For sensitive and accurate gene expression analysis, normalization of gene expression data against housekeeping genes is required. There are conventional housekeeping gene (e.g. ACT) that primarily function as an internal control of transcription. In this study, we performed an in silico analysis of 278 rice gene expression samples (GSM) in order to identify the gene that is most consistently expressed. Based on this analysis, we identified novel candidate housekeeping genes that displayed improved stability among the cross experimental conditions. Furthermore four of the most conventional housekeeping genes were included in our 30 other housekeeping genes among the most stable genes. Therefore, these 30 genes can he used to normalize transcription results in gene expression studies on rice at a broad range of experimental conditions.

TILLING (Targeting Induced Local Lesions IN Genomes) is broadly regarded as an excellent methodology for reverse genetics applications. Approximately 15,000 M3 TILLING lines have been developed via the application of gamma-ray irradiation to rice seeds (cv. Donganbyeo), followed by subsequent selections. In an effort to evaluate the genetic diversity of the TILLING population, we have employed the AFLP multiple dominant marker technique. A total of 96 (0.64%) TILLING lines as well as Donganbyeo were selected randomly and their genetic diversity was assessed based on AFLP marker polymorphisms using 5 primer combinations. An average of 100.4 loci in a range of 97 to 106 was detected using these primer combinations, yielding a total of 158 (31.4%) polymorphic loci between Donganbyeo and each of the 96 lines. A broad range of similarity from 80% to 96% with an average of 89.4% between Donganbyeo and each of the 96 lines was also observed, reflecting the genetic diversity of the TILLING population. Approximately 28 polymorphic loci have been cloned and their sequences were BLAST-searched against rice whole genome sequences, resulting in 20 matches to each of the gene bodies including exon, intron, 1 kb upstream and 1 kb downstream regions. Six polymorphic loci evidenced changes in the coding regions of genes as compared to the rice pseudomolecules, 4 loci of which exhibited missense mutations and 2 loci of which exhibited silent mutations. Therefore, the results of our study show that the TILLING rice population should prove to be a useful genetic material pool for functional genomics as well as mutation breeding applications.

MYB proteins are a superfamily of transcription factors (TF) that play regulatory roles in developmental processes and resistance mechanism in plants. We identified 130 and 109 genes in the MYB superfamily from an analysis of the complete Arabidopsis and rice genome sequence. Although microarray based transcriptome analysis approach allows the investigation of the biological networks of MYB TF in DNA level, the underling mechanisms related to their functional role is not fully understood. In this work, we performed meta-analysis of public microarray data that analyzed with Arabidopsis and rice using co-expression analysis. A phylogenetic comparison of the members of this superfamily were performed with Sorghum bicolour to suggested that MYB super family underwent a rapid expansion their evolutionary times. We identified conserved expression pairs which play important role in transcription. Our comprehensive analysis of this huge transcription factor of Arabidopsis and rice may shed further light on the possible biological roles of the MYB TF in various plants.

TILLING (Targeting Induced local Lesions IN Genomes) is known to be an excellent methodology for reverse genetics approach. About 15,000 M3 TILLING lines have been developed after gamma-ray irradiation to the rice seeds of Donganbye. In order to assess genetic diversity of the TILLING population. we have employed a multiple dominant marker technique, such as AFLP. A total of 96 (0.64%) lines including Dongganbye were randomly selected and their genetic diversity was assessed on the basis of AFLP marker polymorphism by using 5 primer combinations. An average of 100.4 loci with a range of 97 to 106 were detected by using the primer combinations, resulting in 173 (34.6%) polymorphic loci among 96 lines. A broad range of similarities with 80% to 96% was evidenced between Donganbye and each of 96 TILLING lines, reflecting genetic diversity of the TILLING population. About 30 polymorphic loci have been cloned and their sequences have been blasted against rice whole genome sequences. The sequences evidenced highly significant matches to each of genes including exons and introns, upstream sequences and downstream of genes, and intergeneic sequences. Therefore, the TILLING rice population would be valuable genetic sources for rice functional genomics.

All aspects of plant life are controlled by the regulated synthesis of new proteins and the precise degradation of preexisting proteins, predicting up to 50% of total plants protein is replaced every week. The ubiquitin/26S proteosome pathway is known to be one of mechanisms to regulate signal pathways, developmental process and abiotic/biotic stress responses via protein degradations. In the previous study, we have identified a large number of the RING ubiquitin ligase proteins whose functions have been clarified in the protein degradation pathway. Curiously, one RING-H2 finger protein gene evidenced striking differences in expression patterns in response to salt and dehydration stress between leaf and culm-node tissues. Characterization of the gene evidenced its function as E3 ubiquitin ligase activity by using an in vitro ubiquitination assay. We have constructed a library with rice culm-node tissues under salt stress for Yeast two hybrid assay and performed primary yeast two-hybrid screening with the gene as a bait. A total of 13 candidate genes were isolated as positive interacting partners. Gene ontology of most candidate genes appears to be related with various abiotic stresses. Therefore, the RING-H2 finger protein genes might function to regulate plant abiotic stress responses via protein degradation pathways.

Rapid extension of genomic database leads to the remarkable advance of functional genomics. This study proposes a novel methodology of functional analysis using 5-methyltrytophan (5 MT) mutant together with their 2-DE analysis and public microarray database. A total of 24 proteins was changed in 5 MT mutant and four remarkably different expressed proteins were identified. Among them, three spots were converted to Affymetrix probe. A total of 155 microarray samples from Gene Expression Omnibus (GEO) in NCBI was retrieved and followed by constructing gene co-expression networks over a broad range of biological issues through Self-Organising Tree Algorithm. Three co-expressing gene clusters were retrieved and each functional categorization with differential expression pattern was exhibited from 5 MT resistance mutant rice. It was indicated new co-expression networks in the mutant. This study suggests that on investigating possibility which correspond 2-DE to microarray database with their full potential.

Previously, the wheat non-specific lipid transfer proteins (TaLTP), members of a small multigene family, appear to show a complex pattern of expression regulation. For further assessment of expression diversity of the TaLTP genes, we have attempted to evaluate their expression profiles of responses to abiotic stresses via the semi-quantitative RT-PCR method. The expression profiles revealed that the TaLTP genes in group A evidenced highly similar (but not identical) responses against abiotic stresses, whereas much differential expression pattern among genes in each group. The four promoters of TaLTP1, TaLTP7, and TaLTP10 of group A and TaLTP3 of group B were fused to a GUS reporter gene and the recombinant genes were introduced into Arabidopsis. The promoters of TaLTP1, TaLTP7 and TaLTP10 of group A, drove strong but various GUS expression in cotyledons, hypocotyls, epidemic and sub-epidemic cells of young shoots and leaves, floral organs as well as siliques. By contrast, the promoter of TaLTP3 just directed trace expression in cotyledons, young emerged leaves and epidemic cells of flower ovaries. The promoter of TaLTP1 directed the expression in root system whereas the promoters of TaLTP1 and TaLTP10 showed some degree of expression during seed development. The expression diversity of TaLTP genes suggests their multiple physiological functions, evidencing subfunctionalization over evolutionary time.

The C3HC4 zinc RING finger proteins seem to be a family of protein-protein interactions. Little is information regarding the role of the C3HC4 zinc RING finger proteins in rice plant. We have attempted to assess their genome localization, phylogenetic relationship and expression patterns of members via in silico analysis as well as semi-quantitative RT-PCR. A total of 132 genes encoding C3HC4 zinc RING finger proteins appear to be distributed over 12 rice chromosomes, reflecting evolutionary dynamics of the rice genome, e.g. whole genome duplication and tandem duplications. A genome-wide dataset including 155 gene expression omnibus sample (GSM) plates evidenced a high degree of functional specialization of the rice C3HC4 zinc RING finger proteins, especially during developmental stages and against abiotic stresses. We have retrieved co-expression genes with each of the rice C3HC4 zinc RING finger proteins, probably providing some clues on specialized functions of individual genes. Expression patterns of 13 co-expression genes with one gene encoding C3HC4 zinc RING finger protein (Os04g51400) against salt and dehydration stresses were evaluated in crown tissues and leaf tissues, evidencing highly similar patterns among members. These findings might provide clues to shed further light on comprehensive functions of C3HC4 zinc RING finger proteins.

Most gene functions of biochemical pathways were still unexplored, especially interactions of constituent genes. We attempted to uncover interaction network of biochemical pathways via a survey of co-expression clusters, which we have constructed from the NCBI GEO database, and then to define key genes of networks with expression correlations between members. Top 20 pathways with high numbers of individual genes were retrieved from 178 pathways. One pathway, ‘removal of superoxide radicals’ was excluded for further study, evidencing somewhat low degree (16%, 13 out of 79 genes) of mapped probes. We employed expression correlations of random pairs of 1,000 randomly selected genes for determining a cut off r-value for gene networks. Numbers of interactions with a significant expression correlation values between members might evidence that “hub genes” play key roles among a given pathway genes. For example most interactive pathway, ‘tRNA charging pathway’, that is composed of 60 probes corresponding to genes showed 264 positive significant interactions between members of 47 genes while 5 negative interactions between members of 7 genes., evidencing ‘Os10g26050’ (methionyl-tRNA synthetase) gene with highest interactions is suggestive of a hub gene. These findings might provide some clues on evolutionary fate of co-expression genes including each of biochemical pathways, e.g. convergent evolution