| Skeletal muscle,accounting for about 50 % of body weight,is one of the largest and most important tissues in animal.It participates in body movement,protection,and metabolic regulation.The growth performance of livestock and poultry,especially the growth of skeletal muscle,is one of the factors that determine the economic efficiency of farmers.Therefore,the research on skeletal muscle development of livestock and poultry is of great significance in animal husbandry.In China,the production for duck meat is second only to that of chicken,and people’s demand for duck meat is also increasing.In recent years,there are many studies on the development of poultry muscle during embryo stage.The mechanism of muscle fiber formation involves the activation of progenitor cells,the proliferation of progenitor cells into mononuclear myoblasts and the formation of multinuclear myotubes.In addition,many genes,non coding RNAs and transcription factors are also involved in the regulation of muscle proliferation and differentiation.During prenatal and very early postnatal development,muscle growth in vertebrates depends on an increasing number of muscle fibers(hyperplasia).After postnatal growth,the increase in skeletal muscle is mainly due to muscle hypertrophy,accompanied by the proliferation of satellite cells and then the new myonuclei is incorporated into existing myofibers.However,there is still unclear on the internal mechanism of regulating skeletal muscle growth and development in duck.In this study,the skeletal muscles(breast and leg muscles)of Peking duck,Hanzhong Ma duck and black Muscovy duck were used as research objects,and the developmental characteristics of breast and leg muscles at 17,19,21,24 and 27 day of embryonic period(black Muscovy duck also includes 31 and 34 day of embryonic period)and postnatal 6 month-old were compared and analysed.The expression levels of the genes(MyoG,MyoD,Myf5,MRF4,Pax3,Pax7 and MSTN)that related to the growth and development of duck skeletal muscle were detected.The variety differences of skeletal muscle between Peking duck and Hanzhong Ma duck,and the development changes of skeletal muscle in black Muscovy ducks at different growth stages were determined.The differentially expressed genes and related regulatory pathways during the growth and development of skeletal muscle in different duck breeds were screened using RNA-Seq.The tissue expression profile of FKBP5(differentially expressed gene,DEG)and the core activity region of its promoter were determined,and the regulatory mechanism of FKBP5 gene on the proliferation and differentiation of duck myoblasts was studied at the cellular level.In addition,the association between SNPs of FKBP5 gene and body weight of Muscovy ducks was discussed.The results are as follows:1 Study on the expression of genes related to muscle development and the size of skeletal muscle fiber in ducks at different growth stagesThere were differences in the expression of growth related genes in duck skeletal muscles at different growth stages.MRFs(MyoG,MyoD,Myf5 and MRF4),Pax3,Pax7 and MSTN were highly expressed in skeletal muscle of Peking duck,Hanzhong Ma duck and black Muscovy duck at embryonic stage,and the expression of these genes at embryonic stages was higher than that in adult duck,which may be one of the reasons for the differences in skeletal muscle weight and size of ducks.The results of morphological observation showed that the diameter and cross-sectional area of breast/leg muscle fiber increased gradually with the increase of time,while the number of breast/leg muscle fiber per unit area decreased gradually with the increase of time,indicating that the skeletal muscle of duck developed rapidly in the embryonic stage.2 Transcriptome analysis of breast and leg muscles in ducks at different growth stagesThe breast and leg muscles of Peking duck,Hanzhong Ma duck and black Muscovy duck were performed using RNA-Seq.The results showed that the differentially expressed genes,MYL4、SHISA2、CREBL2、RHEB、IGF2BP1、CSRP3、GDF6、SPP1 and KLHL31,as well as LAMB2、LAMA2、ITGB1、OPN、FAF1、RGS8、GRB10、SMYD3 and TNNI2 played an important role in the growth and development of duck skeletal muscles.The regulatory pathways involved in the growth and development of duck skeletal muscle include oxidative phosphorylation,ECM-receptor interaction,focal adhesion,carbon metabolism,biosynthesis of amino acids and MAPK signaling pathway and regulation of the actin cytoskeleton.3 Bioinformatics analysis of CDS region of duck FKBP5 gene and its expression in different tissues at different stagesThe skeletal muscle differentially expressed gene,FKBP5 CDS region and its physicochemical properties of amino acid sequence in Peking duck,Hanzhong Ma duck and black Muscovy duck were predicted,compared and analyzed.The results showed that FKBP5 was an unstable and hydrophilic protein without amino acid signal peptide and transmembrane domain.The results of amino acid homology analysis showed that the amino acid sequence of duck FKBP5 had the highest homology with goose and Muscovy duck(98.69 % and 98.18 %);it also had high homology with chicken(96.20 %),ostrich(94.97 %)and quail(92.66 %),but low homology with pig(83.81 %),cattle(84.03 %),human(82.71 %)and domestic mouse(79.43 %),which indicated that FKBP5 amino acid sequence was highly conserved in the process of biological evolution.Tissue expression profile of duck FKBP5 gene was constructed,and it was found that FKBP5 gene was significantly expressed during embryonic development of Peking duck,Hanzhong Ma duck and black Muscovy duck,and FKBP5 gene was found in the tissues of adult female ducks(except liver)expression is higher than that of male ducks.4 Effect of FKBP5 gene on proliferation and differentiation of duck myoblastsThe constructed FKBP5 overexpression vector could significantly increase the expression of FKBP5 in duck myoblasts.The transfection of FKBP5 overexpression vector showed that FKBP5 could promote the proliferation and differentiation of duck myoblasts.5 Analysis of promoter region of duck FKBP5 gene and its association with body weight of Muscovy duckDouble luciferase assay showed that the core active region of duck FKBP5 gene promoter region was-888 bp/-688 bp,which contained MYF,CCAAT,TATA and Sp1 binding sites.The results of association analysis between FKBP5 gene and body weight of Muscovy duck showed that three SNPs(g.4819252 A >G,g.4821390 G > A,g.4830622 T > G)were significantly associated with body weight of Muscovy duck(P<0.05),which may be used as potential molecular markers in future duck breeding to improve the growth rate of ducks.In conclusion,this study explored the differences of skeletal muscle development between Peking duck,Hanzhong Ma duck and black Muscovy duck at different growth stages from the molecular level.This study provides valuable information for the differential regulation mechanism of duck skeletal muscle growth and development,and also lays a theoretical foundation for the differentially expression gene FKBP5 to regulate skeletal muscle growth and development in ducks. |
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