Phenotypic Genetic Analysis Of MSTN-Edited Meishan Pigs By Integrated Omics

Skeletal muscle is the main source of meat products for carnivores and an important metabolic organ.Therefore,the molecular mechanism of studying skeletal muscle growth and development is of great significance.MSTN is an important muscle regulator that negatively regulates growth and development of skeletal muscle.MSTN is an important muscle factor that can negatively regulate the growth and development of skeletal muscle.MSTN editing Meishan pig is the research object this study,which uses zinc finger nuclease technology(ZFN)to modify the MSTN of Meishan pig,MSTN editing Meishan pigs have obvious double muscle phenotype and have the characteristics which is high lean meat rate,low fat rate and fast growth rate compared with wild Meishan pig.In order to explore the reasons for MSTN editing Meishan pig muscle hypertrophy and muscle fiber transformation,we accomplish RNA-Seq,miRNA-Seq and iTRAQ sequencing analysis of longissimus dorsi muscle of 4 MSTN-edited Meishan pigs and 4 wild Meishan pigs in 65-days of the embryonic period.The differentially expressed genes,miRNAs and differentially expressed proteins identified associated with pig growth and development,and miRNA-95 was verified.The results of the analysis will provide a theoretical basis for further research on the growth and development of skeletal muscle in pigs.The main findings are as follows:1.the results of transcriptome sequencing of the longissimus dorsi muscle showed that 438 differentially expressed genes were identified in MSTN edited Meishan pig group compared with wild Meishan pig group.And functional analysis of these differentially expressed genes found that they are mainly involved in the formation of muscle fiber,muscle fiber contraction,muscle fiber transformation,glucose metabolism process.At the same time,102 differential genes related to muscle growth were identified,which were mainly involved in myocyte differentiation,muscle tissue development,myofibrils and other processes,including XK,ACTC1,IGFBP3,LRP4,METTL8,MYH7 and AIMP2.2.the results of miRNA sequencing of the longissimus dorsi muscle showed that 20 significant differentially expressed miRNAs were identified.Target genes are mainly enriched in signaling pathways such as AMPK,mTOR and TGF-beta.3.Through the integration analysis of mRNA and miRNA,miRNA-208 b,miRNA-499 and miRNA-95,as well as LRP4,METTL8 and AIMP2,which were identified as key miRNAs and key candidate genes related to skeletal muscle growth and development.4.We initially verified miRNA-95 biological function in myoblasts that miRNA-95 can promote myoblast differentiation that miRNA-95 can down-regulate AIMP2 expression and promote C2C12 myogenic differentiation.Overexpression of miRNA-95 post-differentiation,marker gene up-regulated,inhibition of miRNA-95 down-regulates the differentiation marker gene,Through bioinformatics analysis and Dual luciferase reporter gene detection,we found that AIMP2 is a target gene of miRNA-95 in myoblasts,and at the same time,by interfering with the endogenous expression of AIMP2,the negative regulation of AIMP2 in myoblast differentiation is found.5.A total of 2446 proteins were identified by iTRAQ sequencing analysis and which had 66 differentially expressed proteins,including TNNT1,MYH1,MYH7,MYL9,TPM1,TNNI3,TNNI1,TNNC1 and TNNT2,which are mainly enriched in skeletal muscle phylogeny and growth and glycolysis,are likely to be important proteins regulating muscle growth in pigs.6.The integration analysis of the iTRAQ and transcriptome revealed that TNNT1 and MYL9 expression were significantly different and consistently expressed at the transcriptional and protein levels.These two genes are involved in skeletal muscle growth and muscle fiber transformation and may be important candidate genes.In summary,comprehensively analyzed the mRNA,miRNA and protein of the longissimus dorsi muscle at the embryonic stage for 65 days,we identified some differentially expressed genes,differentially expressed miRNAs and differentially expressed proteins related to skeletal muscle growth and development and which provides a new direction and goal for further study about growth of pig.