不同绵羊品种发情期卵巢组织lncRNA和mRNA的差异表达分析

Differential expression analysis of lncRNA and mRNA in ovariesof different sheep breeds during estrus

[目的]本文旨在探究不同绵羊品种发情期卵巢组织中长链非编码RNA(long non-coding RNA,lncRNA)和信使RNA(messenger RNA,mRNA)的差异表达,筛选绵羊繁殖力相关的分子标记。[方法]根据羊场生产记录,挑选2~3岁体型、生理状态相似的空怀蒙古羊(Mon)和湖羊(HBB)各4只,对其进行同期发情处理,发情时密集采血3 h(每次隔12 min),检测血清中促卵泡刺激素(FSH)和促黄体生成素(LH)含量;各组随机挑选3只屠宰,采集卵巢组织用于转录组测序(RNA-seq);从NCBI数据库中下载小尾寒羊(HanBB)和陶赛特羊(Dorset)发情期卵巢RNA-seq数据,整合分析不同绵羊品种卵巢中lncRNA和mRNA的表达特征。[结果]湖羊血清中FSH和LH含量均显著高于蒙古羊(P<0.05);RNA-seq共鉴定到87 874个lncRNA转录本;在5个差异分组均获得一定数量的差异lncRNA和mRNA,并从中筛选与繁殖相关的共有和特有lncRNA和mRNA,去除新基因后,分别构建mRNA-mRNA和lncRNA-mRNA互作网络;筛选到MMP2、CHAD、HOXA10、GNB2、COL4A1、ACTB、COL6A3、CREB3L2和LOC101115805等与繁殖相关的共有差异基因;与繁殖相关的共有差异lncRNA主要注释到受精、卵母细胞成熟和配子发生等条目,富集于PI3K-Akt和Wnt等信号通路;关键基因MMP2(matrix metalloproteinases 2)在蒙古羊和湖羊全身组织中表达趋势一致,均在卵巢组织中高表达,且在湖羊垂体、卵巢和子宫组织中均检测到MMP2蛋白阳性信号。[结论]本研究绘制了湖羊和蒙古羊卵巢组织中差异lncRNA和mRNA表达图谱,筛选到与繁殖相关的lncRNA和mRNA,为绵羊繁殖调控及育种分子标记奠定基础。关键基因MMP2可能通过生殖轴调控绵羊的繁殖性能。

[Objectives]This study aimed to explore the differential expression of long non-coding RNA(lncRNA)and messenger RNA(mRNA)in ovaries of different sheep breeds during estrus,and screened molecular markers related to sheep fecundity.[Methods]According to the production records,four nonpregnant Mongolian sheep(Mon)and four Hu sheep(HBB)aged 2-3 years old with uniform body size and similar physiological status were selected and estrous synchronization.During estrous,intensive blood sampling(12 min/time)was performed for three hours continuously to detect the concentrations of follicle stimulating hormone(FSH)and luteinizing hormone(LH)in serum.Three sheep from each group were chosen randomly for slaughter,and the ovarian tissues from those sheep were taken for RNA-seq analysis.Meanwhile,the ovarian RNA-seq data of Small-tailed Han sheep(HanBB)and Dorset sheep were downloaded from NCBI database,and the expression characteristics of lncRNA and mRNA in ovaries of different sheep breeds were analyzed.[Results]The concentrations of FSH and LH in serum of Hu sheep were significantly higher than those in Mongolian sheep(P<0.05).RNA-seq revealed a total of 87874 lncRNA transcripts.Herein,numerous differentially expressed lncRNAs and differentially expressed mRNA,and fecundity-unique and-common differentially expressed-lncRNA and-mRNA were identified among different comparison groups.The mRNA-mRNA and lncRNA-mRNA interaction networks were constructed after the novel genes were removed.Nine common differentially expressed genes related to female reproduction,including MMP2,CHAD,HOXA10,GNB2,COL4A1,ACTB,COL6A3,CREB3L2 and LOC101115805,were identified.The common differentially expressed lncRNA related to reproduction were mainly annotated into fertilization,oocyte maturation and gametogenesis,and were enriched in PI3K-Akt and Wnt signaling pathways.The expression profile of the key gene MMP2 was similar in Mongolian and Hu sheep tissues,and MMP2 expression was significantly expressed in ovarian tissues,with a positive signal of MMP2 protein discovered in the pituitary,ovary and uterus of Hu sheep.[Conclusions]This study mapped differentially expressed lncRNA and mRNA in ovarian tissues of Hu sheep and Mongolian sheep,and reproduction-associated lncRNA and mRNA were screened,laying the groundwork for sheep reproduction regulation and molecular markers for breeding.Among them,the key gene MMP2 might regulate sheep reproductive performance via the reproductive axis,but the specific mechanism need to be investigated further.