Transcriptiome Analysis And Validation Of Skeletal Muscle Injury And Repair In Megalobrama Amblycephala

Skeletal muscle is the most plastic and athletic tissues in the animal organism.Its growth and development are closely related to the proliferation and differentiation of satellite-like cells.Myogenesis and injury-induced muscle regeneration contribute to muscle formation.Muscle regeneration is regulated by multiple genes and biological pathways.In order to study the molecular mechanism of regeneration after muscle injury,in this study,immunohistochemistry and transcriptome sequencing（RNA-seq）analysis were performed after muscle injury by inserting needle in Megalobrama amblycephala.The main results are as follows:（1）Histopathlological structure of M.amblycephala muscle was observed that the muscle of the control group exhibited a well-organized tubular structure,and myofibers were normally arranged.After 48,144 and 216 hpi（hours post-injury,hpi）of mechanical injury,the muscle exhibited obvious damages with an evident disconnection at the injury sites among myofibers,but injured muscle was repairing and generating as time increase.It was worth mentioning that severe injury showed a larger range of injury than mild injury.（2）To determine whether M.amblycephala Pax7⁺satellite cells were activated in injury sites,injury model of skeletal muscle by needle stick was constructed,and fish were sacrificed at 48～216 hpi to assess whether satellite-like cells were activated after muscle injury.Compared to the control,it was obvious that the cells proliferated at injury sites.Pax7⁺satellite-like cells existed at injury sites,and peaked at 96 to 144 hpi.The proliferation of Pax7⁺satellite-like cells in severe injury group was more obvious than in mild injury group at 96 hpi.Compared to the mild injury group,it could indicate that satellite-like cells responded to stimulation more quickly for severe injury.（3）Statistical assessment of transcripts revealed totally 6729 DEGs were identified,including 2125 up-regulated and 4604 down-regulated genes between mechanical injury and the control group.Go-enriched analysis showed that DEGs were concentrated in intercellular connections,signaling transduction and enzyme activities etc.KEGG enrichment analysis showed that most of the pathways were related to immunity,metabolism and cell related molecules,including actin skeleton regulation,Epstein Barr virus infection and plaque adhesion pathway etc.（4）The protein interaction network showed that actin cytoskeleton-related genes were critical in the entire cytoskeleton network,indicating that the synthesis of muscle fibers is closely related to the regulation of actin.In the lipid metabolism pathway,genes related to glycolysis/gluconeogenesis metabolism were also important during myofibers repair and regeneration.Five modules were identified by the weighted gene co-expression network,and further analysis showed many pathways related to muscle development and energy metabolism were enriched in the Brown module.Visual analysis of Brown module was carried out to screen Hub genes,and revealed that myosin related genes such as mylpf,myl and myh,and lipid and glucose metabolism related genes such as pgm1,pfkma and pfkmb probably acted a crucial effect on the whole network.（5）The knockout zebrafish of the cfl1 gene was constructed by CRISPR/cas9technology.At present,F₂generation individuals with deletion of two base have been obtained and further function analysis was performed.