| MiRNA is a group of recently discovered, small, non-coding RNA, which down-regulate gene expression at the post-transcriptional level. MiRNAs have been proved to play important roles during the process of development, but its function on the chicken sexual differentiation is uncertain. In order to investigate the expression during the early stages of gonad sexual differentiation and explore the effect of miRNA on gonad development in chicken, a body of miRNAs with sexual differentially expression were preliminary selected based on the results of miRNA chips, in which the novel miRNAs were analyzed by bioinformatics methods, and the temporal and spatial expression patterns of candidated miRNAs in female and male gonads of chicken embryo were studied. The results obtained were as follows:1. The chromosomes mapping of new miRNAs were analyzed through chicken genome information, miR-363 and 363*, miR-551b and miR-612 were mapped to the 4 (-),9 (-) and Z (-) and the uknown flanking sequence of miR-363* in chicken genome was obtained by cloning. Secondary structure analysis of precursor showed that all predicted chicken novel miRNAs possessed stem-loop precursor structures, mature sequences located in the stems of the corresponding hairpins, and the lowest free energy was≤-25 kcal/mol. Homology analysis among human, rat, mouse and chicken suggested that the similarities of precursor sequence were all above 80%, and only one or two bases difference existed in mature sequences.2. Temporal expression profiles of 13 miRNAs in female and male chicken embryo gonads at E3.5～6.5 d were studied by RNA tailing and primer extension and stem-loop primer RT-PCR methods, and significant analysis between different sexes at the same stage and different stages in the same sex were carried out. The results indicated that 5 miRNAs with higher expression in femal,4 miRNAs with higher expression in male and 4 miRNAs with scrambled expression were gained. Differential expressions of miR-363, 200b,1810,1599,551b and 196 between both sexes at centain stages reached significant and (or) extremely significant levels and the expressions of miR-363,1456,196,363*, 612,1599,126 and 1810 between some different stages in the same sex existed significant and (or) extremely significant differences.3. Spatial expression pattern analysis of miR-363 and 1810 were done by WISH, the results showed that the expression signals of miR-363 appeared primarily at limb buds, ectoderm, notochord and brain in E4.5 d chicken embryo, and obviously higher in gonads than surrounding kidneys with higher expreesion in female gonads than that in male in E6.5 d UGSs. MiR-1810 expressed mainly at brain and limb buds in E4.5 d chicken embryo, and higher in gonads than surrounding kidneys with higher expression in male gonads than that in female in E6.5 d and 8.5 d gonads.Combined our results in present study with studies of corresponding miRNA in other species, 5 miRNAs (miR-363, miR-551b, miR-1810,1599 and 200b) which might be involved in the process of early chicken embryonic gonadal development were screened as targets for further research. |
