山羊卵巢富集chi-miR-100f-5p的鉴定与表达分析

Characterization and Expression Analysis of Goat Ovarian chi-miR-100f-5p

【目的】探讨山羊卵巢中富集的miR-100及预测的靶基因与山羊卵巢功能及卵泡发育的潜在关系,为山羊繁殖机理研究提供数据。【方法】以山羊卵巢组织为材料,通过miRNA的分离、克隆及测序得到山羊卵巢中表达的miR-100,采用RT-qPCR进行表达检测,用生物信息学方法预测miR-100的靶基因及其功能。【结果】①山羊卵巢的测序结果中,一段成熟体为21nt的序列与果蝇dan-miR-100高度同源,将其命名为chi-miR-100f-5p,其前体位于基因组负(-)链上,长度为59 bp,定位于山羊CM000899.1:33 997 894—33 997 914序列;②chi-miR-100f-5p在山羊卵巢、垂体及心脏等组织中均有表达,说明此miRNA不是卵巢组织所特有;心脏和垂体组织中均有较高表达,其次是卵巢,而在肝脏、脾脏、肺及肾脏中表达显著低于卵巢(P<0.01);在单产羔内蒙古绒山羊卵巢中chi-miR-100f-5p的表达极显著高于高繁殖力大足黑山羊卵巢(P<0.01);③chi-miR-100f-5p主要靶作用于整合蛋白β1亚基基因及垂体特异性转录因子1基因,可能参与整合蛋白信号通路,促进垂体内分泌细胞的生长与分化,调节下丘脑-垂体-性腺轴激素分泌。【结论】山羊卵巢中新发现的chi-miR-100f-5p可能是与繁殖调控有关的重要候选miRNAs之一。

[ Objective ] The relationship between the goat ovarian expressed miR-100 with its predicted target genes and goat ovarian function or follicular development was studied in order to provide data for goat breeding mechanism. [Method] Goat ovarian expressed miR-100 was obtained by cloning and sequencing a small RNA library with pooled RNA from goat ovarian tissue. The expression profiling was detected by RT-qPCR. miR-100 target genes and functional classification were predicted by bioinformatics method. [Result] The chi-miR-100f-5p, at a length of 21 nt, was isolated from goat ovaries. The sequence is highly homologous with dan-miR-100, and has one loci in the genome negative （-） chain, which located in goats sequence CM000899.1： 33997894 to 33997914; The chi-miR-100f-5p was detected in goat ovarian, pituitary, heart and other tissues, showing chi-miR- 100f-5p was not an ovarian-specific miRNA. It was found that chi-miRzl00f-5p expressed at higher level in heart and pituitary,secondly in ovary, and the expression of chi-miR-100f-5p in the liver, spleen, lung, and kidney was significantly lower than in ovary （P〈0.01）. It was alsofound that chi-miR-100f-5p expressed at significantly higher level in single lambing Inner Mongolia cashmere goat ovary than the high fecundity Dazu black goat ovary （P^0.01）; The chi-miR-100f-5p was mainly targeted for integrin 131 subunit and pituitary-specific transcription factor 1 gene, and might be involved in the integrin signaling pathway, promoting the growth and differentiation of pituitary endocrine cells, and regulating the hypothalamic-pituitary-gonadal axis hormonessecretion.[Conclusion] The newly discovered chi-miR-100f-5p in goat ovarian may be an important candidate miRNA in regulation of breeding.