| The freshness in poultry muscle mainly comes from a variety of fresh peptides and nucleotides,among which inosine monophosphate(IMP)is the strongest flavor substance,which has become an important index to evaluate the freshness and freshness of meat.In the process of species development,its character performance is affected by multiple genes.MiRNA plays an important role in regulating the growth and development of skeletal muscle,and indirectly affects muscle quality by mediating the expression of target genes.In this study,the chest and leg muscles of Jingyuan chicken were sequenced by transcriptome to screen non-muscle-specific miR-27b-3p,to predict their target genes and binding sites.293T cells and isolated and cultured myoblasts were used as in vitro models for the study of miRNA function.The targeted regulation mechanism of miR-27b-3p and its target gene SRL was verified and revealed by a double luciferase report experiment,myoblast differentiation culture,overexpression and inhibition,and differential quantitative analysis.The main results are as follows:1.The results of transcriptional sequencing showed that there were 39 miRNA with significant difference between the chest muscle and leg muscle(19 up-regulated and 20 down-regulated),including 32 known miRNA and 7 newly predicted miRNA.The most abundant and significant functional sets of candidate target genes of differential miRNA were single biological process,intracellular and structural molecular activity,respectively,and the target genes were annotated into 133 signal pathways,the main enrichment pathways were purine metabolism,pyrimidine metabolism,Wnt signal pathway,MAPK signal pathway and so on.12 miRNA with significant differences were randomly selected for RT-qPCR verification,and the relative quantitative results were consistent with the trend of transcriptional sequencing.2.The mature sequences of SRL and miR-27b-3p are highly conserved among chicken,human,mouse,and other species.The protein network found that SRL interacts with MYL1,TNNC2,and TNNT3,which regulate the growth and development of skeletal muscle.The expression levels of miR-27b-3p and SRL genes in Jingyuan chicken muscle were lower than those in the leg muscle.The double luciferase reporter gene test showed that SRL was a potential target gene for miR-27b-3p.3.The isolated and purified myoblasts were identified by immunofluorescence after cultured for 2 days.Under a fluorescence microscope,the nucleus was blue,and the myogenic marker protein desmin showed green fluorescence in the periphery of the nucleus and the cytoplasm.Myogenic determinants MYOD1,MYOG,and MYF5 genes were expressed in Jingyuan chicken myoblasts.After induced differentiation of myoblasts,the expression of miR-27b-3p in the differentiation stage was higher than that in the proliferation stage,reached the highest level on the second day of differentiation,and then decreased.When miR-27b-3p was overexpressed,the expression levels of SRL,MYOD1,MYOG,MYF5,TNNT3,TNNC2,and PDLIM7 genes increased,while the expression levels of MRF4 and MYL1 genes decreased.When miR-27b-3p was inhibited,the expression levels of these genes were on the contrary.To sum up,miR-27b-3p participates in the differentiation of myoblasts and affects the growth and development of skeletal muscle and the conversion of muscle fibers by targeting the expression of the target gene SRL.The above results preliminarily elucidate the functional mechanism of specific deposition of target genes mediated by miRNA in IMP of Jingyuan chicken,which provides a theoretical basis for molecular breeding,meat improvement,development,and utilization of local chicken breeds. |
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