梅山猪和杜洛克猪卵泡中差异表达lncRNA克隆鉴定及与miRNAs相关性分析  

Cloning and Identification of Differentially Expressed lncRNAs in Follicles of Meishan Pigs and Duroc Pigs with Their Correlation Analysis with miRNAs

作　　者：张化鹏 张庆泽 何凡 祁梦凡 符彬彬 李清春 李梦寻[1] 马力鹏[1] 刘乙[1] 黄涛[1,2] ZHANG HuaPeng;ZHANG QingZe;HE Fan;QI MengFan;FU BinBin;LI QingChun;LI MengXun;MA LiPeng;LIU Yi;HUANG Tao(College of Animal Science and Technology,Shihezi University,Shihezi 832000,Xinjiang;Xinjiang Pig Seed Industry Engineering Technology Research Center,Changji 831100,Xinjiang)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]新疆生猪种业工程技术研究中心,新疆昌吉831100

出　　处：《中国农业科学》2024年第9期1807-1819,共13页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31960645);新疆维吾尔自治区天山英才青年科技拔尖人才项目(2022TSYCCX0047);新疆维吾尔自治区第七师胡杨河市“揭榜挂帅”项目(QS2023010)。

摘　　要：【目的】通过克隆鉴定梅山猪和杜洛克猪卵泡期第4天M2卵泡中差异表达的lncRNA-ALDBSSCT0000005583,分析其在猪颗粒细胞与miRNAs表达量的相关性,为探究lncRNA调控miRNA在母猪卵泡发育过程中的作用提供理论依据。【方法】对课题组前期在梅山和杜洛克M2卵泡中筛选出的差异表达lncRNA-ALDBSSCT0000005583,进行RT-qPCR验证,并利用RACE克隆其全长序列;根据编码潜力评估工具CPAT、CPC对该lncRNA的编码能力进行预测,原核表达试验进一步鉴定其编码能力;核质分离试验对lncRNA-ALDBSSCT0000005583进行亚细胞定位,RT-qPCR检测其在各组织中的表达水平;利用miRBase网站查找猪的miRNA数据库,结合RNAhybrid、miRanda在线软件预测与lncRNA-ALDBSSCT0000005583有相互作用的物种间保守miRNA,使用TargetScan和miRanda预测与lncRNA-ALDBSSCT0000005583具有相互作用miRNA的靶基因,并对其靶基因进行GO富集和KEGG信号通路分析;通过过表达以及干扰lncRNA验证其对miRNA表达量的影响。【结果】lncRNAALDBSSCT0000005583在杜洛克猪M2卵泡中的表达量显著高于梅山猪,lncRNA 5’RACE和3’RACE片段大小为569 bp和546 bp,测序分析表明lncRNA-ALDBSSCT0000005583大小为588 bp。生物信息学预测其编码潜能较低,同时原核表达试验结果进一步证明其不编码蛋白质。组织表达谱分析表明,lncRNA-ALDBSSCT0000005583在肾上腺、脾肝和卵巢中表达量较高,在下丘脑和心脏中的表达量较低,亚细胞定位结果显示该lncRNA主要存在于细胞质中。生物信息学分析后共筛选出9个物种间保守的miRNA与lncRNA-ALDBSSCT0000005583具有潜在的相互作用,其中有两个与卵巢发育相关的miRNA:miR-193a-5p、miR-361-3p;KEGG和GO富集分析显示miR-193a-5p、miR-361-3p的靶基因与系统进程和细胞与细胞信号传导等生物学过程有关,并显著参与到催产素、Ras、NF-κB、促性腺激素释放激素分泌等通路。随后在颗粒细胞中过表达lnc RNA-【Objective】The objective of this study was to clone and identify the differentially expressed lncRNA-ALDBSSCT0000005583 in M2 follicles on the fourth day of follicular stage in Meishan pigs and Duroc pigs,and to analyze the correlation between the expression of miRNAs in porcine granulosa cells,so as to provide a theoretical basis for exploring the role of lncRNAs in the development of follicles in sows by regulating miRNAs.【Method】Based on the differentially expressed lncRNA-ALDBSSCT0000005583 screened in Meishan and Duroc M2 follicles in our early research,the full-length sequence of ALDBSSCT0000005583 was verified by RT-qPCR and cloned by RACE;the coding ability of this lncRNA was predicted by the coding potential assessment tool of CAPT and CPC,which was further identified by the primary expression test;the coding ability of this lncRNA was identified by the primary expression test;the subcellular coding ability of NA-ALDBSSCT0000005583 was identified by the nucleoplasmic separation experiment and tested to identify its coding ability;the subcellular localization of lncRNA-ALDBSSCT0000005583 by nucleoplasmic isolation assay and its expression level in various tissues were detected by RT-qPCR;miRBase website was used to locate the miRNA database of pigs,and the combination of RNAhybrid and miRanda online software was used to predicte the relationship with the lncRNA-ALDBSSCT0000005583.The inter-species conserved miRNAs that interacted with lncRNA-ALDBSSCT0000005583 were predicted by TargetScan and miRanda,the target genes that interacted with lncRNA-ALDBSSCT0000005583 were predicted by TargetScan and miRanda,and their target genes were subjected to GO enrichment and KEGG signaling pathway analyses;the effects of target genes on miRNA expression were verified by overexpression as well as interference with lncRNA.【Result】The expression level of lncRNA-ALDBSSCT0000005583 in M2 follicles of Duroc pigs was significantly higher than that in Meishan pigs,and the size of lncRNA 5′RACE and 3′RACE fragme

关 键 词：lncRNA-ALDBSSCT0000005583 猪 卵泡 颗粒细胞 miR-193a-5p miR-361-3p 

分 类 号：S828.2[农业科学—畜牧学]