基于全转录组测序的2个不同品种绵羊卵巢ceRNA网络构建及关键miRNA的筛选

doi:10.11843/j.issn.0366-6964.2025.06.021

摘要/Abstract

摘要：

旨在解析非编码RNA (non-coding RNA, ncRNA)对绵羊卵巢发育过程的分子调控机制，筛选提高绵羊繁殖力的关键基因。本研究以多胎型(小尾寒羊)和单胎型(乌珠穆沁羊)绵羊为材料，选取健康成年母羊各3只，构建卵巢组织ncRNA及mRNA表达谱，筛选差异表达(differentially expressed, DE) lncRNAs、circRNAs、miRNAs及mRNAs，并对其进行靶基因预测以及功能和信号通路的富集分析，筛选出繁殖相关通路进一步构建ceRNA (lncRNA-miRNA-mRNA和circRNA-miRNA-mRNA)调控网络，最后总结调控网络中的关键miRNA。结果显示，乌珠穆沁羊和小尾寒羊卵巢组织共筛选出差异表达lncRNAs共1 579个，circRNA共561个，miRNAs共175个及mRNAs共3 095个。GO和KEGG富集分析结果显示，差异表达的靶基因显著富集于胚胎的形态发生、减数分裂的激活等生物过程及粘蛋白型O-聚糖生物合成、鞘糖脂生物合成、精氨酸鸟氨酸代谢等信号通路。筛选繁殖相关通路上富集到的差异表达基因，构建1个ceRNA网络(包含87个lncRNA，27个circiRNA，3个miRNA和3个mRNA)，获得3个关键miRNAs (miR-140、miR-338及miR-423-5p)及对应的靶向调控ceRNA网络。采用qRT-PCR方法对随机选取的9个差异表达lncRNAs、circRNAs、miRNAs、mRNAs进行验证，结果证实了RNA-seq测序的准确性。本研究对DE ncRNAs与对应靶基因的协调调控机制进行分析，为阐明调控绵羊卵巢发育的关键基因及分子机制提供有效理论依据，进而充分了解绵羊的繁殖生物学特性，对于提高繁殖效率具有重要指导意义。

关键词: 乌珠穆沁羊, 小尾寒羊, 卵巢, lncRNA, circRNA, miRNA, ceRNA网络

Abstract:

This study aimed to analyze the molecular regulation mechanism of ncRNA (non-coding RNA) on the development process of sheep ovary, and screen the key genes for improving sheep fertility. In this study, 3 healthy adult ewes were selected from multiparous sheep (Small-tail Han sheep) and single-born sheep (Ujumuqin sheep) to construct ncRNA and mRNA expression profiles of ovarian tissue, the differentially expressed lncRNAs, circRNAs, miRNAs and mRNAs were screened, and their target genes were predicted and GO and KEGG enrichment analysis were performed. Reproductive related pathways were screened out to further construct the ceRNA (lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA) regulatory network, and finally the key miRNAs in the regulatory network were summarized. The results showed that a total of 1 579 lncRNAs, 561 circRNAs, 175 miRNAs and 3 095 mRNAs differentially expressed in the ovarian tissues of Ujumuqin sheep and small-tailed Han sheep were screened. The results of GO and KEGG enrichment analysis showed that the target genes were significantly enriched in biological processes such as embryonic morphogenesis, meiosis activation, and mucin type O-glycan biosynthesis, glycosphingolipid biosynthesis, D-arginine and D-ornithine metabolism and other signaling pathways. The differentially expressed genes enriched in reproductive pathways were screened, and a ceRNA network(contained 87 lncRNAs, 27 circiRNAs, 3 miRNAs and 3 mRNAs)was constructed to obtain three key miRNAs(miR-140, miR-338 and miR-423-5p)and corresponding targeted regulatory ceRNA networks. Real-time fluorescence quantification (qRT-PCR) detection results of 9 differentially expressed lncRNAs, circRNAs, miRNAs, mRNAs, randomly selected were consistent with the trend of high-throughput sequencing results, indicating the reliability of sequencing data. This study analyzed the coordinated regulatory mechanism of DE ncRNAs and corresponding target genes, which provided an effective theoretical basis for elucidating the key genes and molecular mechanisms regulating the development of sheep ovary, and further fully understood the reproductive biological characteristics of sheep, which has important guiding significance for improving the reproductive efficiency.

Key words: Ujumuqin sheep, Small-tailed Han sheep, ovary, lncRNA, circRNA, miRNA, ceRNA network

中图分类号:

S826.3

谷博, 王安琪, 于鑫淼, 郭俊彤, 杨一, 邓祎婕, 姜怀志. 基于全转录组测序的2个不同品种绵羊卵巢ceRNA网络构建及关键miRNA的筛选[J]. 畜牧兽医学报, 2025, 56(6): 2765-2777.

GU Bo, WANG Anqi, YU Xinmiao, GUO Juntong, YANG Yi, DENG Yijie, JIANG Huaizhi. Construction of Ovarian ceRNA Networks and Screening of Key miRNA in Two Different Breeds of Sheep Based on Whole Transcription Sequencing[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2765-2777.

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目的基因 Gene name	引物序列(5′→3′) Sequence information	长度/bp Length	退火温度/℃ Annealing temperature
β-actin	F: GATCTGGCACCACACCTTCTA	115	60
	R: GATCTGGGTCATCTTCTCACG
MSTRG.7755	F: GCGTTGCTCTGTTCTTTATCTCCTG	144	60
	R: ACCTGTGCTGTCTGAGTCTTGTG
MSTRG.25228	F: CCTGATCTTGTCCATCGTCCTATCC	123	60
	R: GGGAACTGACCAAGGATGATAAGGG
MSTRG.28959	F: TGCTAAGCCTATGCCACTAGATGC	128	60
	R: CTCAAAGTCACCAGTTTCCCAAAGG
circRNA4099	F: TGGAGCCTGTAATAACATGTGAC	259	60
	R: AGTTCCGCTGTGTTTGCATC
circRNA2195	F: GGCTGGAGGTTTAAGAAGCG	125	60
	R: TTGAGGAAGGCAGACAGGTC
circRNA15689	F: GTGAGCAAATTGTGGAAGTTGGA	102	60
	R: GGCTTCAAATCTCATGCCCG
CXCR1	F: GCTGACCTGCTCTTCGCCATG	160	60
	R: GGTAGCGGTCCATGCTGATGC
POLR2I	F: AGTGGCAGGACTGGAGTTCGG	145	60
	R: GGTCTTCGGTTCGCGGCAAC
RPS20	F: CCAGCCGCAACGTGAAGTCTC	81	60
	R: GCCGACCTCGCTCAGAACAAG
oar-miR-433-3p	F: AACGGCATCATGATGGGCTCC	65	60
	R: ATCCAGTGCAGGGTCCGAGG
oar-miR-200c	F: AAGCGCCTTAATACTGCCGGG	65	60
	R: ATCCAGTGCAGGGTCCGAGG
oar-mir-221-p5	F: CCTGCTGGACCTGGCATACAAT	65	60
	R: ATCCAGTGCAGGGTCCGAGG
U6	F: ATCCAGTGCAGGGTCCGAGG	65	60
	R: TGAAGCGTGCTCGCTTCGGC

样本 Sample	原始数据 Raw data	有效数据 Valid data	参考基因组比对率 Mapping rate	Q20/%	Q30/%	GC含量/% GC content
Sad_1	94 092 386	91 107 390	86 158 306(94.57%)	99.96	97.78	45.00
Sad_2	97 790 686	94 803 474	89 533 252(94.44%)	99.96	97.71	45.00
Sad_3	89 825 602	86 701 034	80 875 819(93.28%)	99.97	98.16	48.50
C_1	69 188 934	67 121 848	62 676 263(93.38%)	99.84	97.85	46.00
C_2	75 845 456	73 401 010	67 841 183(92.43%)	99.84	97.91	45.50
C_3	77 539 444	75 024 956	69 565 183(92.72%)	99.82	97.81	46.00

样本 Sample	原始数据 Raw data	有效数据 Valid data	参考基因组比对率 Mapping rate	Q20/%	Q30/%	GC含量/% GC content
Sad_1	10 567 624	7 951 578	7 868 882 (98.96%)	97.92	96.91	51.40
Sad_2	9 796 916	6 971 109	6 893 729(98.89%)	97.92	96.96	51.22
Sad_3	10 036 758	6 868 427	6 794 248 (98.92%)	97.29	96.07	51.11
C_1	11 418 221	5 627 660	5 587 704 (99.18%)	99.29	97.37	51.24
C_2	9 754 842	5 237 938	5 189 225 (99.07%)	99.30	97.44	51.07
C_3	10 715 275	7 225 151	7 142 062(98.85%)	99.23	97.18	50.95

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