绵羊季节性发情性状核心基因和关键lncRNA的筛选与分析

doi:10.11843/j.issn.0366-6964.2025.03.026

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (3): 1264-1277.doi: 10.11843/j.issn.0366-6964.2025.03.026

绵羊季节性发情性状核心基因和关键lncRNA的筛选与分析

杨杨¹(), 李良远², 万鹏程¹, 卢守亮¹, 刘长彬¹, 杨华¹, 王立民¹, 代蓉¹^,*(), 周平¹^,*()

1. 新疆农垦科学院, 省部共建绵羊遗传改良与健康养殖国家重点实验室, 石河子 832000
2. 贵州中医药大学中药民族药资源研究院, 贵阳 550025

收稿日期:2024-08-28 出版日期:2025-03-23 发布日期:2025-04-02
通讯作者: 代蓉,周平 E-mail:1010975408@qq.com;dairong1@163.com;zhpxqf@163.com
作者简介:杨杨(1989-), 女, 河南西平人, 硕士, 主要从事动物分子遗传与育种研究, E-mail: 1010975408@qq.com
基金资助:
兵团重点领域科技攻关项目; 兵团农业创新工程(NCG202221);中央引导地方科技发展资金项目; 省部共建绵羊遗传改良与健康养殖国家重点实验室建设(2022CA002)

Screening and Analysis of Core Genes and Key lncRNAs for Seasonal Estrus Traits in Sheep

YANG Yang¹(), LI Liangyuan², WAN Pengcheng¹, LU Shouliang¹, LIU Changbin¹, YANG Hua¹, WANG Limin¹, DAI Rong¹^,*(), ZHOU Ping¹^,*()

1. State Key Laboratory of Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural and Reclamation Science, Shihezi 832000, China
2. Institute of Traditional Chinese Medicine and Ethnical Medicine Resources, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China

Received:2024-08-28 Online:2025-03-23 Published:2025-04-02
Contact: DAI Rong, ZHOU Ping E-mail:1010975408@qq.com;dairong1@163.com;zhpxqf@163.com

摘要/Abstract

摘要：

旨在筛选与季节性发情相关的核心基因和长链非编码RNA(long non-coding RNA，lncRNA)。本研究选取2~3岁空怀的中国美利奴羊和湖羊母羊(乏情期、发情期和发情间期各3只)，采集卵巢组织样本，采用RNA-seq技术筛选差异表达的基因(differentially expressed genes, DEGs)和lncRNAs，并通过GO、KEGG富集分析、PPI分析及ceRNA网络构建鉴定与繁殖性能相关的核心基因和lncRNAs。结果，构建了6个不同生理状态下的RNA文库，筛选得到1 495个差异表达基因(differentially expressed genes, DEGs)和454个差异表达lncRNAs，其中共性DEGs和lncRNAs分别为313个和435个，特有DEGs和lncRNAs分别为1 182个和19个。GO和KEGG富集分析显示，共性DEGs在细胞分裂和细胞周期等方面显著富集，而差异表达的lncRNAs靶基因富集于细胞内信号转导及负调控RNA聚合酶Ⅱ启动子转录等。特有DEGs在细胞迁移的正向调节中富集，lncRNAs的靶基因集中于细胞间粘附和整合素介导的信号通路等。通过PPI筛选出19个核心基因，主要富集在卵母细胞减数分裂和细胞周期信号通路。聚类分析结果显示，核心基因在乏情期表达量较高。ceRNA网络进一步揭示基因在细胞周期等信号通路的富集。RT-qPCR验证结果与高通量测序一致。筛选出19个核心基因和28个lncRNAs，并发现42个lncRNA-miRNA-mRNA靶向关系，为理解绵羊季节性繁殖的分子机制提供了潜在调控靶点。

关键词: 绵羊, 季节性发情, RNA-seq, 核心基因, lncRNA

Abstract:

The aim of this study was to screen core genes and long non-coding RNAs related to seasonal estrus. Ovarian tissue were collected from 2 to 3 years old, non-pregnant female Chinese Merino and Hu sheep (3 sheep per group, each in anestrus, estrus and interestrus). RNA-seq technology was employed to identify differentially expressed genes and lncRNAs. GO and KEGG enrichment analyses, protein-protein interaction (PPI) network analysis and ceRNA network construction were performed to identify core genes and lncRNAs related to reproductive performance. RNA libraries for 6 distinct physiological states were constructed. There were 1 495 differentially expressed genes (DEGs) and 454 differentially expressed long non-coding RNAs (lncRNAs) be identified. Among these, 313 DEGs and 435 lncRNAs were common, while 1 182 DEGs and 19 lncRNAs were unique. GO and KEGG enrichment analyses revealed that common DEGs were significantly enriched in the processes related to cell division and the cell cycle, whereas the target genes of differentially expressed lncRNAs were enriched in intracellular signaling pathways and negative regulation of RNA polymerase Ⅱ promoter transcription. Unique DEGs were primarily associated with the positive regulation of cell migration. The target genes of lncRNAs were enriched in cell adhesion and integrin-mediated signaling pathways. There were 19 core genes identified by protein-protein interaction network analysis, which were predominantly enriched in oocyte meiosis and cell cycle pathways. These core genes exhibited higher expression level during the anestrus phase than those in interestrus. Further analysis of the ceRNA network highlighted the enrichment of genes in cell cycle-related signaling pathways. RT-qPCR results were consisted with the RNA-seq results. A total of 19 core genes and 28 lncRNAs were identified, along with 42 lncRNA-miRNA-mRNA interactions, providing potential regulatory targets for understanding the molecular mechanisms of seasonal reproduction in sheep.

Key words: sheep, seasonal estrus, RNA-seq, core gene, lncRNA

中图分类号:

S826.3

杨杨, 李良远, 万鹏程, 卢守亮, 刘长彬, 杨华, 王立民, 代蓉, 周平. 绵羊季节性发情性状核心基因和关键lncRNA的筛选与分析[J]. 畜牧兽医学报, 2025, 56(3): 1264-1277.

YANG Yang, LI Liangyuan, WAN Pengcheng, LU Shouliang, LIU Changbin, YANG Hua, WANG Limin, DAI Rong, ZHOU Ping. Screening and Analysis of Core Genes and Key lncRNAs for Seasonal Estrus Traits in Sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1264-1277.

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基因 Gene	序列(5'→3') Primer sequence	产物大小/bp Product size	参考序列号 Accession number
A2M	F: CGGGCACAGTTCTCCAT R: GGCAAGGGCTCAAGGTA	185	ENSOARG00000000950
E114	F: TGTGAGACCGTCAAATGG R: CAGAACAAAGGCTGTAAGG	144	ENSOARG00000000114
FSHR	F: TGACACCAAGATAGCCAAGCG R: GGTAGAACAGGACCAGGAGGA	149	NM_001009289.1
GAPDH	F: CTGACCTGCCGCCTGGAGAAA R: TAGAAGAGTGAGTGTCGCTGTT	149	NM_001190390.1

品种 Breed	样品 Sample	质控后序列 Clean reads	匹配/reads Mapped on reference	GC/%	Q30/%
中国美利奴羊 Chinese Merino sheep	M_A	105 030 108	100 750 493(95.93%)	46.66	92.99
	MBE	87 990 548	83 968 497(95.43%)	54.39	92.91
	MBD	83 665 146	78 978 206(94.40%)	54.83	92.86
湖羊 Hu sheep	HBE	87 506 822	83 480 433(95.40%)	53.15	93.09
	HAE	90 475 066	86 278 630(95.36%)	45.40	92.94
	HAD	76 988 138	73 770 028(95.82%)	49.78	93.08

分组 Group	lncRNA		DEGs
分组 Group	up	down	up	down
M_A vs. MBD	235	231	1 396	744
M_A vs. MBE	209	254	1 510	600
M_A vs. HAD	209	266	1 876	1 206
M_A vs. HAE	236	235	556	537
M_A vs. HBE	233	240	1 400	774

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绵羊季节性发情性状核心基因和关键lncRNA的筛选与分析

Screening and Analysis of Core Genes and Key lncRNAs for Seasonal Estrus Traits in Sheep

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