营养诱导舍饲哈萨克羊非繁殖季节卵巢组织中miRNAs差异表达分析

摘要

旨在利用Solexa测序技术分析、挖掘营养诱导非繁殖季节绵羊发情期和乏情期卵巢组织micro RNAs(miRNA),为进一步研究特定miRNA参与非繁殖季节绵羊发情的分子调控机制提供理论基础.以不同营养水平饲喂的哈萨克绵羊作为研究对象,在非繁殖季节前后分别屠宰已鉴定的3只发情(EN)和乏情(AN)母羊,分别采集卵巢(O)组织,构建两个小片段RNA文库(OEN和()AN),进行高通量测序和生物信息学分析,实时荧光定量PCR(qRT-PCR)验证差异表达的miRNAs在哈萨克羊卵巢中表达水平.结果显示,两文库分别获得8 665 978(发情期)和9 071 102条(乏情期)clean reads,且绝大多数小RNA序列长度为21～23 nt.两文库相比较,发现有9个已知和104个未知miRNAs表达差异显著(P＜0.01).利用qRT-PCR随机选择的6个显著差异表达的miRNAs进行验证,其表达水平和测序分析结果一致.KEGG通路分析表明,这些差异miRNAs的靶基因主要参与了甘氨酸、丝氨酸和苏氨酸代谢,氨基糖和核苷酸糖代谢,精氨酸和鸟氨酸代谢及甘油磷脂代谢等营养调控通路.结合靶基因预测及通路富集分析,推测差异表达的miRNAs是通过营养代谢通路对绵羊的非季节性发情起重要作用.%To further explore the theoretical basis of the microRNAs in regulating the non-breeding season estrous in sheep,characterization of ovarian microRNAs were constructed and analyzed in estrous and anestrous of non-breeding season induced by nutrition by Solexa sequencing.In this study,the Kazakh sheep with different nutrition levels were used as the research object.In non-breeding season,3 estrous (EN) and 3 anestrous (AN) sheep were slaughtered with their ovary tissue(O) collected,respectively.Two miRNA libraries were constructed and analyzed by the bioinformatics methods.The real-time quantitative PCR (qRT-PCR) was used for validating the differentially expressed miRNAs betweent the 2 periods in ovary.The results showed that the clean reads of 8 665 978 (OEN) and 9 071 102 (OAN) were obtained in the two libraries,and most of the small RNA sequences were between 21 and 23 nt.Compared the two ovary (OEN vs OAN) libaries,the 9 known and 104 novel miRNAs had significant expression difference (P＜0.01).The expression level of 6 randomly selected miRNAs by qRT-PCR was the same as the result by Solexa sequencing.KEGG pathway analysis showed that these target genes of difference miRNAs were primarily involved in nutrition regulatory related pathways,such as glycine,serine and threonine metabolism,amino sugar and nucleotide sugar metabolism,D-arginine and D-ornithine metabolism,glycerol phospholipid metabolism,and so on.The differentially expressed miRNAs may play an important role in non-breeding season estrous of sheep through nutrition metabolism pathway by the analysis of predicted target and enriched pathway analysis.