Expression Analysis Of Rice U-Box Protein At Different Developmental Stages

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 generation of specific antibodies against rice U-Box proteins to investigate the expression profile of U-Box proteins at different developmental stages and accumulate basic information for functional studies.Materials and methods:Five rice U-Box genes were chosen as their U-Box domains were 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 the 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.The U-Box genes were cloned.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 weight of two U-Box proteins(os06g01304 and osl2g38210) were consistent with predicted size, while the other three proteins(os01g66130,os02g49950and os08g01900) with apparent molecular weight 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.Conclusions:Five 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.