MC4R基因沉默对牛成纤维细胞内基因表达的影响

发布时间：2018-06-02 06:01

本文选题：MCR基因 + 基因敲降　；参考：《畜牧兽医学报》2017年03期

【摘要】：旨在研究黑素皮质激素4受体基因(Melanocortin-4receptor,MC4R)沉默对牛成纤维细胞内能量与脂肪代谢相关通路上基因表达的影响,进一步揭示MC4R基因与肉牛胴体品质以及能量平衡与脂肪代谢之间的关系。本试验使用MC4R被敲降的牛成纤维细胞系,借助深度测序技术寻找试验组(MC4R敲降)和阴性对照组(MC4R未敲降)中表达量差异显著的基因。利用GO注释聚类分析和KEGG通路富集分析对涉及能量代谢、脂肪运输及相关调控通路的差异基因进行了深入的富集分析。随机选取差异表达基因中的10个基因,利用实时定量PCR对深度测序结果进行验证。此外,本研究借助牛SNP芯片对上述目标基因的单核苷酸多态性(SNP)进行了检测,并对136头西门塔尔牛进行了基于目标基因各SNP的基因型检测。基于各SNP的分型数据,本研究进一步利用PLINK软件对SNP与牛各胴体性状的关联性进行分析。通过深度测序,在两组阳性MC4R被敲降的牛成纤维细胞系中共发现7 799个差异表达基因,其中上调基因1 703个,下调基因6 096个。显著性GO分类在两组不同干扰水平处理组间共选出与能量代谢调节、脂肪运输过程相关的差异表达基因265个,功能聚类分析进一步筛选出与能量代谢相关的基因29个。KEGG通路富集分析发现,与能量代谢、脂肪运输等过程相关的265个差异表达基因共涉及到40个不同的通路,其中与MC4R基因及能量代谢调控有密切关联的Leptin、AGRP、NPY和POMC等基因同属于脂联素信号通路。MC4R敲降引起该通路上22个基因的表达发生显著变化。实时定量PCR结果表明,所选择的10个基因(表达量上调基因:CFB、RSAD2、IFIT3、CHI3L1和BOLA-N;表达量下调基因:UPK1B、IER2、LASS3、TGFβ3和AXUD1)的mRNA表达水平与测序结果无显著差异,从而证实了深度测序数据的准确性和可重复性。此外,借助50K密度的牛SNP芯片,本研究共发现了16个SNPs位点,共涉及11个目标基因。SNPs与牛各胴体性状的关联分析结果表明,10号染色体上的TGFβ3对西门塔尔牛眼肌面积、胴体重、腰部肉厚、大腿肉厚和外脊重有显著影响(P0.05),对眼肉重、里脊重有极显著影响(P0.01);同样位于10号染色体的RHOJ基因对西门塔尔牛眼肌面积也有显著影响(P0.05)。19号染色体上的KRT17基因和7号染色体上VCAN基因均显著影响西门塔尔牛肉剪切力(P0.01,P0.05)。1号染色体的MASP1基因对肉色有显著影响(P0.05)。结果显示,MC4R基因沉默显著影响牛成纤维细胞内参与能量代谢、脂肪代谢和能量稳态平衡调控过程相关基因的表达,进一步证实了MC4R基因能调控肉牛能量平衡和脂肪代谢以及影响肉牛胴体品质。
[Abstract]:To study the effect of melanocortin-4 receptor MC4R silencing on gene expression in the pathway related to energy and fat metabolism in bovine fibroblasts. The relationship between MC4R gene and carcass quality, energy balance and fat metabolism in beef cattle was further revealed. In this experiment, the bovine fibroblast cell line with MC4R knockdown was used to find the genes with significant difference in the expression of MC4R knockdown between the test group and the negative control group by deep sequencing technique. Go annotation cluster analysis and KEGG pathway enrichment analysis were used to analyze the differential genes involved in energy metabolism, fat transport and related regulatory pathways. Ten genes of differentially expressed genes were randomly selected and the results of deep sequencing were verified by real-time quantitative PCR. In addition, single nucleotide polymorphisms (SNPs) of the target genes were detected by bovine SNP microarray, and genotypes based on SNP of the target genes were detected in 136 Simmental cattle. Based on the classification data of SNP, the relationship between SNP and carcass traits in cattle was further analyzed by PLINK software. By deep sequencing, a total of 7 799 differentially expressed genes were found in two groups of bovine fibroblast cell lines with positive MC4R knockdown, including 1 703 up-regulated genes and 6 096 down-regulated genes. Significant go classification selected 265 differentially expressed genes related to the regulation of energy metabolism and fat transport between the two groups at different interference levels. Functional cluster analysis further screened 29 genes related to energy metabolism. KEGG pathway enrichment analysis showed that 265 differentially expressed genes related to energy metabolism and fat transport were involved in 40 different pathways. The expression of 22 genes in the MC4R gene and the regulation of energy metabolism, which is closely related to the MC4R gene and the regulation of energy metabolism, belongs to the adiponectin signaling pathway. MC4R knockdown causes a significant change in the expression of 22 genes in this pathway. The results of real-time quantitative PCR showed that there was no significant difference between the mRNA expression levels of the 10 genes (up-regulation gene RSAD2IFIT3, CHI3L1 and BOLA-Nand down-regulated gene UPK1BER2LASS3TGF- 尾 3 and AXUD1), which confirmed the accuracy and repeatability of the deep sequencing data. In addition, with the help of 50K density bovine SNP chip, a total of 16 SNPs loci were found in this study, involving 11 target genes SNPs and carcass traits. The results showed that TGF 尾 3 on chromosome 10 had eye muscle area and carcass weight in Simmental cattle. The thickness of waist, thighs and outer ridges had a significant effect on the weight of eye meat. The KRT17 gene on chromosome 19 and the VCAN gene on chromosome 7 have a significant effect on the eye muscle area of Simmental cattle, and the RHOJ gene also located on chromosome 10 has a significant effect on the area of eye muscle of Simmental cattle. Both the KRT17 gene on chromosome 19 and the VCAN gene on chromosome 7 have significant effects on the beef of Simmental cattle. The MASP1 gene of chromosome 1 has a significant effect on meat color. The results showed that MC4R gene silencing significantly affected the expression of genes involved in energy metabolism, fat metabolism and energy homeostasis regulation in bovine fibroblasts. It was further confirmed that MC4R gene could regulate energy balance, fat metabolism and carcass quality of beef cattle.
【作者单位】：中国农业科学院北京畜牧兽医研究所;洛阳师范学院生命科学学院;
【基金】：转基因生物新品种培育重大专项(2013ZX08007-2;2014ZX08007-2) 中国农业科学院科技创新工程(ASTIP-IAS03)
【分类号】：S823

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