水稻U-Box蛋白质在不同发育时期的表达分析

Expression Analysis of Rice U-Box Proteins at Different Developmental Stages

真核生物基因组中广泛存在U-Box基因,其编码蛋白大部分是泛素系统中决定底物特异性识别的E3蛋白,其构象与RING-finger极其相似.U-Box蛋白质能促进底物蛋白泛素化降解,对细胞内异常蛋白的降解及质量控制方面发挥着重要的作用.水稻基因组中有77个U-Box蛋白质,系统了解它们的表达可为功能研究提供数据.制备针对水稻U-Box蛋白质的抗体,了解水稻中U-Box蛋白质在不同发育时期的表达信息,为功能研究积累数据.选取了4个水稻U-Box蛋白质,其共同结构特点为U-Box结构在N端,C端有ARM结构.用计算机软件预测抗原决定簇,细菌体系体外表达、纯化U-Box蛋白质的片段,免疫动物制备多克隆抗体,用Westernblotting检测U-Box蛋白质在水稻品种93-11苗期地上部和地下部、分蘖期根和茎、孕穗期剑叶和幼穗、开花期剑叶和穗子、成熟期剑叶和种子中的表达,并与EST数据库中公布的U-Box蛋白质EST数据进行了比较分析.体外克隆表达后,获得了纯化的蛋白质,制备的抗体特异性强,蛋白质印迹(Westernblotting)检测可见一条明显的主带,其中Os06g01304和Os12g38210两个蛋白质的表观分子质量与预测分子质量相符,Os01g66130和Os08g01900两个蛋白质的表观分子质量低于预测分子质量.4个U-Box蛋白质在水稻生长发育的不同时期或部位基本上是组成型表达,且表达量接近.对NCBI上公布的来自274个文库100万条以上的EST进行分析,可以看出4个U-Box蛋白质EST的数量分布大致均匀,与Westernblotting结果揭示的组成型表达平行,与ATPase、HSP81-3、EGF-1alpha和RuBisCo等对照基因相比,U-Box基因的EST数目相对很少,说明它们属于低丰度转录的基因.选取了4个水稻U-Box蛋白质,通过抗原决定簇预测,表达片段蛋白,制备了特异性抗体,证明了这一技术路线的可行性.利用抗体对水稻不同发育时期材料进行蛋白质表达谱研究,发现这些U-Box蛋白质呈组成型表达,与EST数据揭示的结果具有平行性.所制备的抗体也为相关功能研究,如免疫共沉淀、ChIP-on-chip、Pull-down以及在抗病、抗逆反应中U-Box蛋白质的表达等,积累了资源.

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. coli. 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 panicle at heading stage, flag leaf and panicle 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. coll. 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 1 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.