Transcriptome And Dna Methylation Analyses Of The Molecular Mechanisms Underlying With Longissimus Dorsi Muscles At Different Stages Of Development In The Yak

As one of the livestock breeds living on the plateau,yak's meat products are high in protein,low in fat and rich in various unsaturated fatty acids.However,yak's special living environment and seven-month dry grass period make yak grow and gain weight in waves.Skeletal muscle development is the focus of yak weight gain and genetic selection.In this study,yaks were taken as the research object,and two experimental studies were carried out by using modern biological technologies such as transcriptome sequencing(RNA-seq),methylation modification dependent endonuclease sequencing(methyl-rad),real-time fluorescence quantitative PCR(Q-pcr),Western blot,gene cloning,cell culture and so on.this research have two aspects: first,yak were studied in different growth period of skeletal muscle protein coding RNA(m RNA)and long chain non-coding RNA(LncRNA)at the genome level expression,DNA methylation level changes,preliminarily explores the regulatory mechanism of DNA methylation in muscle growth,and according to the protein-coding RNA and DNA methylation data to find six key genes affect muscle development.Two genes related to muscle growth and development(RXRA and BIN1)were studied in the apparent transcriptional regulation patterns of mouse myoblast C2C12 differentiation.The results of this study provide a new perspective for further understanding the molecular mechanism of yak muscle development and provide a reference for screening and identification of candidate genes for yak meat producing traits.The main research contents and results are as follows:1.The differential protein coding genes of the longest dorsal muscle of yak were analyzed at different development stages,and the threshold value was set as | log2 FC | > 1.2,padj < 0.01.The results showed that: 7079 differentially expressed genes were screened in the 6-month-old group and the fetal cow group(M-vs.-E).The 1237 differentially expressed genes were screened in the 6-month age group and the adult group(M-vs.-A).6,401 differentially expressed genes were screened in the adult and fetal cow groups(A-vs.-E).There were 3096 up-regulated and 3983 down-regulated genes in the m-vs.-e comparison group,517 up-regulated and 720 down-regulated genes in the m-vs.-a comparison group,and 3017 up-regulated and 3348 down-regulated genes in the a-vs.-e comparison group.2.According to the analysis of protein-coding RNA data,it was found that there were 381co-expressed differentially expressed genes belonging to the three comparison groups.Moreover,functional annotation was carried out on the genes screened above,and many GO entries and KEGG pathways were found to be related to energy metabolism,muscle cell differentiation and fat deposition.For example GO: 0014706(striated muscle tissue development)? GO:0030049(muscle filaments sliding)? GO:0003012(muscle system process)?GO:0006941(striated muscle contraction)?GO:043292(elastic fiber)? GO:0042692(muscle cell differentiation)? GO:0060537(muscle tissue development)?GO: 0001666(reaction to hypoxia)?Bom04068(FoxO signaling pathway)Bom04530(tightly connected)?Bom01100(metabolic pathway)?Bom03320(PPAR signal pathway)?Bom00561(glycerol metabolism)and bom04010(MAPK signaling pathway).3.According to the results of muscle genome-wide methylation,we preliminarily obtained the methylation map of the longest dorsal muscle of yak at different development stages,and analyzed some key methylation regions affecting muscle development.In order to better understand the methylation sequencing data,The two sets of data were integrated and the negative correlations between DEGs and differentially methylated promoters(DMPs)analyzed,which confirmed that TMEM8 C,IGF2,CACNA1 S,MUSTN1 RXRA and BIN1 were methylated in the promoter region and that expression of the modified genes was negatively correlated.It was initially determined that gene expression may be regulated by DNA methylation levels.4.Lnc RNA expression during skeletal muscle development in yak was analyzed by RNA sequencing at three development stages: three years(group A),six months(group M)and 90-day-old fetuses(group E).A total of 1,180 LncRNA were identified in the three development stages.Compared with group E,154 were upregulated and 130 were downregulated in group A.Compared with group A,31 were upregulated and 29 were downregulated in group M.Compared with group E,147 were upregulated and 149 were downregulated in group M(padj < 0.001,|log2FC|>1.2).In addition,functional annotation analysis based on gene ontology(GO)and the Kyoto protocol encyclopedia of genes and genomes(KEGG)database showed that differentially expressed LncRNA were cis-trans target genes.The results showed that differentially expressed LncRNA were mainly involved in PI3K-Akt signaling,Rap1 signaling,regulation of lipolysis in adipocytes,FoxO signaling and p53 signaling.Furthermore,RTL1,IGF2,MEF2 C,Pax7 and other well-known muscle development regulators were included in a co-expression network of differentially expressed target genes and LncRNA.These data will help to further clarify the function of LncRNA in the different stages of skeletal muscle developmental in yak.5.Based on the protein-coding mRNA transcriptome and genome-wide methylation data,candidate genes were selected,and two key genes,RXRA and BIN1,were selected for verification in a mouse myoblast cell line.The degree of inversion is inversely proportional,suggesting that the expression of the two genes in myoblast C2C12 is affected by the degree of methylation in the promoter region.The results of our study showed that there were a large number of genes with DNA methylation modification and mRNA/LncRNA expression differences in yak muscle tissue at different development stages,which were mainly related to the growth,differentiation and metabolism of muscle cells.Identified at the cell and the individual level is associated with muscle growth and development of functional genes RXRA and BIN1,studies these two key gene promoter methylation of regulation at a cellular level,thus systematically from aspects of the genetic and epigenetic regulation and muscle growth related gene function and regulation,In addition,the functional genes and regions with important value in breeding will cultivate new varieties of transgenic yak and its industrialization to provide genetic resources and technical support.