Molecular cloning, in silico characterization and expression analysis of axonemal protein 66.0 in Pacific abalone, Haliotis discus hannai

Abstract

Axonemal protein 66.0 is one of the major axonemal dynein protein involved in activation of sperm motility. The axonemal protein 66.0 gene was successfully cloned and characterized from testis tissue in the Pacific abalone, Haliotis discus hannai. The cloned gene was named Hdh-Axp66.0 based on the predicted molecular mass. The full-length cDNA of Hdh-Axp66.0 was 2023 bp, with a coding region of 1694 bp; it encoded 564 deduced amino acids with a predicted molecular mass of 65.42 kDa and an isoelectric point of 8.68. In silico analysis prediction suggested this gene has two cAMP-dependent protein kinase phosphorylation sites, seven predicted protein kinase C phosphorylation sites, three N-linked glycosylation sites, a tyrosine kinase phosphorylation motif, three coiled-coil domains, and several Ca2+ binding sites, which revealed the possible involvement of Hdh-Axp66.0 in activation of sperm motility. Tissue distribution analysis indicated that Hdh-Axp66.0 was distributed in testis, heart, gill and mantle tissues. However, quantitative real-time PCR (qPCR) revealed that Hdh-Axp66.0 mRNA was highly expressed in testis tissue. Further, highly elevated relative mRNA expression was observed in testis tissue during the ripening stage compared with different testicular developmental stages. These results suggest the possible involvement of Hdh-Axp66.0 in testicular development and reproduction.