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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2013, Vol. 39 Issue (4): 360-368    DOI: 10.3785/j.issn.1008-9209.2012.11.181
生物科学与技术     
水稻泛素缀合酶样蛋白基因OsCROC-1A的克隆与表达分析
王亚1,2， 刘建平1， 徐梦云1， 季为捷1， 凃巨民1， 张晓波1*
(1.浙江大学农业与生物技术学院农学系，杭州 310029;2.河南省农业科学院粮食作物研究所，郑州 450002)
Cloning and expression profile of an ubiquitin-conjugating enzyme-like gene OsCROC-1A in rice (Oryza sativa L.)
WANG Ya1,2, LIU Jianping1, XU Mengyun1, JI Weijie1, TU Jumin1, ZHANG Xiaobo1*
(1. Department of Agronomy, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310029, China; 2. Cereal Crops Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China)
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摘要： 为研究水稻泛素缀合酶样蛋白的序列特征、基因表达模式及亚细胞定位模式，采用逆转录聚合酶链式反应（reverse transcriptionpolymerase chain reaction, RT-PCR），从水稻日本晴中鉴定并分离到1个泛素缀合酶样蛋白基因即OsCROC-1A的开放阅读框.序列分析结果表明，该基因编码1个具有146个氨基酸的蛋白质，含UBC保守功能域和催化位点D.比对分析结果表明，OsCROC-1A蛋白与其他CROC-1蛋白之间具有很高的相似性(42.61%~84.14%).聚类分析结果也显示出良好的物种属性.实时荧光定量PCR分析结果表明，OsCROC-1A在水稻各组织中均有表达，其中，在叶片、根、外稃中的表达量相对较高，在内稃、茎、雌蕊、愈伤组织中的表达量次之，而在雄蕊中的表达量相对最低.经4 ℃低温、20 mmol/L H2O2、0.01%甲基磺酸甲酯等胁迫处理使胚性愈伤中的OsCROC-1A表达量显著上调，经300 mmol/L甘露醇、100 μmol/L脱落酸、200 mmol/L NaCl胁迫处理则使愈伤组织中的OsCROC-1A表达量降低.OsCROC-1A与EGFP的融合蛋白表达于细胞质及细胞核中.这些结果为进一步研究该基因的生物学功能及其调控机制奠定了基础.
Abstract: 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 (Oryza sativa, 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 H2O2 and 0.01% MMS, whereas it was repressed by 300 mmol/L mannitol, 100 μmol/L abscisic 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.
出版日期: 2013-07-20
CLC:  Q 785  
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引用本文:

王亚1,2， 刘建平1， 徐梦云1， 季为捷1， 凃巨民1， 张晓波1*. 水稻泛素缀合酶样蛋白基因OsCROC-1A的克隆与表达分析[J]. 浙江大学学报(农业与生命科学版), 2013, 39(4): 360-368.

WANG Ya1,2, LIU Jianping1, XU Mengyun1, JI Weijie1, TU Jumin1, ZHANG Xiaobo1*. Cloning and expression profile of an ubiquitin-conjugating enzyme-like gene OsCROC-1A in rice (Oryza sativa L.). Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(4): 360-368.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.11.181    或    http://www.zjujournals.com/agr/CN/Y2013/V39/I4/360

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