Molecular cloning and expression analysis of rad51 gene associated with gametogenesis in Chinese soft-shell turtle (Pelodiscus sinensis)

Rad51 is a recA-like recombinase that plays a crucial role in repairing DNA double-strand breaks through homologous recombination during mitosis and meiosis in mammals and other organisms. However, its role in reptiles remains largely unclear. In this study, we aimed to investigate the physiological role of the rad51 gene in reptiles, particularly in Pelodiscus sinensis. Firstly, the cDNA of rad51 gene was cloned and analyzed in P. sinensis. The cloned cDNA contained an open reading frame (ORF) of 1020 bp and encodeed a peptide of 339 amino acids. The multiple alignments and phylogenetic tree analysis of Rad51 showed that P. sinensis shares the high identity with Chelonia mydas (97.95%) and Mus musculus (95.89%). Secondly, reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that rad51 mRNA was highly expressed in both ovary and testis, while being weak in the somatic tissues examined in this study. Furthermore, chemical in situ hybridization (CISH) was performed to examine the expression profile of rad51 mRNA in germ cells at different stages. In the testis, rad51 mRNA expression was found to be stronger in the germ cells at early stages, specifically in spermatogonia and spermatocytes, but it was undetectable in spermatids. In the ovary, rad51 mRNA exhibited a uniform distribution in the cytoplasm of oocytes at early stages. The signal intensity of rad51 mRNA was highest in primary oocytes and gradually declined during oogenesis as the oocytes developed. These results suggest that rad51 plays a vital role in the development of germ cells, particularly during the early stages of gametogenesis in P. sinensis. The dynamic expression pattern of rad51 mRNA provides insights into the mechanisms underlying germ cell development and differentiation into gametes in turtles, even in reptiles.