[show abstract][hide abstract] ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are 20-21 nucleotide RNA molecules that suppress the transcription of target genes and may also inhibit translation. Despite the thousands of miRNAs identified and validated in numerous plant species, only small numbers have been identified from the oilseed crop plant Brassica napus (canola) - especially in seeds. RESULTS: Using next-generation sequencing technologies, we performed a comprehensive analysis of miRNAs during seed maturation at 9 time points from 10 days after flowering (DAF) to 50 DAF using whole seeds and included separate analyses of radicle, hypocotyl, cotyledon, embryo, endosperm and seed coat tissues at 4 selected time points. We identified more than 500 conserved miRNA or variant unique sequences with >300 sequence reads and also found 10 novel miRNAs. Only 27 of the conserved miRNA sequences had been previously identified in B. napus (miRBase Release 18). More than 180 MIRNA loci were identified/annotated using the B. rapa genome as a surrogate for the B.napus A genome. Numerous miRNAs were expressed in a stage- or tissue-specific manner suggesting that they have specific functions related to the fine tuning of transcript abundance during seed development. miRNA targets in B. napus were predicted and their expression patterns profiled using microarray analyses. Global correlation analysis of the expression patterns of miRNAs and their targets revealed complex miRNA-target gene regulatory networks during seed development. The miR156 family was the most abundant and the majority of the family members were primarily expressed in the embryo. CONCLUSIONS: Large numbers of miRNAs with diverse expression patterns, multiple-targeting and co-targeting of many miRNAs, and complex relationships between expression of miRNAs and targets were identified in this study. Several key miRNA-target expression patterns were identified and new roles of miRNAs in regulating seed development are suggested. miR156, miR159, miR172, miR167, miR158 and miR166 are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development. miR156 may regulate the developmental transition to germination.
[show abstract][hide abstract] ABSTRACT: Transcriptome profiling was conducted to detect genes whose expression is significantly changed in an Arabidopsis mutant deficient in S-adenosylhomocysteine hydrolase1 (SAHH1) during early seedling development when mutant phenotypes could be clearly observed. A total of 2,040 differentially expressed genes were identified, representing approximately 6.7% of the 30,385 DNA oligonucleotide targets on the microarray. Among these differential expressed genes, many were mapped to pathways essential to plant growth and development including those of primary, secondary and hormone metabolisms. A significant proportion of up-regulated genes encoded transposable elements which were mapped to the centromeric and pericentromeric regions of the Arabidopsis chromosomes that were analyzed. A number of down-regulated genes were found to be involved in root hair formation, which might have contributed to the root hair defective phenotype of the mutant. Analysis of genes encoding transposable elements and those associating with root hair development indicated that these genes were highly co-expressed during seedling development. Despite SAHH1 deficiency, the expression of genes encoding methyltransferase remained largely unchanged in the sahh1 mutant. Bisulfite sequencing analysis of the transposable elements and the FWA gene revealed that their sequences in the mutant were deficient of 5-methylcytosines. Analysis of mutant genomic DNA using restriction endonucleases that were unable to cut methylated DNA suggested a genome-wide hypomethylation had occurred in the mutant. These results indicated that SAHH1 plays a critical role in methyl homeostasis, and its deficiency is a major contributing factor to the change of global gene expression, metabolic pathways and activation of transposable elements in the sahh1 mutant.
[show abstract][hide abstract] ABSTRACT: Computational gene regulation models provide a means for scientists to draw biological inferences from time-course gene expression data. Based on the state-space approach, we developed a new modeling tool for inferring gene regulatory networks, called time-delayed Gene Regulatory Networks (tdGRNs). tdGRN takes time-delayed regulatory relationships into consideration when developing the model. In addition, a priori biological knowledge from genome-wide location analysis is incorporated into the structure of the gene regulatory network. tdGRN is evaluated on both an artificial dataset and a published gene expression data set. It not only determines regulatory relationships that are known to exist but also uncovers potential new ones. The results indicate that the proposed tool is effective in inferring gene regulatory relationships with time delay. tdGRN is complementary to existing methods for inferring gene regulatory networks. The novel part of the proposed tool is that it is able to infer time-delayed regulatory relationships.
EURASIP Journal on Bioinformatics and Systems Biology 01/2009;
[show abstract][hide abstract] ABSTRACT: Brassica species represent several important crops including canola (Brassica napus). Understanding of genetic elements that contribute to seed-associated functions will impact future improvements in the canola crop. Brassica species share a very close taxonomic and molecular relationship with Arabidopsis thaliana. However, there are several subtle but distinct seed-associated agronomic characteristics that differ among the oil seed crop species. To address these, we have generated 67,535 ESTs predominately from Brassica seeds, analyzed these sequences, and identified 10,642 unigenes for the preparation of a targeted seed cDNA array. A set of 10,642 PCR primer pairs was designed and corresponding amplicons were produced for spotting, along with relevant controls. Critical quality control tests produced satisfactory results for use of this microarray in biological experiments. The microarray was also tested with specific RNA targets from embryos, germinating seeds, and leaf tissues. The hybridizations, signal intensities, and overall quality of these slides were consistent and reproducible. Additionally, there are 429 ESTs represented on the array that show no homology with any A. thaliana annotated gene or any gene in the Brassica genome databases or other plant databases; however, all of these probes hybridized to B. napus transcripts, indicating that the array also will be useful in defining expression patterns for genes so far unique to Brassica species.