奶牛乳脂代谢关键miRNAs的筛选及鉴定

doi:10.11843/j.issn.0366-6964.2022.12.011

摘要/Abstract

摘要： 旨在探究影响中国荷斯坦奶牛乳脂代谢过程的关键miRNAs及靶mRNAs。本研究选择泌乳中后期(150～220 d)的第一胎次且乳脂率具有极端差异的8头宁夏荷斯坦奶牛，根据乳脂率的差异分为两组：高乳脂组和低乳脂组，每组4个重复。采集8头奶牛的奶样分离乳腺上皮细胞，进行转录组测序和生物信息学分析，筛选出差异表达miRNA-mRNA并与乳脂率进行相关性分析。通过实时荧光定量PCR验证miRNAs和mRNAs表达。结果表明，8个small RNA (sRNA)文库测序获得11 976 914~16 235 680的clean reads，占总raw reads的比例均在97%以上，Q30达到91%以上，sRNA片段长度主要分布在21~24 nt。在高乳脂组和低乳脂组间共鉴定了34个差异表达miRNAs (16个上调，18个下调)。GO和KEGG富集分析表明，差异表达miRNAs的靶基因在细胞内信号转导、单一生物体过程、激酶结合和磷酸转移酶活性等功能条目显著富集，并且显著富集到TNF信号传导途径、MAPK信号传导途径、Ras信号传导途径、Rap1信号通路和催产素信号传导途径等乳脂代谢相关通路。通过miRNA-mRNA综合分析，共获得16个miRNA-靶基因对，其中miR-1343-3p和MTM1与乳脂率显著负相关，miR-370、miR-2285cb和SRRM2与乳脂率显著正相关。RT-qPCR验证差异表达miRNA和mRNA与转录组测序表达趋势一致。这些结果从miRNA和mRNA水平探究了宁夏地区荷斯坦奶牛乳脂代谢的功能机制，鉴定出miR-370-MTM1、miR-1343-3p-DIS3L2、miR-2285cb-XLOC-122799和miR-2387-SRRM2互作对可能是调控奶牛乳脂代谢的关键因子。

关键词: miRNA, 乳脂, 中国荷斯坦奶牛, 转录组学, miRNA-mRNA互作

Abstract: This study aimed to investigate the key miRNAs and target mRNAs affecting the process of milk fat metabolism in Chinese Holstein cows. In this study, 8 Ningxia Holstein cows with extreme differences in milk fat rate in their first lactation at mid to late lactation stage(150-220 d) were selected and divided into two groups according to differences in milk fat rate: high and low milk fat groups, with 4 replicates in each group. Milk samples from the 8 cows were collected to isolate mammary epithelial cells for transcriptome sequencing and bioinformatics analysis to screen out differentially expressed miRNA-mRNA, and their correlation with milk fat rate was analyzed. The miRNAs and mRNAs expression were verified by real-time fluorescence quantitative PCR. The results showed that 8 small RNA (sRNA) libraries were sequenced to obtain 11 976 914-16 235 680 clean reads, accounting for more than 97% of the total raw reads, with Q30 reaching more than 91%. The length of sRNA fragments were mainly distributed in the range of 21-24 nt. A total of 34 differentially expressed miRNAs (16 up-regulated and 18 down-regulated) were identified between the high and low milk fat groups. GO and KEGG enrichment analysis showed that the target genes of differentially expressed miRNAs were significantly enriched in functional items of intracellular signaling transduction, single organism processes, kinase binding and phosphotransferase activity, and significantly enriched in TNF signaling pathway, MAPK signaling pathway, Ras signaling pathway, Rap1 signaling pathway and oxytocin signaling pathway. A total of 16 miRNA-target gene pairs were obtained by comprehensive miRNA-mRNA analysis. miR-1343-3p and MTM1 were significantly negatively correlated with milk fat rate, and miR-370, miR-2285cb and SRRM2 were significantly positively correlated with milk fat rate. The expression trend of differentially expressed miRNAs and mRNAs verified by RT-qPCR were consistent with the result of transcriptome sequencing. These results explored the functional mechanisms of milk fat metabolism in Holstein cows in the Ningxia region at the miRNA and mRNA levels and identified miR-370-MTM1, miR-1343-3p-DIS3L2, miR-2285cb-XLOC-122799 and miR-2387-SRRM2 interaction pairs as possible key factors regulating milk fat metabolism in cows.

Key words: miRNA, milk fat, Chinese Holstein cows, transcriptomics, miRNA-mRNA interaction

中图分类号:

S823.91

刘佳敏, 禹保军, 母童, 张迪, 冯小芳, 张娟, 王影, 温万, 顾亚玲. 奶牛乳脂代谢关键miRNAs的筛选及鉴定[J]. 畜牧兽医学报, 2022, 53(12): 4244-4257.

LIU Jiamin, YU Baojun, MU Tong, ZHANG Di, FENG Xiaofang, ZHANG Juan, WANG Ying, WEN Wan, GU Yaling. Screening and Identification of Key miRNAs for Milk Fat Metabolism in Dairy Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4244-4257.

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