Project description:Small RNAs of 20 to 25 nucleotides in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the small RNA component of more than 40 plant species. Here, use deep sequencing and molecular methods to report the first inventory of small RNAs in olive (Olea europaea). Small RNAs of 24 nts dominate the small RNA transcriptome and atypically accumulate to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. By contrast, small RNAs of 20 to 22 nts were poorly represented in the population at levels lower than those found in most plant species tested. A total of 14 known miRNA families were identified in two libraries prepared from growing and dormant lateral buds. We found that some known miRNAs showed tissue- and/or developmental-specific expression. Also, seven novel, olive-specific miRNA candidates were found in our sequenced set of which 1 were supported by their star strands. Potential precursors for these miRNA candidates with intramolecular folding capacities were found in the olive EST database. Target mRNAs of conserved miRNAs and new olive-specific miRNA were computationally predicted among the olive EST collection and experimentally validated through endonucleolytic cleavage assays.
Project description:The delineation of the olive pollen proteome and its allergogram can improve the clinical management of patients with this pollinosis. We here integrated the recently described wild olive genomic data in a comprehensive proteomic approach to get the annotated olive (Olea europaea) pollen proteome and complete its complex allergogram. Olive pollen proteins were identified by LC-MS/MS using predicted protein sequences from its genome. GO annotation, KEGG Pathway analysis and identification of allergen families were performed by bioinformatics. Recombinant DNA, protein expression and purification, and immunological analyses were used to characterize putative allergens. A total of 1,907 proteins were identified. 60% of the proteins were predicted to possess catalytic activity and be involved in metabolic processes. 203 proteins belonging to 47 allergen families were found, with 37 non-previously described in olive pollen. Of four potential allergens produced in Escherichia coli, a peptidyl-prolyl cis-trans isomerase -cyclophilin-, masked in the protein extract by the major allergen Ole e 1, was found as a new olive pollen allergen (Ole e 15). 63% of the Ole e 15-sensitized patients were children and showed strong IgE recognition of the allergen. Ole e 15 shared high sequence identity with other plant, animal and fungal cyclophilins and a high IgE cross-reactivity with pollen, plant food and animal extracts. Taken together, the combination of available genomic data with proteomics permitted the profiling of the olive pollen proteome, revealing the spectrum of allergen families and cyclophilin as a new relevant allergen implicated in cross-reactivity.