新吉细毛羊和小尾寒羊差异表达miRNAs筛选与调控网络分析

doi:10.16431/j.cnki.1671-7236.2023.09.004

摘要/Abstract

摘要： 【目的】筛选新吉细毛羊和小尾寒羊体内差异微小RNAs(microRNAs,miRNAs),研究miRNAs和靶基因对羊毛纤维机制表达的调控模式。【方法】选取两种毛细度存在显著差异的新吉细毛羊(XFWS)和小尾寒羊(SXW),采集血浆中外泌体进行转录组测序,构建文库并鉴定miRNAs,使用edgeR包对miRNAs进行差异表达分析,使用miRanda和Targetscan软件预测miRNAs的靶基因,对靶基因进行GO功能和KEGG通路富集分析,对差异miRNAs靶基因进行miRNAs-mRNA互作调控网络分析,使用实时荧光定量PCR对转录组数据中miRNAs进行验证。【结果】新吉细毛羊和小尾寒羊血浆外泌体中存在1 019个miRNAs,其中已知miRNAs 112个,新预测miRNAs 907个,经筛选存在差异表达miRNAs 364个,其中与羊毛细度呈正相关性基因62个,呈负相关性基因302个。经多数据库比对获得靶基因的注释信息18 996个,GO功能富集分析显示,17 193个注释靶基因富集到6 608个条目中;KEGG通路分析显示其富集在276个通路之中,经蛋白互作分析找到与羊毛纤维机制表达相关miRNAs 5个及靶基因42个。实时荧光定量PCR验证差异miRNAs结果显示,oar-miR-218a、oar-miR-370-3p、oar-miR-133、oar-miR-29a、oar-miR-27a、oar-miR-485-3p、oar-miR-22-3p、oar-miR-23a、oar-miR-148a、oar-miR-412-3p在新吉细毛羊体内表达量均显著高于小尾寒羊(P＜0.05),oar-miR-26a、oar-miR-29b、oar-miR-329b-3p、oar-miR-409-3p、oar-miR-30a-3p在小尾寒羊体内表达量均显著高于新吉细毛羊(P＜0.05),与转录组测序结果一致。【结论】新吉细毛羊和小尾寒羊体内存在oar-miR-218a、oar-miR-370-3p、oar-miR-133等364个差异miRNAs,其中oar-miR-133、oar-miR-150、oar-miR-127、oar-miR-23a、oar-miR-370-3p与其42个靶基因共同参与羊毛纤维机制表达,FGFR2、NOTCH1、SHH参与多通路调节,在联级调节中起重要作用。

关键词: 新吉细毛羊; 小尾寒羊; 血浆; 外泌体; miRNAs

Abstract: 【Objective】 This study was aimed to screen the differential microRNAs (miRNAs) in Xinji Fine wool sheep (XFWS) and Small-tailed Han sheep (SXW),and study the regulatory mode of miRNAs and target genes on the mechanism expression of wool fiber.【Method】 Two kinds of Xinji Fine wool sheep and Small-tailed Han sheep with significant differences in capillary were selected,exosomes in plasma were collected for transcriptome sequencing,libraries were constructed and miRNAs were identified,miRNAs were differentially expressed using edgeR packs,miRanda and Targetscan were used to predict miRNA target genes,GO function and KEGG pathway enrichment of target genes were performed,miRNAs-mRNA interaction regulatory network for differential miRNAs target genes were analyzed,and miRNAs in transcriptome data were validated using Real-time quantitative PCR.【Result】 There were 1 019 miRNAs in the plasma exosomes of Xinji Fine wool sheep and Small-tailed Han sheep,including 112 known miRNAs,907 newly predicted miRNAs,and 364 differentially expressed miRNAs after screening,including 62 genes positively correlated with wool fineness and 302 negatively correlated genes.After multi-database comparison,18 996 annotated target genes were obtained,and GO function enrichment analysis showed that 17 193 annotated target genes were enriched into 6 608 biological processes.KEGG pathway enrichment analysis was enriched in 276 pathways,and 5 miRNAs and 42 target genes related to the expression of wool fiber mechanism were found by protein interaction analysis.Real-time quantitative PCR verified differential miRNAs,the expression of oar-miR-218a,oar-miR-370-3p,oar-miR-133,oar-miR-29a,oar-miR-27a,oar-miR-485-3p,oar-miR-22-3p,oar-miR-23a,oar-miR-148a and oar-miR-412-3p in Xinji Fine wool sheep were significantly higher than those of Small-tailed Han sheep (P<0.05), the expression of oar-miR-26a,oar-miR-29b,oar-miR-329b-3p,oar-miR-409-3p and oar-miR-30a-3p in Small-tailed Han sheep were significantly higher than those of Xinji Fine wool sheep (P<0.05),which were consistent with transcriptome sequencing results.【Conclusion】 There were 364 differential miRNAs in Xinji Fine wool sheep and Small-tailed Han sheep,including oar-miR-218a,oar-miR-370-3p,oar-miR-133,etc.,among which differential miRNAs,oar-miR-133,oar-miR-150,oar-miR-127,oar-miR-23a,oar-miR-370-3p and their 42 target genes were involved in the mechanism expression of wool fiber,FGFR2,NOTCH1 and SHH participated in multi-channel regulation and played an important role in joint regulation.

Key words: Xinji Fine wool sheep; Small-tailed Han sheep; plasma; exosomes; miRNAs

中图分类号:

张新玉, 王丹, 杨霞, 付佳棋, 曹阳, 张立春, 孙福亮. 新吉细毛羊和小尾寒羊差异表达miRNAs筛选与调控网络分析[J]. 中国畜牧兽医, 2023, 50(9): 3480-3489.

ZHANG Xinyu, WANG Dan, YANG Xia, FU Jiaqi, CAO Yang, ZHANG Lichun, SUN Fuliang. Differentially Expressed miRNAs Screening and Regulatory Network Analysis of Xinji Fine Wool Sheep and Small-tailed Han Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3480-3489.

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