Functional Analysis And Association Studies Of Bovine MYOT Gene With Meat Quality

The main objective of the present studywas to study the molecular mechanisms ofMYOT gene expression and its associations with beef production traits.Land for agriculture is reducing while the population of the world is increasing atunprecedented rate, this necessitates researches to increase production rate per animal bothfrom quality and quantity standpoint. Myot gene is a multifunctional gene involving in muscledevelopment, which makes it a candidate gene for the improvement of meat quantity andquality. Understanding the molecular mechanisms and association study of the gene isexpected to contribute in expediting genetic improvement by Marker assisted selection andother molecular techniques.In the current study, the full length of bovine MYOT gene cDNA spanning “2234” bpwassuccessfully cloned using skeletal muscle tissue and we have conducted sequential ofmethods to investigate the effect of the gene on muscle cells.1.Real-time PCR analysisrevealed that the gene is highly expressed in skeletal muscle followed by heart and heart fattissues.2.Bioinformatics analysis showed that bovine MYOT protein contain “8” differentconserved functional domains including two potential Ig-like domains, three N-glycosylationsites, eight protein kinase C phosphorylation sites, six potential Casein kinase IIphosphorylation sites, one tyrosine kinase phosphorylation site, seven N-myristoylation sites,one Endoplasmic reticulum targeting sequence and one cell attachment sequence site.Furthermore, amino-acid sequences comparison of the gene showed higher phylogeneticsimilarity among mammals.3. Polymorphism analysis of the gene sequence showed three SNPsnamed T152C(intron5), A142C (exon2) and T120C (exon8).Both A142C and T120CSNPs aresynonymous mutation and could affect phenotypictraits. Association analysis of the SNPsrevealed significant associations with loin muscle area, back fat thickness and Intra-muscularfat (P <0.05) in Chinese Qinchuan cattle breed with C allele (T152C) and T allele(T120C)being the beneficial contributor to the gene pool.4. We have further investigated the longitudinal expression profile of the MYOT gene and its corresponding cell differentiation process, during myotube formation. Thedifferentiation process was carried out using a cell line we established ourselves from abovine muscle sample. Two different culturing media were used to induce the cells’differentiation and the medium containing2%of horse serum was found to be more efficient.Real-time PCR analysis revealed initiation of expression of MYOT gene on day3ofdifferentiation and scored a peak on day6. This is identical to the expression pattern exhibitby three other myotube markers included in this experiment (MYOG, MYL1and MYF6).Hence, MYOT gene may play a pivotal role in myoblast fusion and could also be consideredas myotube marker. As showed by western blot analysis protein expression of the gene startedfrom day6of differentiation and increased slightly with the increase in the number of days.5. Lentivirus-mediated down regulation of MYOT has inhibited significantly theexpression of type I (slow-oxidative) and type IIA (fastoxidative) muscle fiber types.However the inhibition of the gene has lead to the increase in expression of type IIX (fastglycolytic) muscle fiber. These suggest thatMYOT gene could affect muscle fibercharacteristics and subsequently sensory quality of cooked meat.In conclusion our findings showed that Myot gene is essential for muscle regulation andits SNPs can be used as genetic markers to expedite genetic change using marker-assistedselection in cattle breeding.