Molecular Cloning And Expression Analysis Of LGR8 And CSW3 Function Verification In The Half-smooth Tongue-sole, Cynoglossus Semilaevis

This experiment in half smooth tongue sole on the basis of the sequencing of entire genome and transcriptome, we cloned LGR8 gene using the RACE method. The full-length of LGR8 is 2768 bp, which encoding 734 amino acids. The fluorescence quantitative PCR results showed LGR8 mainly expressed in male fish in brains and testises,and there is no significant difference among the different tissues of females. Moreover, the expression quantity in males is significantly higher than that of females; Based on the different periods of gonads, real-time fluorescent quantitative PCR results showed that LGR8 expression began to rise after 80 days and 150 d to 210 d reach to the maximum amount, and the expression quantity in males is all higher than that of females, which suggests that LGR8 play a significant role in the development of sex determination and gland development in half smooth tongue sole.This experiment according to observing the genderrelated gene expression regulation, such as Dmrt1, Sox9, Amh and P450 after CSW3 recombinant protein injection in gonad to explore the role of sex determination and differentiation of CSW3 in half smooth tongue sole.The real-time quantitative PCR analysis results showed that after the injection, the expression of Dmrt1, Sox9, Amh, P450 varied in different degrees,which proved CSW3 protein has a certain activity and maybe play a significant role during sex deter- mination.