Cloning, Expression And Functional Verification Of SYCE1 Gene Of Male Sheep

Synaptonemal complex central element protein 1(SYCE1)is the main member of the Synaptonemal complex(SC)and has a very important effect on animal reproductive development.The reproductive system of male animals is relatively complex.The functions of secreting hormones,producing sperm,and maturation are mainly supported by the hypothalamic-pituitary-testicular axis,and are closely related to various regulatory proteins involved.In this study,male sheep were used as the research object.The SYCE1 gene sequence of sheep was cloned and analyzed by RT-PCR;qRT-PCR,Western blotting,immunohistochemistry and other techniques were used to analyze SYCE1 in the hypothalamic-pituitary-testicular reproductive axis And different developmental stages(40-day-old,3-month-old,and 12-month-old)testis expression;using sheep testicular sertoli cells as the research object,the function of SYCE1 gene was verified by transfecting pEGFP-C1-SYCE1 vector and siRNA-SYCE1 vector.turn out:1.The sheep SYCE1 gene CDS has a total length of 972 bp,is a non-transmembrane soluble unstable protein,and encodes 274 amino acid residues;the SYCE1 gene is highly conserved during the evolution of different species.2.SYCE1 is expressed in various tissues of sheep epididymis(caput,corpus,cauda),testis,hypothalamus and pituitary,and its mRNA and protein expression in testis is significantly higher than other tissue(P <0.01).3.SYCE1 is expressed in the testis tissues of sheep at different ages(40 days old,3months old,12 months old),and with the age of sheep,the expression of SYCE1 mRNA and protein levels are increasing.The highest expression at 12 months of age(P <0.01).4.By constructing the pEGFP-C1-SYCE1 overexpression vector and synthesizing the siRNA-SYCE1 interference vector,and transfecting the sertoli cells respectively,the expression levels of pEGFP-C1-SYCE1 mRNA and protein are increased at each point,and the mRNA and The protein expression decreased at all time points.Cell proliferation was detected by flow cytometry and CCK8.The proliferation rate of sertoli cells at each time point in the overexpression group was significantly higher than that in the empty vector group.Serum cell proliferation rate in the silent group was lower than that in the NC group at each time point,but the difference was not significant.Proliferating cell nuclear antigenand Cyclin D1 mRNA expression levels increased and decreased with the overexpression and silencing of the SYCE1 gene in sheep testicular sertoli cells,respectively.The above results indicate that SYCE1 protein is related to the growth and development of sheep testis and spermatogenesis,and it is inferred that it participates in the regulation of the reproductive axis of male sheep,which provides a scientific basis for further research on the relationship between SYCE1 and germ cells and spermatogenesis and its mechanism of action.