Identification And Validation Of Reference Proteins In Rice

Background: With the completion of the Rice Genome Project, the next step forward is to define the biological function of target proteins. In order to compare changes in translational and transcript level, appropriate internal reference control for data normalization is needed to correct for variability resulted from the various steps of the experimental procedure, such as differences in amount of sample loaded. This renders the identification of reference proteins highly significant in biology studies. Ideally, a "good" housekeeping gene is expected to show a constant level of expression presence across all tissue samples and various experimental designs throughout many biological contexts.However, the housekeeping genes used in research were all evaluated under specific conditions and confined to looking at gene expression at mRNA level. To date, the expression stabilities of reference protein markers have not yet been examined systemically in rice.Aim: The generation of specific antibodies against reference proteins of rice will help identify the internal reference control in rice translational research and investigate the expression profile of reference proteins at different developmental stages.Materials and methods: A set of 9 candidate genes were selected. Epitopes were predicted and the immunogens were got by the means of the proteins expressed in E. coli. system or peptide synthesized. Antibodies were generated by rabbit immunization and 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. GeNORM and Microcal Origin 6.0 software were used to analyze the expression stability of tested genes in various tissue samples and ranked them accordingly. The expression stability of the reference proteins was further tested in tissues of circadian rhythm and tissues inoculated with Xoo. Comparison analysis between the profiling of gene transcription and translation was carried out. The concentrations of reference proteins in rice were calculated and the lowest detection limit of reference in rice was determined.Results: The housekeeping genes were cloned and their specific antibodies were obtained by rabbit immunization of recombinant proteins expressed in E. coli system or peptide synthesized. Western blotting results indicated that the reference proteins were constitutively expressed in tested tissues except UBC,TUB and eIF-4a. HSP and eEF-1a were determined as the most stable reference proteins across different developmental stages of rice by geNORM and Microcal Origin 6.0 software. Further study found that the expression levels of HSP and eEF-1a in the tissues inoculated with Xoo and tissues of circadian rhythm did not change, demonstrating the stability of these two reference proteins. Comparison analysis between the profiling of gene transcription and translation proved that the HSP and eEF-1a proteins are the best internal reference genes in both translational and transcript levels. Using the linear region of standard curves, the concentration of HSP and eEF-1a proteins in rice was about 0.10%. The detection limit of the reference protein HSP and eEF-1a in rice was about 0.24ng and 0.06ng.Conclusions: The protein HSP and eEF-1a were determined as the internal reference proteins suitable for the protein research in rice. Using standard curves generated, the concentration of HSP and eEF-1a proteins in rice and the lowest detection limit of them in rice were calculated. The antibodies will also provide resources for functional studies, such as co-immunoprecipitation, ChIP-on-chip, Pull-down and stress response etc.