Cloning and characterization of rec8 gene in orange-spotted grouper (Epinephelus coioides) and Dmrt1 regulation of rec8 promoter activity

Abstract

Meiosis is a specialized type of cell division critical for gamete production during sexual reproduction in eukaryotes. The meiotic recombination protein Rec8 has been identified as an important factor in germ cell meiotic initiation in vertebrates; however, its equivalent role in teleosts is poorly characterized. In this study, we cloned and sequenced the rec8 gene from orange-spotted grouper (Epinephelus coioides). The cDNA sequence consisted of 2244 base pairs (bp), including a 5' untranslated region (UTR) of 198bp and a 3'UTR of 284bp. The open reading frame of grouper rec8 was 1752bp, encoding 584 amino acids. Expression levels of rec8 were higher in the ovary, intersex gonad, and testis. A neighbor-joining phylogenetic tree based on the deduced amino acid sequence indicated a common origin for grouper and other teleost rec8 molecules. Immunohistochemistry using a polyclonal anti-Rec8 antibody localized the protein in the oogonia and primary oocytes in the ovary and in spermatogonia and spermatocytes in the intersex gonad and testis, suggesting that Rec8 may play an important role in the meiotic division and the development of grouper germ cells. In addition, we found that the transcription factor Dmrt1 increased rec8 promoter activity through the second binding site, based on dual-luciferase assays. Together, these results suggest that Rec8 plays a crucial role in meiosis and may be regulated by Dmrt1 to affect meiosis in groupers.