Abstract
Calcium-dependent protein kinases (CDPKs), as an important calcium sensor in plants, are widely involved in the signal transmission process of growth and development, pathogen defense response and biotic or abiotic stress. In this study, we cloned in mulberry leaves, the full-length CDS sequence of MaCDPK1, which encodes 573 amino acids with an isoelectric point of 5.36. Bioinformatics analysis found MaCDPK1 have a classical kinase domain and an EF-hands domain. We obtained the wild-type and the mutant-type of MaCDPK1(CDPK1 and mCDPK1) by prokaryotic expression and purification. Through kinase assay, it was found that CDPK1 has intact enzymatic activity and exhibits Mg2+ and Ca2+ dependence with a Km of 30 µM and a Vmax of 100,000 [RLU]/min/µg. However, the mCDPK1 has no activity, which means that aspartic acid at position 234th is critical for the activity. The 1500 bp upstream of the start codon of the MaCDPK1 gene contains core promoter elements TAAT-box and CAAT-box, and contains response elements associated with adverse signals such as light, drought, and abscisic acid. The tissues expression level of MaCDPK1 gene in different tissues of mulberry trees under different stress conditions reflect complex expression patterns, indicating that the MaCDPK1 gene responded to stress tolerance. These results are likely to indicate that MaCDPK1 positively regulates salt and drought stress in Morus atropurpurea Roxb.
Key Message
This research explored the basic properties of the MaCDPK1 protein, discovered a key amino acid site associated with enzyme activity, and found its corresponding anti-reversal process.
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Acknowledgements
The authors are very thankful to Jingchen Sun and Huichao Yan for the technical assistance and everyone involved in the current study. The study was supported by China Agriculture Research System (CARS-18), Sericulture Research System of Guangdong Province, China (Grant Number 2019KJ124). The authors thank South China Agricultural University 2018 University Student Innovation and Entrepreneurship Training Program Project (201810564047), South China Agricultural University Graduate Student Overseas Study Program, China (Grant Number 2019GWFX040). The authors thank Dr. Main Muhammad Awais and Dr. Yuyi Zhong for correcting the language in the manuscript.
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Conception and design: JS, ZC, HY. Analysis and interpretation of the data: ZC, QH. Drafting of the manuscript: ZC, QH, JS, HY. Critical revision of the manuscript for important intellectual content: ZC, QH, JY, MMA, JS, HY. All of the authors have read and approved the manuscript. Requests for materials and data should be addressed to correspondence author J.S (cyfz@scau.edu.cn).
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Communicated by Francisco de Assis Alves Mourão Filho.
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Fig. S1
Experiment principle of Kinase-Glo® luminescent kinase assay. Supplementary material 2 (TIF 18121.2 kb)
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Cao, Z., He, Q., Wang, P. et al. Functional characteristics of a calcium-dependent protein kinase (MaCDPK1) enduring stress tolerance from Morus atropurpurea Roxb.. Plant Cell Tiss Organ Cult 141, 131–143 (2020). https://doi.org/10.1007/s11240-020-01773-x
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DOI: https://doi.org/10.1007/s11240-020-01773-x