence detection and protoplast observation, displayed that the fluorescence signals were concentrated on the cell walls of the root, stem, leaf, and vascular bundle sheath in rice. It indicated that the CBM could mediate the red fluorescent protein anchoring on the cell wall, and the cell wall may be a good subcellular structure for accumulation of exogenous proteins in future.
Key message
The fusion gene SP-CBM11-mCherry was transformed into rice, and the subcellular localization of fusion protein indicated the red fluorescent protein mCherry anchored on the cell wall.
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Funding
This research was jointly supported by Hainan Yazhou Bay Seed Lab (Grant No. B21HJ0107), National Science and Technology Major Project on Breeding of New Genetically Modified Organisms (2016ZX08001003-001) and Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA24030201).
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HL designed, did the experiments and wrote the manuscript; GX conceived the project, secured the funding and revised the manuscript; DL, WL, LJ and YJ contributed to the planting management in the field. All authors have read and agreed to the published version of the manuscript.
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Li, H., Deng, L., Weng, L. et al. The carbohydrate-binding module mediates mCherry protein anchoring on the cell wall in rice. Plant Cell Tiss Organ Cult 154, 527–539 (2023). https://doi.org/10.1007/s11240-023-02471-0
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DOI: https://doi.org/10.1007/s11240-023-02471-0