摘要：

旨在通过分析布莱凯特黑牛大小睾丸转录组数据,挖掘与精子发生相关的关键基因和microRNAs(miRNAs),为探究精子发生分子机制和改善畜牧生产中公畜精液品质下降问题提供理论依据。以布莱凯特黑牛为试验动物,采集6头12月龄黑牛睾丸组织,根据睾丸质量、长径和短径分为大小睾丸两组,构建全转录组文库,以|log₂(Fold Change)|≥1且FDR<1为阈值筛选差异表达基因(DEGs),以|log₂(Fold Change)|≥1且P≤0.05为阈值筛选差异miRNA(DEMs),GO和KEGG富集分析筛选影响精子发生的生物学条目及通路,加权基因共表达网络分析(WGCNA)筛选与睾丸性状关联的基因群,整合蛋白互作网络图谱(PPI),最后筛选出与精子发生相关候选基因和预测靶向的DEMs。结果表明,转录组文库共筛选出3 935个DEGs(2 049个上调,1 886个下调)。GO功能注释差异基因在信号转导、MAPK级联等通路内显著富集;KEGG显示FGFR3、NFKB1、MAP3K5和TAB1在MAPK级联、鞘脂信号通路等与精子发生相关的标志通路内显著富集,WGCNA显示分别有6 762,295个基因与睾丸大小关联性强。整合PPI蛋白互作确定20个差异基因连接度高。结合小RNA文库,最终筛选上述4个候选基因存在靶向miRNA,即FGFR3(Bta-mir-296-5p)、NFKB1(Bta-mir-15b)、MAP3K5(Bta-mir-375)和TAB1(Bta-mir-15b、Bta-mir-11988和Bta-mir-7180)。实时荧光定量PCR验证显示,Bta-mir-15b、Bta-mir-375、TAB1和MAP3K5在睾丸中的表达量较高,其他基因和miRNA在睾丸中表达量较低。综上,筛选出了与精子发生相关的20个候选基因和5个候选miRNA,其中TAB1与Bta-mir-15b、MAP3K5与Bta-mir-375可能是影响精子发生的重要候选基因及miRNA。

关键词: 布莱凯特黑牛, 睾丸, 转录组, mRNA, miRNA

Abstract:

This study aimed to explore the molecular mechanism of spermatogenesis and address the decline of semen quality in male animals by analyzing the transcriptome data of the testicles of large and small Blcattle black cattle and mining the key genes and microRNAs(miRNAs)related to spermatogenesis.Six 12-month-old black cattle testicular tissues were collected from Blcattle black cattle as experimental animals,and the testicles were divided into large and small testis groups according to testicular weight,length diameter and short diameter.The whole transcriptome library was constructed,and the differential expression genes(DEGs)were screened with |log₂(Fold Change)|≥1 and FDR<1 as the threshold,and the differential expressed microRNAs(DEMs)were screened with |log₂(Fold Change)|≥1 and P≤0.05 as the threshold.GO and KEGG enrichment analysis screened the biological items and pathways that affect spermatogenesis,weighted correlation network analysis (WGCNA)screened the gene groups associated with testicular traits,integrated protein protein interaction(PPI),and finally screened out candidate genes related to spermatogenesis and predicted to target DEMs.The results showed that a total of 3 935 DEGs(2 049 up-regulated,1 886 down-regulated)were screened in the transcriptome library,and the differentially annotated genes of GO function were significantly enriched in signal transduction and MAPK cascade.KEGG showed that FGFR3,NFKB1,MAP3K5 and TAB1 were significantly enriched in the MAPK cascade and sphingolipid signaling pathway,and 6 762 and 295 genes were strongly correlated with testicular size,respectively.The integration of PPI protein-protein interaction identified 20 differential genes with high connectivity.Combined with the small RNA library,the above four candidate genes were screened for targeting miRNAs,namely FGFR3(Bta-mir-296-5p),NFKB1(Bta-mir-15b),MAP3K5(Bta-mir-375)and TAB1(Bta-mir-15b,Bta-mir-11988 and Bta-mir-7180).Real-time PCR verification showed that the expression levels of Bta-mir-15b,Bta-mir-375,TAB1 and MAP3K5 were higher in the testis,and the expression of other genes and miRNAs was lower in the testis.In summary,20 candidate genes and five candidate miRNAs related to spermatogenesis were screened,among which TAB1 and Bta-mir-15b,MAP3K5 and Bta-mir-375s may be important candidate genes and miRNAs affecting spermatogenesis.

Key words: Blcattle black cattle, Testicles, Transcriptome, mRNA, miRNA

中图分类号: 

• S813.1遗传

• S823