Cloning and Bioinformatics Analysis of CERS4 Gene in Hetian Sheep and the Effect of Androgens on Its Expression Distribution in the Skin
[Objective]The aim of this study was to investigate the effect of androgens on the skin structure of Hetian sheep,and understand the role of ceramide synthases 4(CERS4)gene and protein in the influence of androgens on the skin structure of Hetian sheep.[Method]15 2-year-old Hetian ewes were selected and randomly divided into 5 groups.Every three days,0,1,2,3 and 4 mg/kg of testosterone were injected into the muscles of Hetian ewes in the control group and experimental groups 1,2,3 and 4.The treatment lasted for 42 days,and the back skin tissue was collected on the 42nd day.Firstly,the skin samples from control group were selected for cloning and sequencing of the CDS region of the CERS4 gene.A phylogenetic tree of the amino acid sequence of the CERS4 protein in Hetian sheep was constructed,and the physicochemical properties and protein structure of the CERS4 protein in Hetian sheep were analyzed.Afterwards,by making skin tissue slices and conducting Masson staining,the variations in skin structure after different doses of testosterone treatment were investigated in this study.Real-time quantitative PCR was used to detect the expression of CERS4 gene in Hetian sheep skin treated with different doses of testosterone,and immunohistochemistry was used to observe the distribution of CERS4 protein in Hetian sheep skin.[Result]The CDS of CERS4 gene in Hetian sheep had been successfully cloned,with a length of 1 182 bp,encoding 393 amino acids.Similarity analysis showed that CERS4 gene of Hetian sheep had the highest similarity with Ovis aries.The results of the phylogenetic tree analysis showed that Hetian sheep belonged to the same branch of evolution as Ovis aries,and had the farthest genetic relationship with Homo sapiens and Mus musculus.Bioinformatic analysis showed that CERS4 protein of Hetian sheep was a hydrophilic protein,with no signal peptide and might have 5 transmembrane domains.The CERS4 protein of Hetian sheep was mostly located in the endoplasmic reticulum and cytoplasmic membrane,with the least content in the Golgi apparatus,nucleus,and mitochondria.This protein had a correlation of 95％or more with W5Q2R1SHEEP,ACER3,and SGMS1.It was found that the number and area of sebaceous glands in Hetian sheep evidently expanded after treatment with testosterone(P＜0.05).The CERS4 gene expression of the testosterone-treated skin samples in Hetian sheep were outstandingly higher compared with the control skin samples(P＜0.05).CERS4 protein was expressed in the skin mainly in the sebaceous gland site.[Conclusion]The CERS4 gene of Hetian sheep was been successfully cloned.Androgen treatment promoted an extend in sebaceous gland area and quantity in Hetian sheep,and prominently enhanced the expression of skin CERS4 gene.This study provided a theoretical basis for further understanding the impact and mechanism of androgens on the proliferation and differentiation of Ovis aries sebaceous gland,and had theoretical reference significance for solving skin health problems caused by androgens.
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