Biological Effects Of CKM Gene On Proliferation And Differentiation Of Chicken Myoblasts

In recent years,the study of skeletal muscle growth and development has been regarded as a breakthrough point for animal breeding improvement in animal husbandry.As an important economic tissue in meat producing animals,the proper development of skeletal muscle is positively correlated with the increase of meat production,and meat producing traits have been a hot spot in chicken genetics and breeding research.Studies have shown that muscle-specific Creatine Kinase(LOC107051134,Creatine kinase M-type-like)is located on chicken chromosome 5 and is directly related to intracellular energy transport,muscle contraction and ATP regeneration.CKM is most abundant in skeletal muscle.The abundance of CK-M in skeletal muscle means that it is the most important CK subtype in energy regulation during exercise,and its role in cellular energy homeostasis makes it an important candidate gene.Therefore,CKM gene plays an extremely important role in the growth and development of skeletal muscle,but its role in skeletal muscle development of chicken is still unclear.Therefore,in order to explore whether THE same function of CKM exists in chickens,this study mainly focused on AA Broilers(AA).Firstly,the expression of CKM gene in different tissues and different periods of chicken was investigated.At the cellular level,chicken primary myoblasts were isolated and cultured in vitro and induced differentiation.The effects of CKM gene on proliferation,apoptosis and ifferentiation of chicken myoblasts were analyzed by using overexpression and RNA interference techniques.Finally,the differentially expressed genes in interfering CKM myoblasts were identified by transcriptome sequencing,and the differentially expressed genes were analyzed by KEGG and GO to further explore the effects of CKM gene on skeletal muscle development during chicken growth and development.The main results of this study are as follows:Experiment 1:Study on expression characteristics of specificity highly expressed genes in chicken muscle tissueThrough the integration and analysis of expression data of different tissues in chicken,it was found that CKM(LOC107051134,Creatine kinase M-type-like)was specificity highly expressed in chicken muscle tissue.The full length sequence of CKM was 1321bp by RACE.The CDS region containing 395bp 5 ’UTR and 1146bp could encode 38 amino acid residues with 674bp 3’ UTR.Specific primers were designed and verified.Real-time quantitative PCR was used to detect the high expression of CKM gene in muscle tissue of 1WAA chickens,and the highest expression was found in leg muscle.In embryonic stage(E10,E12,E14,E16,E18),the pectoralis muscle and leg muscle showed an upward trend,but showed a downward trend after embryonic stage(1W,3W).The expression level was highest in E16 period.Experiment 2:Study on the regulation of CKM gene on proliferation and apoptosis of chicken myoblastsChicken primary myoblasts were isolated and verified in vitro,and overexpressed CKM vector was constructed to synthesize small RNA interference(siRNA)fragments with high interference efficiency and specific targeting of CKM gene.After overexpression of CKM,CCK8 results showed that CKM group was significantly lower than CKM-PC3.1 group over time(P<0.05).EDU results showed that the number of cells in CKM group was significantly lower than that in CKM-PC3.1 group.Flow cytometry showed that compared with the control group,cells in G1 phase were up-regulated and cells in S phase were down-regulated.Flow cytometry showed that apoptosis was significantly up-regulated after overexpression of CKM(P<0.01).Fluorescence quantitative PCR results showed that CCNB2,CCND1,PCNA gene expression was significantly down-regulated(P<0.05),P21,Caspase3 gene expression was significantly up-regulated(P<0.01),and Caspase9 expression was significantly increased(P<0.05).The opposite effect was observed after interference with CKM.Experiment 3:Study on the regulation of CKM gene on differentiation of chicken myoblastsIsolation and differentiation of chicken primary myoblasts;RT-qPCR was used to detect the mRNA expression changes of cells growing to 50%,100%and 1,2,3,4,5 and 7 days after differentiation.The results showed that the overall expression trends of CKM and MyHc genes were consistent,showing a trend of first decreasing,then increasing and then decreasing,and the expression levels reached the lowest at day 1 of differentiation.CKM gene and MyHc gene peaked at day 4 and day 3 of differentiation,respectively.After overexpression of CKM gene,the results of RT-qPCR showed that MYOD,MYOG and MyHc were significantly increased(P<0.01).Desmin immunofluorescence results showed that the number of differentiation of myoblasts into myoducts was significantly increased after CKM treatment,and the formation of muscle bundles was more obvious.Western Blot analysis showed that the protein expression levels of CKM and MyHc were significantly increased after overexpression.The opposite effect was observed after interference with CKM.Experiment 4:Transcriptome sequencing was used to verify the effect of CKM on myoblast developmentA total of 39.08Gb Clean Data were obtained from chicken myocytes which interfered with CKM gene.438 differentially expressedgenes(DEGs)were screened,including 81 upregulation and 357 downregulation.CKM is also one of the down-regulated genes;The transcriptome sequencing results were verified by RT-qPCR,and the results showed that the changes of mRNA relative expression were consistent with the sequencing results.Twelve differentially expressed genes CAPN3,PDK4,RBM24,SMYD1,KLHL41,KLHL40,STAC3,FAM65B,LMOD3,ANKRD1,EGF and CAMK2A,were screened out in this study.GO enrichment analysis showed that the differential genes were mainly enriched in cardiac myofibrils,myofibrils,creatine phosphate biosynthesis,skeletal muscle fiber development,the regulation of myoblast differentiation and creatine kinase activity.KEGG analysis showed that the protein was mainly enriched in MAPK signaling pathway,ErbB signaling pathway,calcium signaling pathway,Apelin signaling pathway,actin cytoskeleton regulation,and other related signaling pathways involved in the regulation of skeletal muscle development.In conclusion,the specific expression of CKM in chicken muscle can promote the apoptosis and differentiation of chicken myoblasts by inhibiting the proliferation of chicken myoblasts.Meanwhile,transcriptome analysis was used to further verify the expression of differentially expressed genes involved in muscle growth and development.This study provides theoretical and experimental basis for further elucidating the molecular mechanism of CKM gene regulating chicken muscle growth,and provides reference for improving and developing molecular breeding of broilers.