Cloning, Expression And Function Analysis Of SOX3 In Two Loaches

The Sox gene superfamily was a class with a SRY(sexdetermination region of Y chromosome) gene homology, the code region contained DNA combination of high mobility group(High-mobility group, HMG box). Sox3 belonged to the SoxB1 subfamily of the Sox family, which was the only gene located on the X chromosome and the highest homology gene with SRY/Sry.In this study, we obtained the full length cDNA of Sox3 from two loaches by RACE. The full length c DNA of Sox3 in Misgurnus anguillicaudatus was 1863 bp, containing the 295 bp 5’-untranslated region, 674 bp 3’-untranslated region(including polyA) and encoding a putative protein of 297 amino acids, the molecular weight was 33109.5 KD, theoretical isoelectric point was 9.63. The full length cDNA of Sox3 in Paramisgurnus dabryanus was 1865 bp, containing the 295 bp 5’-untranslated region, 676 bp 3’-untranslated region(including polyA) and encoding a putative protein of 297 amino acids, the molecular weight was 33095.5 KD, theoretical isoelectric point was 9.63. The two fragments which isolated from the two loaches contained the conservative sequences in HMG-box and the Soxp motif of SoxB subfamily.Semi-quantitative RT-PCR and fluorescent real-time RT-PCR were conducted to determine the expression of Sox3 gene in different stages during embryonic development and adult tissues of the two loaches. During embryonic development in Misgurnus anguillicaudatus, Sox3 gene showed the highest expression in the neurula stage, then the expression decreased; In Paramisgurnus dabryanus, Sox3 gene in the early gastrula stage has the highest expression level, then the expression decreased. In adult tissues, the expression level of Sox3 varied among tissues, and had the similar expression patterns in the two loaches. Sox3 with the highest expression detected in both ovary and testis, the stronger expression level were in brain, heart, liver; the expression level in the kidney was the lowest.In situ hybridization results demonstrated that Sox3 genes expressed weakly in primary spermatocytes in testes, but had strong expression in ovarian primary oocytes, suggesting important roles of Sox3 gene in gonad development, especially in ovary. A strong positive signal was detected in the brain, it implied that Sox3 may probably play roles in the development of nervous system. Whole mount in situ hybridization results showed that the zygotic expression of Sox3 in two loaches all occured in the early embryonic development stage, somitogenesis, strong positive hybrid signals could be detected in the spine. After hatching period, the major tissues expressing Sox3 included head, spine and internal organs.Our study showed that Sox3 gene played important roles in the early embryonic development, the formation and development of the nervous system, and gonad development, especially ovary. We know that the Sox3 gene played an important role in the formation of the hypothalamus pituitary(Hypothalamic-pituitary, HP), then fish gonad development was regulated dominantly by the hypothalamic pituitary gonadal(Hypothalamic-pituitary-gonad, HPG) axis of the neuroendocrine system. Thus, Sox3 gene played a very important role in the process of individual development, especially with complex regulatory mechanisms in the development process associated with the HPG axis.