Different Expression Of Energy Metabolism Associated Gene In Bovine Fibroblast Cells With MC4R Gene Silencing

With the high speed development of the molecular biotechnology and achievement of more and more genome drafts, Post-genome era is coming. Marker assistant selection and whole genome wide selection is attracting much attention, and the screening and research of function gene which control economic traits become focus. MC4R played a pivotal role in inhibition of controlling meal size, fat deposition and growth, and adjustment of energy homeostasis in mammals, but the regulation and molecular mechanism of MC4R is unclear. The herd of 245 cattle was used to validate the effect of MC4R in economic traits. The expression of MC4R was validated to be relative with backfat thickness in 30 simmenta cattle selected from 136 simmenta cattle. The expression of MC4R was inhibited by lentivirus-mediated RNA Interference in bovine fibroblast cells. The expression profiles of MC4R in different inhibition level were drew by RNA deep sequencing, and the data of SNP chip was used to validate the effect of different expression gene in the herd of 136 cattle. The results were as follows:1. 5 SNPs were found in MC4R, one of them located in CDS region and 4 located in 3’ UTR region. The polymorphism of G1069C locus was significantly associated with backfat thickness, and the cattle with GG and GC genotype has more thickness than the one with CC genotype (P<0.01). The result of Real-time PCR indicated that MC4R was high expression in testis and lymph, the moderate expression in heart, kidney, muscle, adipose and thymus, and the low expression in liver, spleen, lung and intestinal. MC4R mRNA abundances in adipose were negative correlative with backfat thickness (P<0.05) and had no correlation with carcass weight (P>0.05).2. Lentivirus particles including MC4R were constructed successfully and packaged. They were transfected efficiently into bovine fibroblast cells. The result of Real-time PCR indicated that MC4R was significantly knockdown and the expression of POMC was also down-regulated, the expression of AGRP and MC3R were up-regulated.3. The result of RNA deep sequencing and GO annotation cluster analysis indicated that, 29 different expression gene of energy metabolites and regulation process were screened with different MC4R-knockdown level in bovine fibroblast cells. And these different expression genes referred to many biology processes including cellular hormone metabolic process, multicellular organismal catabolic process, positive regulation of macromolecule biosynthetic process, GTPase activity, sugar binding. For validating the reliability of RNA-seq result, 10 genes which all differently expressed in different MC4R-knockdown level sequencing group were picked out randomly. The result of Real-time PCR indicated that all the 10 genes change entirely consisted with the tendency of RNA-seq, which was proved to be accurated and reliable. The 14 SNPs of 8 genes had significant effect on carcass traits by the blast of those 29 different expression genes with the data of SNP chip. TGFB3 had significant effect on carcass weight, loin meat thickness, thigh meat thickness, lion-eye area, ribeye weight, striloin weight, tenderloin weight. KRT17 had significant effect on shearing force and carcass weight. MASP1 had significant effect on meat color, carcass depth and ham girth. COLEC12 had significant effect on ribeye weight and striloin weight. RAB15 had significant effect on carcass depth, RHOJ had significant effect on lion-eye area, and VCAN had significant effect on shearing force.In our research, plenty of candidate genes related with growth and meat quality traits were screened, which were implied to have relation with fat deposition and energy homeostasis. And the new method of energy homeostasis regulation molecular mechanism research was bringing forward.