Expression Characteristics And Function Analysis Of Goat Beta-defensin 104a Gene In Bucks

Beta-defensins was a family of antimicrobial canonic peptides with lower molecular weights, which played an important role in innate immunity of mammals and in biological process of reproduction, such as sperm motility and capacitation in male mammals. The goat p-defensin 104a (gBD104a) is one of epididymal beta-defensins family. However, the expression profiles of gBD104a and its function were unclear. Therefore, the aims of this study were conducted to predict the characteristics of gBD104a protein, to investigate the expression profiles of gBD104a in various organs of bucks and localization of gBD104a in reproduction organs of adult bucks, to detect the expression of gBD104a on surface of sperm before and after capacitation. The results will enrich reproductive theory and provide enough information for further research of reproductive biology.Online softwares were used to analyse the bioinformatics of gBD104a protein sequence; Testis and epididymis of bucks were collected at the age of 7 days,2 months,3 months,4 months,6 months,9 months and adult. Twenty-three organs and tissues were collected from the adult bucks. The relative expression of gBDlO4a mRNA and gBD104a protein was detected by QRT-PCR technique and Western blotting technique respectively. The localization of gBD104a protein was determined in testis and epididymis of adult bucks by immunohistochemistry. Semen was collected for the localization of gBD104a on surface of sperm by immunofluorescence. The results were as follows:(1) The bioinformatics analysis showed that the complete coding sequence of gBD 104a contains 324bp, encoding 107 amino acids, which had a signal peptide at 1-19 amino acids and a potential P-defensin modify at the 27-55 amino acids. There were seven potential O-glycosylation sites at the C-terminus.(2) QRT-PCR results showed that gBDIMa. mRNA was widely expressed in twenty-three organs and tissues, and highly expressed in reproductive organs. The relative expression of gBD 104a mRNA in the trachea, abomasum, jejunum, ileum were higher than that in other epithelial tissues. The expression of gBD 104a mRNA in epididymal caput and corpus were higher than that in other organs and tissues. The trend of gBD104a mRNA expression in the caput was decreased from the age of 7d to adult, while was increased in the corpus. Western-blotting results showed that the expression of gBD 104a protein had a similar trend with that of expression of mRNA in epididymal caput and corpus at different ages. Immunofluorescence results showed that strong positive signal of gBD104a were detected in pseudostratified ciliated columnar epithelium cells of caput and corpus, as well as in ciliated columnar epithelial cells of cauda. The spatiotemporal specific expression of gBD104a indicated that gBD104a may play a role in sperm maturation.(3) Sperm immunofluorescence assays showed gBD104a protein coated on sperm acrosome and middle mitochondria of sperm tail except the principal and end piece. gBD104a protein was dropped off from sperm acrosome after the capacitation, which showed that gBD104a may involve in sperm capacitation.