绵羊尾脂沉积关键基因筛选及lncRNA-miRNA-mRNA调控网络分析

doi:10.7668/hbnxb.20195458

华北农学报 ›› 2025, Vol. 40 ›› Issue (3): 190-198. doi: 10.7668/hbnxb.20195458

所属专题： 畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

绵羊尾脂沉积关键基因筛选及lncRNA-miRNA-mRNA调控网络分析

张羽欣¹^,², 张龙¹^,³, 郭妍岩¹^,², 石笑迪¹, 李博禹¹^,³, 阮维斌², 姚大为¹^,, 张效生¹^,

¹ 天津市农业科学院畜牧兽医研究所,天津市畜禽分子育种与生物技术重点实验室,天津市畜禽健康养殖工程技术中心,天津 300381
² 南开大学生命科学学院,天津 300071
³ 天津农学院动物科学与动物医学学院,天津 300384

收稿日期:2024-09-27 出版日期:2025-07-04
通讯作者:
姚大为(1987—),女,河北涿州人,副研究员,博士,主要从事反刍动物乳腺脂代谢研究。
张效生(1978—),男,宁夏石嘴山人,研究员,硕士,主要从事动物繁殖与遗传育种技术研究与推广工作。
作者简介:
张羽欣(2002—),女,河北唐山人,在读硕士,主要从事动物遗传育种与繁殖研究。
张羽欣、张龙为同等贡献作者。
基金资助:
科技创新2030—农业生物育种重大项目(2022ZD04013); 畜禽生物育种全国重点实验室2024年度开放课题(2024SKLAB6-4); 畜禽种质资源创新及高效繁育技术研究项目(2024ZYCX012); 天津市自然科学基金项目(23JCYBJC00260)

Screening of Key Genes in Tail Fat Deposition and Analysis of lncRNA-miRNA-mRNA Network in Sheep

ZHANG Yuxin¹^,², ZHANG Long¹^,³, GUO Yanyan¹^,², SHI Xiaodi¹, LI Boyu¹^,³, RUAN Weibin², YAO Dawei¹^,, ZHANG Xiaosheng¹^,

¹ Institute of Animal Science and Veterinary,Tianjin Academy of Agricultural Sciences,Tianjin Key Laboratory of Animal Molecular Breeding and Biotechnology,Tianjin Engineering Research Center of Animal Healthy Farming,Tianjin 300381,China
² College of Life Sciences,Nankai University,Tianjin 300071,China
³ College of Animal Science and Animal Medicine,Tianjin Agricultural University,Tianjin 300384,China

Received:2024-09-27 Published:2025-07-04

摘要/Abstract

摘要：

为筛选不同尾型绵羊尾部脂肪组织之间的差异表达mRNAs、miRNAs和lncRNAs,构建参与调控绵羊尾部脂肪沉积的重要竞争性内源RNA(ceRNA)调控网络,随机选取2岁(24月龄)杜泊羊和天津大尾羊各3只,取尾部脂肪组织进行H&E染色和全转录组测序,运用生物信息学筛选差异表达的RNAs,并进行靶基因预测和功能富集,从调控脂质沉积的角度,进一步分析其调控网络和靶向关系。结果表明,天津大尾羊尾部脂肪细胞面积显著大于杜泊羊尾部脂肪细胞;杜泊羊和天津大尾羊尾部脂肪组织存在差异表达的mRNAs共有1 156个(403个上调表达,753个下调表达),差异表达的miRNAs共有72个(其中46个上调表达,26个下调表达),差异表达的lncRNAs共392个(142个上调表达,250个下调表达);同时,差异表达lncRNAs预测到靶基因216个,这些靶基因参与四氢叶酸代谢过程、GAIT复合体、辅酶分解代谢过程等与脂代谢相关生物学过程以及甘油酯代谢、生物素代谢、脂肪消化和吸收及磷脂酰肌醇信号系统等信号通路;通过预测差异表达的mRNAs和lncRNAs与miRNAs的靶向关系,成功构建一个与绵羊尾脂沉积密切相关的ceRNA网络,其中24个lncRNAs和6个miRNAs被预测可以调控PROX1、LPL及LRP1B与脂代谢密切相关的基因。综上,PROX1、LPL和LRP1B在绵羊尾脂沉积中发挥关键作用,预测到MSTRG.9916.7等4个lncRNAs通过PROX1正向调节脂肪代谢,MSTRG.7563.1和MSTRG.4729.1通过LPL正向调节脂肪代谢。

关键词: 杜泊羊, 天津大尾羊, 尾脂, RNA-seq, ceRNA

Abstract:

The objective of this study was to identify differentially expressed mRNAs,miRNAs,and lncRNAs between different tail types of sheep in their tail fat tissues,and to construct a critical ceRNA(competing endogenous RNAs)regulatory network involved in the regulation of fat deposition in the tails of sheep.Three 2-year-old(24-month-old)Dorper sheep and three Tianjin large-tail sheep were randomly selected.Tail fat tissues were collected for H&E staining and whole transcriptome sequencing.Bioinformatics approaches were employed to screen for differentially expressed RNAs,predict target genes,and conduct functional enrichment analyses.The regulatory network and targeting relationships were further analyzed from the perspective of lipid deposition regulation.The results demonstrated that the tail fat cell area of Tianjin large-tail sheep was significantly larger than that of Dorper sheep.There were a total of 1 156 differentially expressed mRNAs between the tail fat tissues of Dorper sheep and Tianjin large-tail sheep(with 403 upregulated and 753 downregulated),72 differentially expressed miRNAs(46 upregulated and 26 downregulated),and 392 differentially expressed lncRNAs(142 upregulated and 250 downregulated).Additionally,216 target genes were predicted from the differentially expressed lncRNAs.These target genes were involved in biological processes related to lipid metabolism,such as the tetrahydrofolate metabolic process,GAIT complex,and coenzyme catabolic process,as well as signaling pathways like glycerolipid metabolism,biotin metabolism,fat digestion and absorption,and the phosphatidylinositol signaling system.The study successfully constructed a ceRNA network closely associated with sheep tail fat deposition by predicting the targeting relationships between differentially expressed mRNAs and lncRNAs and miRNAs.Among them,24 lncRNAs and 6 miRNAs were predicted to regulate genes closely related to lipid metabolism,including PROX1,LPL,and LRP1B.In summary,PROX1,LPL,and LRP1B play a key role in the deposition of tail fat in sheep.This study predicts that four lncRNAs,including MSTRG.9916.7,positively regulate fat metabolism through PROX1,while MSTRG.7563.1 and MSTRG.4729.1 positively regulate fat metabolism through LPL.

Key words: Dorper sheep, Tianjin large-tail sheep, Tail fat, RNA-seq, ceRNA

中图分类号:

S826.89

张羽欣, 张龙, 郭妍岩, 石笑迪, 李博禹, 阮维斌, 姚大为, 张效生. 绵羊尾脂沉积关键基因筛选及lncRNA-miRNA-mRNA调控网络分析[J]. 华北农学报, 2025, 40(3): 190-198. doi: 10.7668/hbnxb.20195458.

ZHANG Yuxin, ZHANG Long, GUO Yanyan, SHI Xiaodi, LI Boyu, RUAN Weibin, YAO Dawei, ZHANG Xiaosheng. Screening of Key Genes in Tail Fat Deposition and Analysis of lncRNA-miRNA-mRNA Network in Sheep[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 190-198. doi: 10.7668/hbnxb.20195458.

http://www.hbnxb.net/CN/Y2025/V40/I3/190

图/表 7

图1 杜泊羊与天津大尾羊尾部脂肪细胞面积对比不同大写字母表示细胞面积差异极显著(P<0.01)。A—B.杜泊羊尾部脂肪切片H&E染色(20×);C—D.天津大尾羊尾部脂肪切片H&E染色(20×);E.杜泊羊和天津大尾羊尾部脂肪细胞面积差异。

Fig.1 Comparison of tail adipocyte area of DS and TJLTS Different capital letters indicate extremely significant differences in cell area(P<0.01).A—B.H&E staining of DS tail fat sections(20×);C—D.H&E staining of TJLTS tail fat sections(20×);E.The difference of adipocyte area between DS and TJLTS.

表1 杜泊羊与天津大尾羊尾脂细胞RNA测序概况

Tab.1 Overview of RNA sequencing of tail adipocytes in DS and TJLTS

样本 Sample	总测序数据条数/条 Total reads	干净数据条数/条 Clean reads	干净数据占比/% Clean reads rate	Q30/%
DS-1	11 481 551 700	11 025 543 442	96.03	90.74
DS-2	11 514 227 100	11 094 165 042	96.35	91.24
DS-3	11 252 444 700	10 822 169 237	96.18	90.85
TJLTS-1	12 610 019 100	12 106 135 122	96.00	91.32
TJLTS-2	12 387 500 400	12 045 289 806	97.24	93.16
TJLTS-3	11 762 612 400	11 445 769 722	97.31	93.35

图2 杜泊羊和天津大尾羊尾部脂肪组织样本相关性热图样本相关性采用皮尔逊相关性系数度量。

Fig2. Correlation heatmap of gene expression in tail adipose tissue of DS and TJLTS The sample correlation is measured by Pearson correlation coeficient.

图3 差异表达mRNAs(A)、miRNAs(B)和lncRNAs(C)火山图

Fig.3 Volcano plot for differentially expressed mRNAs(A),miRNAs(B)and lncRNAs(C)

图4 lncRNA靶基因功能分析 A.差异表达lncRNA靶基因GO富集条形图:1.通过铁硫簇转移的蛋白质成熟,2.内皮细胞形态发生,3.2铁,2硫簇结合,4.铁硫转移酶活性,5.D-氨基酸代谢过程,6.四氢叶酸代谢GAIT过程,7.GAIT复合体,8.内胚层消化道形态发生,9.结合,10.辅酶分解代谢过程,11.线粒体基质,12.硫胺素激酶活性,13.翻译调节,14.ATP依赖的5'—3'DNA解旋酶活性,15.转录后基因表达调控,16.5'—3'DNA解旋酶活性,17.含叶酸化合物代谢过程,18.逆行蛋白质运输,内质网到细胞质,19.蛋白质脂酰化,20.[4Fe-4S]簇组装;B.差异表达lncRNA靶基因KEGG气泡图:1.叶酸的一碳代谢,2.青霉素和头孢菌素的生物合成,3.D-精氨酸和D-鸟氨酸代谢,4.蛋白酶体,5.阿米巴病,6.甘油酯代谢,7.生物素代谢,8.丙型肝炎,9.逆行内源性大麻素信号传导,10.IL-17信号通路,11.磷脂酰肌醇信号系统,12.Ⅱ型糖尿病,13.甘氨酸、丝氨酸和苏氨酸代谢,14.内质网中的蛋白质加工,15.脂肪的消化和吸收,16.麻疹,17.癌症中的转录调控失调,18.TNF信号通路。

Fig.4 Functional analysis of target genes of lncRNA A.Bar diagram of GO pathway enrichment analysis of differentially expressed target gene of lncRNA:1.Protein maturation by iron-sulfur cluster transfer,2.Endothelial cell morphogenesis,3.2 iron,2 sulfur cluster binding,4.Iron-sulfur transferase activity,5.D-amino acid metabolic process,6.Tetrahydrofolate metabolic GAIT process,7.GAIT complex,8.Endodermal digestive tract morphogenesis,9.Binding,10.Coenzyme catabolic process,11.Mitochondrial matrix,12.Thianmine kinase activity,13.Regulation of translation,14.ATP-dependent 5'—3' DNA belicase activity,15.Posttranscriptional regulation of gene expression,16.5'—3' DNA helicase activity,17.Folic acid-containing compound metabolic process,18.Retrograde protein transport,ER to cytosol,19.Protein lipoylation,20.[4Fe-4S]cluster assembly;B.Bubble diagram of KEGG pathway enrichment analysis of differentially expressed target gene of lncRNA:1.One carbon pool by folate,2.Penicillin and cephalosporin biosynthesis,3.D-Arginine and D-ornithine metabolism,4.Proteasome,5.Amebiasis,6.Glycerolipid metabolism,7.Biotin metabolism,8.Hepatitis C,9.Retrograde endocannabinoid signaling,10.IL-17 signaling pathway,11.Phosphatidylinositol signaling system,12.Type Ⅱ diabetes mellitus,13.Glycine,serine and threonine metabilism,14.Protein processing in endoplasmic reticulum,15.Fat digestion and absorption,16.Measles,17.Transcriptional misregulation in cancer,18.TNF signaling pathway.

图5 miRNA靶基因功能分析 A.差异表达miRNA靶基因GO富集条形图:1.细胞,2.细胞部分,3.细胞内,4.细胞内部分,5.结合,6.细胞内细胞器,7.细胞器,8.蛋白质结合,9.细胞质,10.膜结合细胞器,11.细胞内膜结合细胞器,12.细胞组分的组织,13.细胞器部分,14.发育过程,15.细胞组分的组织或生物发生,16.细胞内细胞器部分,17.单个有机体的发育过程,18.单个multicellular有机体的过程,19.系统发育,20.代谢过程;B.差异表达miRNA靶基因KEGG气泡图:1.代谢途径,2.磷脂酰肌醇信号系统,3.轴突引导,4.癌症中的通路,5.MAPK信号通路,6.CAMP信号通路,7.Rap1信号通路,8.肌醇磷酸代谢,9.紧密连接,10.黏附连接,11.内吞作用,12.Hippo信号通路,13.催产素信号通路,14.Ras信号通路,15.Notch信号通路,16.钙信号通路,17.脂联素信号通路,18.磷脂酶D信号通路。

Fig.5 Functional analysis of target genes of miRNA A.Bar diagram of GO pathway enrichment analysis of differentially expressed target gene of miRNA:1.Cell,2.Cell part,3.Intracellular,4.Intracellular part,5.Binding,6.Intracellular organelle,7.Organelle,8.Protein binding,9.Cytoplasm,10.Membrane-bounded organelle,11.Intracellular membrane-bounded organelle,12.Cellular component organi zation,13.Organelle part,14.Developmental process,15.Cellular component organization or biogenesis,16.Intracellular organelle part,17.Single-organism developmental process,18.Single-multicellular organism process,19.System development,20.Metabolic precess;B.Bubble diagram of KEGG pathway enrichment analysis of differentially expressed target gene of miRNA:1.Metabolic pathways,2.Phosphatidylinositol signaling system,3.Axon guidance,4.Pathways in cancer,5.MAPK signaling pathway,6.CAMP signaling pathway,7.Rap1 signaling pathway,8.Inositol phosphate metabolism,9.Tight junction,10.Adherens junction,11.Endocytosis,12.Hippo signaling pathway,13.Oxytocin signaling pathway,14.Ras signaling pathway,15.Notch signaling pathway,16.Calcium signaling pathway,17.Adipocylokine signaling pathway,18.Phospholipase D signaling pathway.

图6 ceRNA网络的构建

Fig.6 The construction of ceRNA networks

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绵羊尾脂沉积关键基因筛选及lncRNA-miRNA-mRNA调控网络分析

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