Cloning, Identification And Expression Analysis Of Vasa, Dazl, Nanos1 And Dmrt1 Genes In The River Sand Pond Snakehead

Odontobutis potamophila is an important economic fish in China with obvious sexual dimorphism.Males grow 30% faster than females which is ideal model for studying sex determination and differentiation mechanisms.The maintenance of gonadal function requires the expression of multiple genes.The Vasa,Dazl,Nanos1 and Dmrt1 genes are specifically expressed in germ cells and are essential for fish primordial germ cell migration,differentiation and sex determination.In this study,the expression of Vasa,Dazl,Nanos1 and Dmrt1 genes in different developmental stages of gonads was studied by q PCR,western and immunohistochemical techniques in Odontobutis potamophila.The experimental results were showed as follows:1.The full-length c DNA of Vasa,Dazl,Nanos1 and Dmrt1 genes in Odontobutis potamophila were cloned.The full-length c DNA of Vasa is 2357 bp,including the 1944 bp open reading frame encoding 647 amino acids,5’ and 3’ UTR regions is 116 bp and 297 bp,respectively.Dazl gene has a full-length c DNA of 842 bp,including a 648 bp ORF encoding 215 amino acids,a 117 bp 3’UTR and a 77 bp 5’UTR.The c DNA sequence of the Nanos1 gene is 1178 bp in length and contains a 155 bp 5’UTR,a 660 bp open reading frame encoding 219 amino acids and a 363 bp 3’UTR.Amino acid sequence alignment and phylogenetic tree analysis revealed that the Vasa,Dazl,Nanos1 and Dmrt1 genes in Odontobutis potamophila were highly evolutionarily conserved.Tissue expression results showed that Vasa,Dazl and Dmrt1 were specifically expressed in the gonads,and the expression levels of other tissues were negligible.The Nanos1 gene is expressed in both gonads and other adult tissues,but its expression in brain and gonads is significantly higher than other tissues.It is speculated that Nanos1 participates in and regulates the Hypothalamic-pituitary-gonadal axis.2.Quantitative real-time PCR and western blot were performed to measure Vasa,Dazl,and Nanos1 m RNA and protein expression during gonad development.The result demonstrated that Vasa,Dazl and Nanos1 showed a decreasing expression trend in testis.Their proteins showed a downward trend in the ovary,while their m RNA decreased and then increased.The distribution of the Vasa,Dazl and Nanos1 observed by immunohistochemistry.The result revealed that Vasa,Dazl and Nanos1 were abundantly expressed in spermatogonia,spermatocytes and every stages of oocyte.No signal can be detected in spermoblast and mature sperm,indicating that Vasa,Dazl,and Nanos1 proteins can be used as new markers for identifying germ cells in the gonads of Odontobutis potamophila.In ovary Vasa,Dazl,and Nanos1 genes have a relatively high m RNA expression level in the stage IV,and maintain high expression in the early embryonic.Vasa,Dazl,and Nanos1 may participate in the generation and migration of primordial germ cells in Odontobutis potamophila as a maternal factors.3.The expressions of Vasa,Dazl,and Nanos1 after treatments with LHRH-A2 and HCG were examined by q RT-PCR.In testis,the expressions of Vasa,Dazl,and Nanos1 in both low-and high-concentration HCG groups were significantly down-regulated compared with the control group.In ovary,the expressions of Vasa and Nanos1 were significantly up-regulated after treatments with HCG and 0.1 μg/g body weight LHRH-A2.However,all three proteins were unable to detect in both testis and ovary after stimulation.It is speculated to participate in the regulation of the HPG axis during gonadal development.4.The full-length c DNA of Dmrt1 gene in Odontobutis potamophila is 2025 bp,including 894 bp open reading frame(ORF),73 bp 5’UTR and 1058 bp 3’UTR。 Quantitative real-time PCR was performed to measure Dmrt1 m RNA during embryonic and gonad development.The results showed that the expression level of Dmrt1 gene first increased and then decreased during embryonic development,and reached the highest in the gastrulae stage.It is speculated that the high expression of Dmrt1 gene in early embryonic development may be related to the production of primordial germ cells.In testis,Dmrt1 were highly expressed in early and later stage of gonadal development(I phase,Ⅳphase)when compared to other developmental stages.Dmrt1 is expressed in low levels during ovarian development.