鸡胚性分化早期性别差异表达miRNAs的鉴定及其特性分析
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摘要
miRNA是近年来新发现的一类在转录后水平下调基因表达的小分子非编码RNA家族。研究发现,IniRNA在动物发育过程中发挥着重要作用,但它在鸡胚性腺分化中的调控作用还未确定。为研究miRNA在鸡胚早期性分化过程中的表达,探讨miRNA在性腺早期发育中的作用,本研究以前期芯片筛选出的差异表达miRNAs为研究对象,对其中新的miRNAs进行生物信息学分析,并分析了候选miRNAs在雌、雄鸡胚早期性腺中的时空表达模式。本研究所取得的结果如下：
1.利用鸡基因组信息对新miRNAs进行定位,发现miR-363和363*、miR-551b和miR-612分别位于鸡染色体4(-)、9(-)和Z(-)上,同时通过克隆获得了miR-363*在鸡基因组上的未知侧翼序列。前体的二级结构折叠结果表明,预测的鸡新miRNAs均具有茎环状前体结构,成熟序列基本位于相应的茎部,且折叠最低自由能均≤-25kcal/mol。miR-363 (363*)和miR-551b在人、大鼠、小鼠和鸡4个物种中的同源性分析结果表明,前体序列的相似性均在80%以上,且成熟序列中仅有个别碱基的差异。
2.利用RNA加尾及引物延伸和茎环状引物的半定量RT-PCR方法分析了13个miRNAs在E3.5-6.5 d雌、雄鸡胚性腺中的时间表达谱,并对它们在同一时期不同性别中以及同一性别不同时期中的差异性进行了显著性分析。结果表明,从总体趋势看,所检测miRNAs中有5个呈雌性高表达和4个呈雄性高表达,其余4个为不规则表达；miR-363、miR-200b、miR-1810、miR-1599、miR-551b和miR-196在所检某些时期两性间的差异表达达到显著或极显著水平；miR-363、miR-1456、miR-196、miR-363*、miR-612、miR-1599、miR-126和miR-1810在同一性别某些时期间的表达存在显著或极显著差异。
3.利用WISH技术对miR-363和miR-1810进行空间表达模式分析,结果表明,miR-363主要在E4.5 d鸡胚的四肢、外胚层、脊索以及脑等部位表达,在E6.5 d性腺部位的表达明显高于肾脏,且在雌性性腺中的表达高于雄性；miR-1810主要在E4.5 d鸡胚的脑和四肢表达,在E6.5及E8.5 d性腺中表达高于肾脏,且雄性性腺中的表达高于雌性。
结合本研究结果及其它物种中相应miRNA的研究,筛选了5个(miR-363、miR-551b、miR-1810、miR-1599和miR-200b)可能参与早期鸡胚性腺发育过程的miRNAs作为下一步研究的对象。
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.
1.利用鸡基因组信息对新miRNAs进行定位,发现miR-363和363*、miR-551b和miR-612分别位于鸡染色体4(-)、9(-)和Z(-)上,同时通过克隆获得了miR-363*在鸡基因组上的未知侧翼序列。前体的二级结构折叠结果表明,预测的鸡新miRNAs均具有茎环状前体结构,成熟序列基本位于相应的茎部,且折叠最低自由能均≤-25kcal/mol。miR-363 (363*)和miR-551b在人、大鼠、小鼠和鸡4个物种中的同源性分析结果表明,前体序列的相似性均在80%以上,且成熟序列中仅有个别碱基的差异。
2.利用RNA加尾及引物延伸和茎环状引物的半定量RT-PCR方法分析了13个miRNAs在E3.5-6.5 d雌、雄鸡胚性腺中的时间表达谱,并对它们在同一时期不同性别中以及同一性别不同时期中的差异性进行了显著性分析。结果表明,从总体趋势看,所检测miRNAs中有5个呈雌性高表达和4个呈雄性高表达,其余4个为不规则表达；miR-363、miR-200b、miR-1810、miR-1599、miR-551b和miR-196在所检某些时期两性间的差异表达达到显著或极显著水平；miR-363、miR-1456、miR-196、miR-363*、miR-612、miR-1599、miR-126和miR-1810在同一性别某些时期间的表达存在显著或极显著差异。
3.利用WISH技术对miR-363和miR-1810进行空间表达模式分析,结果表明,miR-363主要在E4.5 d鸡胚的四肢、外胚层、脊索以及脑等部位表达,在E6.5 d性腺部位的表达明显高于肾脏,且在雌性性腺中的表达高于雄性；miR-1810主要在E4.5 d鸡胚的脑和四肢表达,在E6.5及E8.5 d性腺中表达高于肾脏,且雄性性腺中的表达高于雌性。
结合本研究结果及其它物种中相应miRNA的研究,筛选了5个(miR-363、miR-551b、miR-1810、miR-1599和miR-200b)可能参与早期鸡胚性腺发育过程的miRNAs作为下一步研究的对象。
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.
引文
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