In analysing protein expression, the use of the correct reference proteins has a direct impact on the reliability of the results. Thus, the identification of reference proteins and the validation of their application range are quite important. In this study, a set of nine candidate proteins were selected and western blotting analysis was carried out in order to detect their expression in a set of 10 samples representing different rice tissues/organs at different developmental stages. GeNorm and Microcal Origin 6.0 software were used to analyse the expression stability of the tested proteins. The results indicate that HSP and eEF-1α are the most stable rice reference proteins.
GeNorm operates on the principle that the expression ratio of two ideal reference genes is identical in all samples. In other words, changes in the expression ratio of two candidate reference genes indicate a decline in the stability of one or both of the genes. The gene expression stability was ranked by M value, and the two genes with the lowest M value, and therefore the most stable expression, were selected for normalization. This algorithm has been widely used for the identification of reference genes. Microcal Origin 6.0 calculates the minimum, maximum, and median expression level of reference proteins and enables researchers to compare the expression stability visually by examining the extent of variation evident in the box plot. Although the ranks generated by the two approaches differ in this study, both HSP and eEF-1α were shown to be the most stable reference proteins. In addition, both HSP and eEF-1α provided a wide range of applications as they were expressed constantly in rice materials resistant to bacterial blight, taken at different times during a single day, taken from different plants, and taken from different rice varieties.
HSPs are highly conserved among different organisms, and play important roles in protein folding, assembly, transportation, and degradation. Based on their molecular weight, HSPs can be grouped into five families: HSP100, HSP90, HSP70, HSP60, and small HSP (Schmitt et al., 2007
). Because of their importance in eukaryotes, they are usually constitutively expressed and thus are used as internal controls. For example, a member of the HSP60 family has been used as a reference protein for liver tissues and hepatocellular carcinoma (Sun et al., 2009
). The HSP identified as a reference protein in this study has a predicted mol. wt of 94kDa and thus belongs to the HSP90 family. Members of the HSP90 family were reported to be involved in resistance gene-mediated innate immune responses in plants (Hubert et al., 2003
; Takahashi et al., 2003
; Liu et al., 2004
). Most of the HSPs are highly expressed. HSP90α and HSP90β, two major members of this family, account for 1–2% of all cytosolic proteins (Sreedhar et al., 2004
eEF-1α is a protein factor that plays an important role in activating the elongation of amino acid chains in ribosomes and in regulating protein synthesis. eEF-1α is highly expressed and widely present in eukaryotic cells; for example, it accounts for 5% of the total protein in wheat embryos (Browning et al., 1990
). eEF-1α is usually encoded by multiple genes to ensure its sufficient and reliable translation under stress conditions. For example, two copies of eEF-1α are in charge of translation in soybeans (Aguilar et al., 1991
), and four copies are found in both the A. thaliana
(Axelos et al., 1989
) and rice (Kidou and Ejiri, 1998
) genomes. In this study, HSP and eEF-1α proteins were found to be highly expressed in rice, each accounting for 0.12% of total rice proteins.
The western blotting results showed that most of the commonly used housekeeping genes, such as ACT and TUB, displayed considerable expression variability at the translation level. In fact, ACT and TUB have also displayed unstable expression at the transcription level. For instance, a preliminary study showed that the expression of ACT and GAPDH varied up to 2-fold among different cultivars of rice (Jain et al., 2006
). Another study showed that UBQ5 and eEF-1α were the most reliable reference genes for normalization of real-time PCR data in rice, while ACT2 and β-TUB varied considerably in different rice tissues/organs (Zhang et al., 2008
). Recently, 25 novel reference genes have been identified in a systematic analysis of the whole-genome microarray data for the various stages of vegetative and reproductive development in rice (Jain, 2009
). Among the identified novel reference genes, HSP, the best internal control gene in the present study, was ranked as the first candidate. The eEF-1α gene, ranked second (Os03g08010) and fifth (Os03g08050) in the list, have the same amino acid sequence as the gene used in the present study. In the same study, the authors also analysed the relative expression levels of some conventional housekeeping genes, such as ACT and TUB, that have the same locus number as the genes in the present study. All these genes exhibited highly variable expression patterns in various developmental stages, a result consistent with the findings of the present study. Another systematic analysis of the genome-wide dynamic transcriptome throughout the life cycle of rice plants also identified 19 novel internal control genes (Wang et al., 2010
). Among them, the UBQ (Os06g46770) ranked ninth has the same locus number as the gene used in the present study, and also had stable expression at the translation level. The qualitative comparison among protein expression, MPSS, and EST data also showed that HSP and eEF-1α were uniformly expressed during both translation and transcription, while ACT and TUB varied at these levels. These results provided further evidence for the existence of a correlation between transcription and translation.
Most of the antibodies generated in this study showed acceptable specificity and sensitivity. The lower limits of detection for HSP and eEF-1α in rice are ~0.24ng and 0.06ng, respectively. In order to obtain more uniform antibodies with stable performance, the recombinant HSP protein was used to generate a monoclonal antibody. The results of the western blotting analysis demonstrated the consistency of monoclonal and polyclonal antibodies. The monoclonal HSP antibody routinely used in our laboratory showed stable performance (data not shown). In addition, the UBQ antibody can detect a ladder at higher molecular weight positions, and thus may be used to detect protein ubiquitination if the specificity can be validated.
Western blotting is widely performed to reveal the protein expression profile and the function of target proteins, a method that has been considered as an antibody-based proteomics (AbP) strategy (Uhlen and Ponten, 2005
; Uemura et al., 2009
). Compared with the conventional 2DE-MS-based strategy, AbP is easy to perform, more sensitive, and provides intuitive and quantitative results. By using this strategy, researchers can systematically investigate the target protein or protein families involved in a specific biological process. With the accumulation of rice protein-specific antibodies, the AbP strategy will be applied in more laboratories, contributing to a larger scope to the interpretation of the function of proteins and the mechanisms of biological processes.