两品种牛脂肪组织miRNA的鉴定及bta-miR-320a的生物信息学分析

发布时间：2019-01-07 11:41

【摘要】：日本黑毛和牛与荷斯坦牛同为世界知名牛种,但是在脂肪沉积方面二者却形成了显著差异,这就为牛脂肪沉积相关研究提供了优良的素材。因此,本实验旨在利用RNA-Seq方法对两个品种牛背部皮下脂肪组织miRNA表达情况进行比较研究,以期鉴定出调控牛脂肪沉积的miRNAs,为牛脂肪沉积机理的研究和肉牛种质资源利用提供理论基础。分别采集健康、体况良好、品种内不同个体体重相近的处于屠宰期(约34月龄)5头日本黑毛和牛和5头荷斯坦牛的背部皮下脂肪组织,构建两个品种small RNA的c DNA混合文库W和H,然后对其进行Illumina测序,进而对测序数据进行生物信息学分析。从W和H文库中分别获得10974628(88.32%)和9417372(85.96%)条clean reads,鉴定出bta-miR-30f、bta-miR-196b和bta-miR-2887等14个上调表达miRNA,3个下调表达miRNA bta-miR-320a、bta-miR-874和bta-miR-1247-3p,预测出15个新miRNA。通过对17个已知差异表达miRNA的靶基因进行预测共获得1345个可能靶基因,其中包括APOA1、APOA5、ANGPTL4、PPARα、RXRα和CDK11B等已报道参与脂肪细胞分化和脂代谢调控的基因,这些靶基因富集在脂肪酸代谢和细胞周期调控等生物过程以及甘油磷脂代谢和PPAR等信号通路中。由此推测:bta-miR-30f、bta-miR-196b、bta-miR-320a和bta-miR-874等差异表达miRNA通过对其靶基因的抑制作用,调节了脂肪酸代谢和细胞周期等生物过程,参与了甘油磷脂代谢和PPAR等信号通路的信号转导,进而影响了牛的脂肪沉积。实验第二部分旨在对鉴定出的在日本黑毛和牛中下调表达且丰度较高的bta-miR-320a进行生物信息学预测和分析,探索其影响牛脂肪沉积的作用机制。分别利用Promoter Scan、TargetScan、DAVID、Cytoscape等生物信息学软件和miRBase、Ensemble、NCBI、mi RWalk等数据库对bta-miR-320a进行转录因子结合位点预测、保守性分析、靶基因预测、基因本体论富集分析和信号通路富集分析。结果表明,miR-320(a)在各物种间非常保守,bta-mi R-320a启动子区域有SP1等多个转录因子结合位点,获得的84个靶基因主要参与负调控细胞分化、细胞周期、负调控生长等多个生物学过程中,涉及p53、细胞周期和MAPK等信号转导通路。由此我们推测:bta-miR-320a受到SP1等多种转录因子调控,它可能通过对MAPK、细胞周期和p53信号通路中靶基因TP53、MAPK1等的抑制作用调控了牛脂肪细胞分化,进而影响了牛的脂肪沉积。
[Abstract]:Japanese black hair and Holstein cattle are both world-famous cattle breeds, but there is a significant difference in fat deposition between them, which provides a good material for the study of bovine fat deposition. Therefore, the purpose of this study was to compare the expression of miRNA in subcutaneous adipose tissue of two cattle by using RNA-Seq method, in order to identify the miRNAs, that regulates the deposition of bovine fat. It provides a theoretical basis for the study of bovine fat deposition mechanism and the utilization of beef germplasm resources. The subcutaneous adipose tissue in the back of 5 Japanese black hair and cattle and 5 Holstein cattle with similar body weight in different breeds were collected during slaughter period (about 34 months old). The c DNA hybrid library W and H of two varieties of small RNA were constructed and sequenced by Illumina, and then the sequence data were analyzed by bioinformatics. Bta-miR-30f,bta-miR-196b and bta-miR-2887 were identified by 10974628 (88.32%) and 9417372 (85.96%) clean reads, from W and H libraries, respectively. 14 up-regulated miRNA, and 3 down-regulated miRNA bta-miR-320a, were identified. Bta-miR-874 and bta-miR-1247-3p, predict 15 new miRNA. A total of 1345 possible target genes were obtained by predicting 17 known differentially expressed miRNA target genes, including APOA1,APOA5,ANGPTL4,PPAR 伪, RXR 伪 and CDK11B, which have been reported to be involved in adipocyte differentiation and lipid metabolism regulation. These target genes are enriched in biological processes such as fatty acid metabolism and cell cycle regulation as well as signal pathways such as glycerol phospholipid metabolism and PPAR. It is inferred that differential expression of miRNA such as bta-miR-30f,bta-miR-196b,bta-miR-320a and bta-miR-874 regulates fatty acid metabolism and cell cycle by inhibiting its target genes. It is involved in the signal transduction of glycerol phospholipid metabolism and PPAR signaling pathway, thus affecting the fat deposition of cattle. The second part of the experiment aimed to predict and analyze the bioinformatics of the identified bta-miR-320a, which was down-expressed and abundant in Japanese black hair and cattle, and to explore the mechanism of its effect on bovine fat deposition. Bioinformatics software such as Promoter Scan,TargetScan,DAVID,Cytoscape and database such as miRBase,Ensemble,NCBI,mi RWalk were used to predict transcription factor binding sites, conservative analysis and target gene prediction of bta-miR-320a. Gene ontology enrichment analysis and signal pathway enrichment analysis. The results showed that miR-320 (a) was conserved among species. There were many transcription factor binding sites such as SP1 in the bta-mi R-320a promoter region. The 84 target genes were mainly involved in the negative regulation of cell differentiation and cell cycle. Many biological processes, such as negative regulation of growth, involve p53, cell cycle and MAPK signal transduction pathways. Therefore, we speculate that bta-miR-320a is regulated by many transcription factors such as SP1, which may regulate the differentiation of bovine adipocytes by inhibiting the cell cycle of MAPK, and the target gene TP53,MAPK1 in p53 signaling pathway. In turn, it affects fat deposition in cattle.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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