| In some countries,snakes are a valuable source of important protein for the human diet and their economic value depends mostly on muscle production.The king ratsnake(Elaphe carinata)is a large-sized,oviparous colubrid snake found in China and other countries.The growth and development of king ratsnake embryonic muscles are rarely reported.There are different growth phenomena of king ratsnakes under the same feeding conditions.Which genes,pathways and miRNAs regulate muscle differential growth,is unclear.The purpose of this study:(1)Identification of genes and pathways related to muscle growth and development at different developmental stages of embryo and post-embryo based on the transcriptome.(2)A growth curve is constructed for the 6 months after the commencement of eating to determine the time at which the weights of king ratsnakes diverge.The morphological characteristics of the muscle tissue of the fast-growing and slow-growing king ratsnakes are compared.(3)The miRNA and mRNA transcriptomes of the king ratsnake muscle tissues are determined with next-generation sequencing(NGS)technology.The differential expression profiles of the mRNAs and miRNAs,the candidate genes and miRNAs related to muscle growth,and the pathways that regulate muscle growth and development in the fast-growing and slow-growing snakes are preliminarily determined.The main results are as follows:1.Analysis of mRNA expression profile of skeletal muscle development at different developmental stages(1)Differentially expressed genes related to muscle growth and development at different developmental stages.A total of 3254 DEGs were identified in the comparison of muscle samples between the myotube fusion stage(S11)and the primary muscle fiber formation stage(S12)(SZEM11 VS SZEM12).178 genes related to muscle growth and development were screened by GO enrichment,including MYOD1,MYOG,IGFN1,MYH7 B and MYOZ3.Seven genes have higher expression in the S11,171 genes were up-regulated in the S12.These genes may play a key role in myotubes' differentiation and formation of primary muscle fibers of king ratsnake embryos.2362 DEGs were identified in the comparison of muscle samples between the primary muscle fiber formation stage(S12)and the secondary muscle fiber formation stage(S13)(SZEM12 VS SZEM13).131 genes related to muscle growth and development were screened by GO enrichment,including MYH6,MYH9,MYOC,MYH2 and EP300.Twenty-two showed higher expression in the S12,109 genes were up-regulated in the S13.These genes may play a key role in the myotubes differentiation and the formation of secondary muscle fibers of king ratsnake embryos.4330 DEGs were identified in the comparison of muscle samples between the secondary muscle fiber formation stage(S13)and the myofibers formation stage(S14)(SZEM13 VS SZEM14).169 genes related to muscle growth and development were screened by GO enrichment,including MYH2,MYH1 B,MYH4,MYH1 and PLN.The 54 genes showed higher expression in the S13.115 genes were up-regulated in the S14.These genes may play a key role in formation of different types of muscle fibers of king ratsnake muscle.4876 DEGs were identified in the comparison of muscle samples between the myofibers formation stage(S14)and stage 15(S15)(SZEM14 VS SZEM15).206 genes related to muscle growth and development were screened by GO enrichment,including TNNT2,MYH3,MYH9,EP300,and MYH11.142 genes showed higher expression in the S14.64 genes were upregulated in the S15.These genes may affect the differentiation of myotubes and the further maturation of muscle fibers.9124 DEGs were identified in the comparison of muscle samples between stage 15(S15)and the stage to be hatched(S16)(SZEM15 VS SZEM16).370 genes related to muscle growth and development were screened by GO enrichment,including MYOC,FBLIM1,PLN,ABRA and MYH2.129 genes showed higher expression in the S15,241 genes were up-regulated in the S16.6474 DEGs were identified in the comparison of muscle samples between the stage to be hatched(S16)and one month after hatching(SL1)(SZEM16 VS SZLM1).281 genes related to muscle growth and development were screened by GO enrichment,including MYH1,COL11A1,MYH16,TRIM63,and MYO18 A.165 genes showed higher expression in the S16.116 genes were up-regulated in the SL1.5001 DEGs were identified in the comparison of muscle samples between one month after hatching(SL1)and six months after hatching(SL6)(SZLM1 VS SZLM6).221 genes related to muscle growth and development were screened by GO enrichment,including MYH3,MYOD1,COL11A1,SRPK3,and COL5A1.97 genes showed higher expression in the SL1,124 genes were up-regulated in the SL6.These genes may promote myotube differentiation and further hypertrophy of muscle fibers to promote muscle growth and enhance athletic performance.(2)Pathways related to muscle growth and development at different developmental stages.34 KEGG pathways obtained in the comparison of muscle samples between the myotube fusion stage(S11)and the primary muscle fiber formation stage(S12)(SZEM11 VS SZEM12).Among them,7 KEGG pathways were related to muscle growth and development,including ECM-receptor interaction,focal adhesion,tight junction,MAPK signaling pathway,and oxytocin signaling pathway.These pathways may affect the myotubes differentiation and the formation of primary muscle fibers of king ratsnake embryos.27 KEGG pathways obtained in the comparison of muscle samples between the primary muscle fiber formation stage(S12)and the secondary muscle fiber formation stage(S13)(SZEM12 VS SZEM13).Among them,6 KEGG pathways were related to muscle growth and development,including ECM-receptor interaction,focal adhesion,tight junction,TGF-beta signaling pathway,and PI3K-Akt signaling pathway.These pathways may play a key role in the myotubes differentiation and the formation of secondary muscle fibers of king ratsnake embryos.29 KEGG pathways obtained in the comparison of muscle samples between the secondary muscle fiber formation stage(S13)and the myofibers formation stage(S14)(SZEM13 VS SZEM14).Among them,5 KEGG pathways were related to muscle growth and development,including ECM-receptor interaction,focal adhesion,p53 signaling pathway,PPAR signaling pathway,and TGF-beta signaling pathway.These pathways may play a key role in formation of different types of muscle fibers of king ratsnake muscle.19 KEGG pathways obtained in the comparison of muscle samples between the myofibers formation stage(S14)and stage 15(S15)(SZEM14 VS SZEM15).Among them,3 KEGG pathways were related to muscle growth and development,including ECM-receptor interaction,tight junction,and TGF-beta signaling pathway.These pathways may affect the differentiation of myotubes and the further maturation of muscle fibers.37 KEGG pathways obtained in the comparison of muscle samples between stage 15(S15)and the stage to be hatched(S16)(SZEM15 VS SZEM16).Among them,7 KEGG pathways were related to muscle growth and development,including the PPAR signaling pathway,tight junction,focal adhesion,Wnt signaling pathway,and ECM-receptor interaction.14 KEGG pathways obtained in the comparison of muscle samples between the stage to be hatched(S16)and one month after hatching(SL1)(SZEM16 VS SZLM1).Among them,2KEGG pathways were related to muscle growth and development,including ECM-receptor interaction,and focal adhesion.21 KEGG pathways obtained in the comparison of muscle samples between one month after hatching(SL1)and six months after hatching(SL6)(SZLM1 VS SZLM6).Among them,3KEGG pathways were related to muscle growth and development,including ECM-receptor interaction,glycolysis/gluconeogenesis,and tight junction.These pathways may promote myotube differentiation and further hypertrophy of muscle fibers to promote muscle growth and enhance athletic performance.ECM-receptor interaction was enriched in each stage.Other pathways differ between different expression profiles and play specific regulatory roles at different developmental stages.2.Transcriptome analysis of muscle growth between the two traits fast-growing individuals(FGIs)and slow-growing individuals(SGIs)of 3-month-old king ratsnakes(1)Muscle differential growth and morphological characteristics of king ratsnakeThere were no significant differences in the weight of the one-month-old king ratsnake,but at the age of 3-month-old,there were significant differences in physical indexes between traits,and the FGIs have significantly high weight than the SGIs.H&E-stained sections showed the anatomic structures of the muscles,and the myolins of the FGI and SGI muscles were all in the form of myofibers and myotubes.At 3-month-old,the muscle fiber cross-section area and diameter of skeletal muscle in the FGI were larger than SGI.(2)Analysis of DEGs related to muscles development608 DEGs were identified in the comparison of muscle growth differences between fastand slow-growing king ratsnakes(SZFM VS SZMM).Among them,48 of which were related to muscle growth and development.The 37 DEGs in the fast-growing individuals show significantly higher expression levels than those in the slow-growing individuals,including COL11A1,COL1A2,SRPK3,BCAN and EP300.This DEGs upregulated in FGIs may be related to muscle growth in the king ratsnake.The expression levels of 11 DEGs were significantly higher in the SGIs than in the FGIs,including OBSCN,NEB,TP63,TSC1,and MYO18 A.These genes may cause SGIs to grow slowly.(3)Analysis of DEGs related to protein-synthesis and degradationProtein synthesis and degradation processes are associated with the regulation of skeletal muscle mass.When the rate of protein synthesis exceeds then the rate of protein degradation,the protein accumulates and the muscle fiber volume increases.Six hundred eight DEGs were enriched in 14 KEGG pathways(P < 0.05),including PI3K–AKT–m TOR signal pathway.Seven DEGs are enriched on this pathway.The expression levels of TSC2 and TSC1 were higher in the SGIs than in the FGIs.ADAMTS2,MFAP4,ANGPT4,ANGPTL2,and ANGPTL5 were higher in the FGIs.It is implied that this pathway may promote the synthesis of protein in FGIs.The ubiquitin-proteasome pathway is the major protein degradation pathway and regulates the degradation of up to 80% of cellular proteins.Six hundred eight DEGs were enriched in 248 GO terms(P < 0.05),including ubiquitin-proteasome pathway.Eleven DEGs were associated with protein degradation.The expression levels of 10 DEGs(except RNF115)related to the ubiquitin-proteasome pathway,were significantly higher in the SGIs than in the FGIs,including USP25,PSMD14,ATG3,PSMD4,and TRIM63.It is suggested that the specific ubiquitination pathway may promote the degradation of muscle proteins in SGIs.Consequently,faster protein accumulation in FGIs results in faster growth.(4)Identification of differentially expressed miRNAs and miRNA–mRNA interaction analysis are associated with muscle growth698 miRNA were identified,including 125 novel miRNAs.63 differentially expressed miRNA were screened(P < 0.05)between the fast and slow-growing individuals.Among them,27 miRNAs were significantly up-regulated and 36 were down-regulated in the SGI,including miR-125,miR-92,let-7b,miR-10,miR-22,miR-26 and miR-24.A total of 950 miRNA–mRNA interaction sites correlated negatively with the 63 DEMs and 608 DEGs were predicted.Among these,113 miRNA–mRNA interaction sites correlated negatively with the 63 DEMs and 48 DEGs that are associated with muscle growth;24 miRNA–mRNA interaction sites correlated negatively with the 63 DEMs and seven DEGs associated with protein synthesis;and 33 miRNA–mRNA interaction sites correlated negatively with the 63 DEMs and 11 DEGs associated with protein degradation.It is implied that miRNAs regulate DEGs associated with muscle development in the king ratsnake and provide a theoretical molecular basis for breeding and parent selection in these reptiles and for further study of muscle growth.The above research results are analyzed from the perspective of different development stages and differential growth traits in the same stage of a species,which provides basic data for revealing the general laws of reptile muscle growth and development,and lays the foundation for the subsequent in-depth study of king ratsnake phenotype differences and the selection of parents of breeding. |
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