畜牧兽医学报2017，Vol.48Issue(3)：403-415,13.DOI:10.11843/j.issn.0366-6964.2017.03.003
MC4R基因沉默对牛成纤维细胞内基因表达的影响
Effects of MC4R Gene Silencing on Gene Expression in Bovine Fibroblast Cells
摘要
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 adipone(c)tin 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:UPK 1B,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.关键词
MC4R基因/基因敲降/mRNA表达量/相关性分析Key words
MC4R gene/gene knockdown/mRNA expression/correlation analysis分类
农业科技引用本文复制引用
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):403-415,13.基金项目
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) （ASTIP-IAS03）
