【摘要】：日本黑毛和牛與荷斯坦牛同為世界知名牛種,但是在脂肪沉積方面二者卻形成了顯著差異,這就為牛脂肪沉積相關(guān)研究提供了優(yōu)良的素材。因此,本實(shí)驗(yàn)旨在利用RNA-Seq方法對(duì)兩個(gè)品種牛背部皮下脂肪組織miRNA表達(dá)情況進(jìn)行比較研究,以期鑒定出調(diào)控牛脂肪沉積的miRNAs,為牛脂肪沉積機(jī)理的研究和肉牛種質(zhì)資源利用提供理論基礎(chǔ)。分別采集健康、體況良好、品種內(nèi)不同個(gè)體體重相近的處于屠宰期(約34月齡)5頭日本黑毛和牛和5頭荷斯坦牛的背部皮下脂肪組織,構(gòu)建兩個(gè)品種small RNA的c DNA混合文庫W和H,然后對(duì)其進(jìn)行Illumina測序,進(jìn)而對(duì)測序數(shù)據(jù)進(jìn)行生物信息學(xué)分析。從W和H文庫中分別獲得10974628(88.32%)和9417372(85.96%)條clean reads,鑒定出bta-miR-30f、bta-miR-196b和bta-miR-2887等14個(gè)上調(diào)表達(dá)miRNA,3個(gè)下調(diào)表達(dá)miRNA bta-miR-320a、bta-miR-874和bta-miR-1247-3p,預(yù)測出15個(gè)新miRNA。通過對(duì)17個(gè)已知差異表達(dá)miRNA的靶基因進(jìn)行預(yù)測共獲得1345個(gè)可能靶基因,其中包括APOA1、APOA5、ANGPTL4、PPARα、RXRα和CDK11B等已報(bào)道參與脂肪細(xì)胞分化和脂代謝調(diào)控的基因,這些靶基因富集在脂肪酸代謝和細(xì)胞周期調(diào)控等生物過程以及甘油磷脂代謝和PPAR等信號(hào)通路中。由此推測:bta-miR-30f、bta-miR-196b、bta-miR-320a和bta-miR-874等差異表達(dá)miRNA通過對(duì)其靶基因的抑制作用,調(diào)節(jié)了脂肪酸代謝和細(xì)胞周期等生物過程,參與了甘油磷脂代謝和PPAR等信號(hào)通路的信號(hào)轉(zhuǎn)導(dǎo),進(jìn)而影響了牛的脂肪沉積。實(shí)驗(yàn)第二部分旨在對(duì)鑒定出的在日本黑毛和牛中下調(diào)表達(dá)且豐度較高的bta-miR-320a進(jìn)行生物信息學(xué)預(yù)測和分析,探索其影響牛脂肪沉積的作用機(jī)制。分別利用Promoter Scan、TargetScan、DAVID、Cytoscape等生物信息學(xué)軟件和miRBase、Ensemble、NCBI、mi RWalk等數(shù)據(jù)庫對(duì)bta-miR-320a進(jìn)行轉(zhuǎn)錄因子結(jié)合位點(diǎn)預(yù)測、保守性分析、靶基因預(yù)測、基因本體論富集分析和信號(hào)通路富集分析。結(jié)果表明,miR-320(a)在各物種間非常保守,bta-mi R-320a啟動(dòng)子區(qū)域有SP1等多個(gè)轉(zhuǎn)錄因子結(jié)合位點(diǎn),獲得的84個(gè)靶基因主要參與負(fù)調(diào)控細(xì)胞分化、細(xì)胞周期、負(fù)調(diào)控生長等多個(gè)生物學(xué)過程中,涉及p53、細(xì)胞周期和MAPK等信號(hào)轉(zhuǎn)導(dǎo)通路。由此我們推測:bta-miR-320a受到SP1等多種轉(zhuǎn)錄因子調(diào)控,它可能通過對(duì)MAPK、細(xì)胞周期和p53信號(hào)通路中靶基因TP53、MAPK1等的抑制作用調(diào)控了牛脂肪細(xì)胞分化,進(jìn)而影響了牛的脂肪沉積。
[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 偽, RXR 偽 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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823

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相關(guān)期刊論文 前1條

1 Jian Yao;Lin-hui Liang;Yu Zhang;Jie Ding;Qi Tian;Jin-jun Li;Xiang-huo He;;GNAI1 Suppresses Tumor Cell Migration and Invasion and is Post-Transcriptionally Regulated by Mir-320a/c/d in Hepatocellular Carcinoma[J];Cancer Biology & Medicine;2012年04期

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本文編號(hào)：2403617