Expression Analysis of Rice U-Box Proteins at Different Developmental Stages
Expression Analysis of Rice U-Box Proteins at Different Developmental Stages
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水稻U-Box蛋白在不同发育阶段的表达分析
DOI：
10.3724/sp.j.1206.2009.00075
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发表时间：
2009-09-01
影响因子：
0.3
通讯作者：
Liu Guo-Zhen
中科院分区：
文献类型：
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作者：
Chen Hao;Li Li-Yun;Liu Guo-Zhen
U-Box结构域蛋白与RING finger蛋白结构相似，在真核生物中具有高度保守性，它们大多属于泛素/蛋白酶体系统，作为E3泛素蛋白连接酶，U-Box结构域蛋白在识别和选择泛素化靶蛋白及其降解过程中起着关键作用，这是活细胞中存在的蛋白质维持和质量控制过程。水稻基因组中共有77个U-Box基因，对其表达的系统研究将为U-Box基因的功能分析提供基础信息。制备针对水稻U-Box蛋白的特异性抗体，研究U-Box蛋白在不同发育阶段的表达谱，为功能研究积累基础信息。选择了4个水稻U-Box基因，因为它们的U-Box结构域位于n端，在c端具有ARM重复序列。利用计算机软件进行表位预测，并在大肠杆菌中表达和纯化目标蛋白片段。系统。兔免疫制备多克隆抗体。对不同发育阶段收集的水稻材料进行Western blot分析，包括苗期的茎和根、分蘖期的根和茎、抽穗期的旗叶和幼粒、开花期的旗叶和颗粒、灌浆期的旗叶和种子。与EST测序数据进行对比分析。重组蛋白在大肠杆菌中表达，经兔免疫获得特异性抗体。Western blotting检测水稻U-Box蛋白发现，2个U-Box蛋白（Os06g01304和Os12g38210）的表观分子质量与预测值一致，2个蛋白（Os01g66130和Os08g01900）的表观分子质量小于预测值。Western blotting结果显示，U-Box蛋白在被试组织中组成性表达，丰度接近。基于NCBI EST数据库274个文库的100万条EST分析，U-Box基因转录闭合数与蛋白质组成表达结果一致。然而，与ATPase、HSP81-3、EGF-1 α和RuBisCo的表达相比，U-Box蛋白的est数量要少得多，表明U-Box基因的转录丰度较低。选择4个水稻U-Box蛋白通过预测表位片段的表达产生特异性抗体，证明了该工艺的可行性。Western blotting分析表明，4种U-Box蛋白在水稻不同发育阶段的组织中均有组成性表达，这与EST测序结果一致。该抗体将为共免疫沉淀、ChIP-on-chip、拉下和应激反应等功能研究提供资源。
The U-Box domain proteins, with similar configuration to RING finger proteins, are highly conserved among eukaryotic organisms and most of them belong to the ubiquitin/proteasome system as E3 ubiquitin protein ligases, U-Box containing proteins play a key role in the recognition and selection of abnormal proteins targeted for ubiquitination and subsequent degradation, a process for the maintenances and quality control of proteins exist in living cells. There are 77 U-Box genes in rice genome and the systematic investigation of their expression will provide basic information for the functional analysis. The specific antibodies against rice U-Box proteins was prepared to investigate the expression profile of U-Box proteins at different developmental stages and accumulate basic information for functional studies. Four rice U-Box genes were chosen as their U-Box domains are located at the N-terminal and posses ARM repeats at C-terminal. Epitopes prediction were carried out by computer software and the target protein fragments were expressed and purified in E. colt. system. Polyclonal antibodies were generated by rabbit immunization. Western blotting analysis were carried out for rice material collected at different developmental stages including shoot and root at seedling stage, root and stem at tilling stage, flag leaf and young particle at heading stage, flag leaf and particle at flowering stage, flag leaf and seed at filling stage. Comparison analysis was carried out with EST sequencing data. Specific antibodies were obtained by rabbit immunization of recombinant proteins expressed in E. coli. One major band were observed for Western blotting detection of rice U-Box proteins, the apparent molecular mass of two U-Box proteins (Os06g01304 and Os12g38210) were consistent with predicted size, while the other two proteins (Os01g66130 and Os08g01900) with apparent molecular mass smaller than that of predicted. Western blotting results indicated that the U-Box proteins were constitutively expressed with close abundance in tested tissues. EST analysis based on I million ESTs derived from 274 libraries from NCBI EST database revealed closed numbers of U-Box gene transcription, which is parallel with the results of constitutively expression of proteins. However, compared with the expression of ATPase, HSP81-3, EGF-1 alpha and RuBisCo, the number of ESTs for U-Box proteins were much lower, suggest the low abundance transcription of U-Box genes. Four rice U-Box proteins were chosen for the generation of specific antibodies via the expression of predicted epitope fragments, demonstrated the feasibility of the process. Western blotting analysis indicated that four U-Box proteins were constitutively expressed among tested rice tissues at different developmental stages, which is parallel with EST sequencing data. The antibodies will provide resources for functional studies, such as co-immunoprecipitation, ChIP-on-chip, Pull-down and stress response etc.