| As an important economic animal,sheep is one of the sources of meat,milk and fur in human society.Our country is the largest mutton producing country and also the largest consumer of mutton in the world,however,the production level of sheep has not yet reached the advanced level in the world.Slow growth speed and low average carcass weight are all problems in sheep production.At present,studies on the early development of sheep mainly focus on skeletal muscle growth and development,hair follicle genesis and tail lipid deposition,and few studies have been conducted on the development pattern of embryonic myocardium and the comparison between embryonic skeletal muscle and myocardium.Therefore,this study conducted a preliminary analysis on the expression of related genes during the embryonic muscle growth and development of Tan sheep.In addition,the function of CCNY gene at the cellular level was explored,and the main work contents and results were as follows:(1)Embryonic period is the key period of muscle growth and development in sheep.Skeletal muscle and myocardial tissue of embryo of Tan sheep at 50 d(E50),55 d(E55),60 d(E60),66 d(E66)and 72 d(E72)were selected as research objects.Real-time PCR was used to detect the expression rules of growth and development related genes,such as EYA2,CCNY,PPARG,FTO,LPL,MTPN,MEF2 B and MSTN,and the expression differences in different muscle tissues during the same embryonic period.The results showed that the above genes were expressed in both skeletal muscle and myocardium.EYA2 and MEF2 B genes showed the same expression trend in skeletal muscle and myocardium,and had a certain conservative expression in tissues.The expression level of MTPN gene was significantly different in myocardium and skeletal muscle,and was much higher in skeletal muscle than in myocardium,while PPARG gene was higher in myocardium than in skeletal muscle.The expression of CCNY and FTO genes in skeletal muscle was significantly higher than that in myocardium.The expression of CCNY and FTO genes increased in skeletal muscle and decreased in myocardium.The expression level of LPL gene in E50,E55,E60 and E66 was basically unchanged,and there was no difference in skeletal muscle and myocardial tissue.While the expression level of LPL gene in skeletal muscle was significantly increased in E72.The expression level of MSTN gene in myocardial was greater than skeletal muscle in the first three stages,and began to decrease with the development of embryo.Amount of MSTN gene expression during the first three myocardial is greater than the skeletal muscle,along with the development of embryo in myocardial expression began to reduce.(2)In this study,adipose derived stem cells(ADSCs)of Tan sheep were successfully isolated.Through the q RT-PCR found that the expression of CCNY gene gradually increased during the differentiation of ADSCs into adipocytes.In the study of CCNY gene,CCK8,MTT and Alarm Blue results showed that interference CCNY gene expression could significantly inhibit ADSCs proliferation(P<0.05).(3)Bioinformatics analysis of the nucleotide and amino acid sequences of sheep CCNY showed that the full length of the CDs region of CCNY gene was 1126 bp,encoding 314 amino acids.It is relatively conservative in the evolutionary process,has high homology with other animals,and has similar biological functions on different species.The prediction of physical and chemical properties and structure of the protein showed that CCNY was an unstable hydrophilic protein with no transmembrane structure and no signal peptide sequence.It was neither a membrane receptor protein nor a secretory protein.Three glycosylation sites and 34 phosphorylation sites were predicted,which may be important for CCNY protein to regulate cellular pathways and participate in signal transduction.Protein interaction network analysis showed that CCNY protein may be mainly associated with ELF4 H,CMTM4,TMEM186,NHSL2 and CDK14 proteins in cell development regulation,transcriptional activation and signal transduction. |
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