Screening Of MRNA,lncRNA,miRNA And Partial Functional Analysis Of Chicken Pectoralis Major Muscle And Sassinius Muscle Fiber Types

Muscle fiber is the basic constituent unit of skeletal muscle.Different types of muscle fiber make skeletal muscle have specific physiological characteristics and functions.Studies have shown that muscle fiber type is closely related to meat quality.The higher level of oxidative muscle fiber(slow muscle)and the lower level of glycolytic muscle fiber(fast muscle)contributes to the better main quality index of muscle,such as p H,flesh color,water holding capacity,intramuscular fat(IMF)and flavor compounds.However,the regulation mechanism of different muscle fiber types has not been well clarified.In order to study the regulation of muscle fiber types,the glycolytic muscle pectoralis major(PMM)and oxidative muscle sartorius(SART)of Qingyuan partridge chickens were used as the research materials.RNA-seq technique was used to screen differentially expressed mRNA,lncRNA and microRNAs,which were used to build interactive networks,and that we can find the important factors for the next step of functional verification.The main results as following:1.MRNA and lncRNA sequencing were performed in PMM and SART muscles.The results showed that there are 3457 differentially expressed mRNA including 2346up-regulated and 1093 down-regulated in SART.Functional enrichment analysis showed that differentially expressed mRNAs that mainly involved in biological processes such as energy metabolism,blood circulation,muscle development and contraction,as well as involved in the variety of energy metabolism-related pathways,including oxidative phosphorylation,carbon metabolism and glycolysis/gluconeogenesis.A total of 365 differentially expressed lncRNA were screened out in PMM and SART,among which 169 were up-regulated and 196 were down-regulated in SART.Functional enrichment analysis illustrated that differentially expressed lncRNA was mainly involved in GO entries and MAPK signaling pathways related to muscle growth and development.According to mRNA-lncRNA interaction networks analysis,results demonstrated that XR?003077811.1,XR?003072304.1,XR?001465942.2,XR?001465741.2,XR?001470487.1,XR?003077673.1 ? XR?003074785.1 may play an important role in the regulation of muscle fiber type.2.MiRNA sequencing was performed in PMM and SART muscles.Results showed that a total of 698 miRNA were screened in PMM and SART,among which 189 were new miRNAs.Differentially expression analysis illustrated that there are 67 differentially expressed miRNAs including 42 up-regulated and 25 down-regulated in SART between the two groups.Functional enrichment analysis revealed that differentially expressed miRNAs were involved in energy metabolism,muscle contraction,PPAR,insulin and adipocytokine signal transduction pathways.Four candidate miRNA-gene pairs,which were gga-mi R-193a-3p/PPARGC1 A,gga-mi R-221-5p/CSRP3,gga-mi R-196-5p/CALM1 and gga-mi R-499-5p/SOX6,that may affect muscle fiber traits were identified by miRNA-mRNA interaction analysis and dual-luciferase reporter gene assay.The lncRNA-miRNA-mRNA interaction network was further constructed,and a total of 44 pairs of regulatory relationships were screened out,such as ln CXR?003074785.1/mi R-193-3p/PPARGC1 A and XR?001468684.2/GGA-mi R-499-5p/SOX6.The results of protein interaction network showed that CSRP3 interacts with MYOM,MYOZ2,PRKAG2,MYBPC1,TNN1,MUSTN1,MYH7 B,MYH1D,MYHIE,MYL10 and other muscle-related genes,suggesting that CSRP3 may play an important role in muscle development.3.The function of PPARGC1 A gene and gga-mi R-193a-3p was verified in chicken primary myoblast cells.After overexpression of PPARGC1 A gene,the mRNA and protein expressions of PPARGC1 A gene were up-regulated,and the expression of differentiation key genes such as PAX7,Myo D and Myo G,calcineurin signaling pathway genes such as CNAA and MEF2 C and myosin heavy chain gene MYH7 B were up-regulated.The expression of myosin heavy chain gene My H1 F was down-regulated.After the expression of PPARGC1 A gene was interfered,the results were contrary to the results of overexpression,the expression of key differentiation genes such as PAX7,Myo D and Myo G,the calcineurin signaling pathway genes such as CNA? and MEF2 C,and the expression of myosin heavy chain gene My H7 b in slow muscle were all down-regulated,while the expression of myosin heavy chain gene My H1 F in fast muscle was up-regulated.After overexpression of gga-mi R-193 a in chicken primary myoblast cells,the expression of its target gene PPARGC1 A was inhibited,and the expression of related differentiation genes such as MYOD and MYOG,calcineulin phosphatase signaling pathway genes such as CNAA and MEF2 C was down-regulated,which was contrary to the results of PPARGC1 A.These results showed that PPARGC1 A was involved in the regulation of myocyte differentiation,possibly regulating muscle fiber type specificity through calcineurin phosphatase signaling pathway.Gga-mi R-193 a may be involved in the regulation of myocyte differentiation and muscle fiber type specificity by regulating the expression of PPARGC1 A gene.4.The function of the CSRP3 gene was verified by interfering in chicken primary myoblast cells.The expression of CSRP3 increased with the progression of cell differentiation in myoblasts.After interfering with the expression of CSRP3,the expressions of key differentiation genes such as MYOD and MYOG were down-regulated,the expression of myosin heavy chain gene My H1 E was up-regulated,these results showed that CSRP3 gene may be involved in regulating the differentiation of chicken myoblast cells.In conclusion,mRNA,lncRNA and miRNA all play important roles in the skeletal muscle fiber types of chickens.The regulation mechanism of PPARGC1 a,CSRP3 and gga-mi R-193 a on chicken fiber type was proved,which provided theoretical support for the genetic improvement of chicken quality traits.