MC4R基因沉默对牛成纤维细胞内基因表达的影响

doi:10.11843/j.issn.0366-6964.2017.03.003

摘要/Abstract

摘要：

旨在研究黑素皮质激素4受体基因（Melanocortin-4 receptor, 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50 K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（P<0.05），对眼肉重、里脊重有极显著影响（P<0.01）；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（P<0.05）。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（P<0.01，P<0.05）。1号染色体的MASP1基因对肉色有显著影响（P<0.05）。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:

The study aimed to investigate the effect of MC4R gene silencing on gene expression in bovine fibroblast，further reveal the relationship between MC4R gene and carcass quality, energy balance and energy metabolism in beef cattle. Bovine fibroblasts with MC4R-knockdown were used for this study. Differentially expressed genes between experimental group (MC4R knockdown) and control group (MC4R non-knockdown) were identified by deep sequencing. GO annotation cluster analysis and KEGG pathway enrichment analysis were performed to enrich the differentially expressed genes involving in energy metabolism, fat transport processes and their regulation pathway. For validating the reliability of RNA-sequencing, mRNA expression of 10 differentially expressed genes randomly selected were validated by quantitative real-time PCR (qRT-PCR). Moreover, single nucleotide polymorphisms (SNPs) of the targeted genes were tested by the bovine SNP microarray, and SNP-based genotypes were tested for the 136 Simmental cattle. On the basis of genotyped data, further association analysis between SNP and carcass traits were carried out using PLINK software. 7 799 differentially expressed genes in MC4R knockdown cells were detected using deep sequencing, including 1 703 up-regulated genes and 6 096 down-regulated genes. Significant GO annotation analysis identified 265 differentially expressed genes involving in energy metabolism and fat transport processes from 2 different MC4R knockdown level groups. Function cluster analysis further screened out 29 genes associated with the energy metabolism. KEGG pathway enrichment analysis indicated that the 265 differentially expressed genes were involved in 40 different signaling pathways. Of which, MC4R gene and other 4 genes (Leptin, AGRP, NPY and POMC) closely associated with the energy metabolism regulation were all involved in the adiponectin signaling pathway. And, MC4R knockdown significantly influenced the expression of 22 genes in this pathway. As the qRT-PCR data, mRNA expression of the 10 genes (up-regulated genes: CFB, RSAD2, IFIT3, CHI3L1 and BOLA-N; down-regulated genes: UPK1B, IER2, LASS3, TGFβ3 and AXUD1) showed non-significant difference with the results of RNA-sequencing, which was proved that the results of deep sequencing were accurate and reliable. 16 SNPs were detected from 11 genes by the bovine 50 K SNP microarray. The association analysis between SNPs and carcass traits showed that TGFβ3 gene on chromosome 10 significant influenced the eye muscle area, carcass weight, thickness of the waist and thigh meat and striploin weight of Simmental cattle (P<0.05), while extremely significantly influenced the eye meat weight and tendorloin weight (P<0.01). The RHOJ gene on chromosome 10 also had a significant influence on the eye muscle area of Simmental cattle (P<0.05). The KRT17 gene on chromosome 19 and VCAN gene on chromosome 7 could significantly affect the meat shearing force of Simmental cattle (P<0.05 and P<0.01, respectively). Additionally, MASP1 gene on chromosome 1 significantly influenced the meat color (P<0.05).The results indicate that MC4R knockdown significantly influence the expression of genes involving in the processes of energy metabolism, protein metabolism, fat metabolism and energy homeostasis regulation, further MC4R gene regulate the energy balance, fat metabolism and carcass characteristics in beef cattle.

中图分类号:

S823
S813.1

黄萌，贤明，史明艳，陈燕，张路培，高会江，李俊雅，许尚忠，高雪. MC4R基因沉默对牛成纤维细胞内基因表达的影响[J]. 畜牧兽医学报, 2017, 48(3): 403-415.

HUANG Meng, XIAN Ming, SHI Ming-yan, CHEN Yan, ZHANG Lu-pei, GAO Hui-jiang, LI Jun-ya, XU Shang-zhong, GAO Xue. Effects of MC4R Gene Silencing on Gene Expression in Bovine Fibroblast Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 403-415.

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