乏情期发情绵羊卵巢miRNA及相关靶基因筛选  

Screening and preliminary verification of miRNA related to estrus in sheep during the non-breeding season

作　　者：谢梦婷 解一凡 朱梦婷[1] 南颖 方晨辉 江白慧 齐行东 赵宗胜[1] XIE Mengting;XIE Yifan;ZHU Mengting;NAN Ying;FANG Chenhui;JIANG Baihui;QI Xingdong;ZHAO Zongsheng(College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang 832000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000

出　　处：《石河子大学学报（自然科学版）》2023年第5期545-554,共10页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(32160770)。

摘　　要：目的 筛选、鉴定绵羊卵巢差异MicroRNA(miRNA)并研究其调控机制,为绵羊非繁殖季节发情的研究奠定基础。方法 使用Solexa测序技术进行筛选和分析,对发情和乏情绵羊卵巢,差异miRNA的数量和特征进行鉴定,通过实时荧光定量PCR进行检测。还进行了对miR-200c靶基因检测、GO注释和KEGG信号通路富集等的研究工作。并通过TargetScan技术、RNAhybrid技术等,成功找到了MAPK8-3′端非翻译区(3′UTR)与miR-200c的潜在互补结合位点,PCR扩增MAPK8(丝裂原活化蛋白激酶8)基因的3′UTR序列,并成功实现了在psiCHECK2的克隆,成功建立了MAPK8野生型/突变型重组的双荧光素酶报告质粒。接着把野生型psiheck2-MAPK8-3′UTR/突变型psiheck2-MAPK8-mut3′UTR的质粒,与miR-200c mimics/mimics-NC,转染至HEK 293T细胞中,从而通过双荧光素酶的系统,测定双荧光素酶的生物活性。结果 建立了OAN(乏情)和OEN(发情)非繁殖季节绵羊卵巢文库。鉴定出113个差异miRNA,9个为已知miRNA,104个为未知miRNA。在已知miRNA中有6个miRNA显著(P<0.05)上调、3个下调,其中上调倍数最高的为miR-200c。在104个未知的miRNA中,有52个miRNA显著(P<0.05)上调,52个显著(P<0.05)下调。获得候选新miRNA的长度分布主要集中在21~23 nt之间。荧光定量证实,miRNA表达的趋势与测序结果一致。用软件预测到miR-200c的27个靶基因。GO注释发现miR-200c在卵巢组织中介导细胞增殖、迁移、凋亡等过程。KEGG分析表明,miR-200c的靶基因涉及发情相关途径(MAPK信号途径、胰岛素信号通路和GnRH信号途径)以及与卵泡/黄体发育相关的途径,MAPK通路是富集基因最多的通路,共有25个基因被富集。干扰miR-200cmimics,会引起MAPK8基因的Wt型质粒的荧光表达明显减少(P<0.05),而Mut型则没有明显改变。结论 筛选出非繁殖季节发情差异显著(P<0.05)的miRNA有6个。试验研究初步证实了miR-200c与MAPK8的靶向关系,开展了miRNA家Objective Screening and identification of microRNAs(miRNAs)in sheep ovaries and studying their regulatory mechanisms laid the foundation for the study of sheep estrus in the non-breeding season.Methods Screening and analysis using Solexa sequencing technology to identify the number and characterisation of differential miRNAs,by real-time fluorescence quantitative PCR,in oestrus and estrus sheep ovaries.Work was also carried out on miR-200c target gene detection,GO annotation and enrichment of the KEGG signalling pathway.The potential complementary binding site of MAPK8-3′-end untranslated region(3′UTR)and miR-200c was suc-cessfully found by TargetScan technology and RNAhybrid technology,and the 3′UTR sequence of MAPK8(mitogen-activated protein kinase 8)gene was PCR amplified and cloning in psiCHECK2 was successfully achieved,and a dual luciferase reporter plasmid for MAPK8 wild-type/mutant recombination was successfully established.The plasmid of wild-type psiheck2-MAPK8-3′UTR/mutant psi-heck2-MAPK8-mut3′UTR,with miR-200c mimics/mimics-NC,was then transfected into HEK 293T cells,thereby measuring the bi-ological activity of the dual luciferase by a dual luciferase system.Results A library of OAN(absence)and OEN(estrus)non-breed-ing season sheep ovaries was established.113 differential miRNAs were identified,9 were known miRNAs and 104 were unknown miR-NAs.Six of the known miRNAs were significantly(P<0.05)up-regulated and three were down-regulated,with the highest up-regula-tion fold being miR-200c.Of the 104 unknown miRNAs,52 were significantly(P<0.05)up-regulated and 52 were significantly(P<0.05)down-regulated.The length distribution of the obtained candidate new miRNAs was mainly concentrated in the range of 21~23 nt.Fluorescence quantification confirmed that the trend of miRNA expression was consistent with the sequencing results.The software was used to predict the 27 target genes of miR-200c.GO annotation revealed that miR-200c mediates cell proliferation,migration,and apoptosis in ovarian tissue.K

关 键 词：绵羊 卵巢 非繁殖季节发情 MIR-200C MAPK8 

分 类 号：S813[农业科学—畜牧学]