Cloning and expression profile of an ubiquitin-conjugating enzyme-like gene OsCROC-1A in rice (Oryza sativa L.)
Protein ubiquitination is a fundamental post-translational modification event that serves as a signaling function in diverse biological processes. Ubiquitination is accomplished by a series of three enzymatic steps as follows: E1 (ubiquitin-activating enzyme) -E2 (ubiquitin-conjugating enzyme, Ubc)...
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| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Zhejiang University Press
2013-07-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2012.11.181 |
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| Summary: | Protein ubiquitination is a fundamental post-translational modification event that serves as a signaling function in diverse biological processes. Ubiquitination is accomplished by a series of three enzymatic steps as follows: E1 (ubiquitin-activating enzyme) -E2 (ubiquitin-conjugating enzyme, Ubc) -E3 (ubiquitin ligase enzyme). In these processes, the role of Ubc is of pivotal importance. All Ubcs have an active site cysteine (Cys) residue within the highly conserved UBC domain. Ubc-like proteins share high similarities with Ubc but lack the active site Cys which is essential for the conjugation and transfer of ubiquitin to protein substrates, therefore, they have also been designated as UEV (ubiquitin-conjugating E2 enzyme variant) proteins. Ubc-like proteins have been found in all eukaryotes examined such as animal, plant and yeast. To date, the functions of Ubc-like protein have been extensively studied in animal and yeast. However, it remains largely unknown in plant species, especially in monocot plants, so it is necessary to study Ubc-like protein in this field.Rice is a prominent model for monocotyledonous plants and one of the most important food crops. However, no information is available on Ubc-like protein in rice or other monocot plants. Here, we mainly characterized the expression pattern and subcellular localization of a rice Ubc-like protein in order to provide a foundation for the function studies of UEV proteins in monocot plants.The molecular characterization of an Ubc-like protein gene in rice was cloned and analyzed, and was designated as OsCROC-1A in terms of protein sequence analysis using bioinformatics, gene expression via real-time quantitative PCR, and fluorescence signal localization of the fusion protein in transformed tobacco suspension cultures.The open reading frame (ORF) of OsCROC-1A was cloned from rice (Oryz a sativ a, cultivar Nipponbare) via RT-PCR, and was used to analyze the expression pattern under abiotic stresses. OsCROC-1A contained five exons and four introns, and it encoded a UEV homolog of 146 amino acids corresponding to a theoretical molecular mass of 17 ku and pI of 6.42. OsCROC-1A harbored a conserved UBC domain and D catalytic site, and the deduced sequence shared similarities with other homologs ranging from 42.61% to 84.14%. Real-time quantitative PCR was conducted to analyze the spatial expression pattern of OsCROC-1A as well as the changes of its expression level under abiotic stresses. As a result, OsCROC-1A was found to be expressed in all tissues examined, and the abundance level was high in leaf, root, lemma, and low in palea, stem, pistil, callus, and the lowest in stamen, implying that the OsCROC-1A is important to both vegetative growth and reproductive development in rice especially to that of leaf, root, lemma. The expression of OsCROC-1A was induced by low temperature of 4 ℃, 20 mmol/L H<sub>2</sub>O<sub>2</sub> and 0.01% MMS, whereas it was repressed by 300 mmol/L mannitol, 100 μmol/Labscisic acid (ABA) and 200 mmol/L NaCl, suggesting that OsCROC-1A can respond actively to the negative circumstances by changing its transcripts. OsCROC-1A-EGFP was localized to both nucleus and cytosol, indicating that OsCROC- 1A not only can play its role in the nucleus as a transcription factor, but also have function in cytosol.In conclusion, OsCROC-1A encodes an Ubc-like protein. It is constitutively expressed in all the tissues examined and is generally modulated by abiotic stress, and OsCROC-1A has functions in both nucleus and cytosol. These data provide a foundation for investigating the cellular function and molecular mechanism of OsCROC-1A. |
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| ISSN: | 1008-9209 2097-5155 |