The Expression and Application of the Recombinant Protein of the Female Specific Gene CSW3 in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

Abstract:

The half-smooth tongue sole (Cynoglossus semilaevis) is a mariculture fish species in China that has high economic values. The growth of this fish is sexually dimorphic, and females grow 23 times faster than males. The tongue sole exhibits the phenomenon of sex reversal during the growth and has a ZZ-ZW sex-determining system, thus it is a valuable animal model in the study of the mechanisms of fish sexual growth dimorphism. Several sex-specific genes have been cloned from half-smooth tongue sole, but the functions of these genes remain unknown. This research aimed to preliminarily study the function of the female-related gene CSW3. CSW3 was one of the female specific genes in half-smooth tongue sole which was identified from the screening in the whole genome sequencing project of half-smooth tongue sole. The full length of CSW3 cDNA was 1677 bp and contained a 414 bp open reading frame which coded 137 amino acids. The sequence also included a 173 bp 5 noncoding region and a 1090 bp 3 noncoding region that possessed a polyadenylation signal and a poly A tail. We constructed a recombinant expression vector for CSW3 and the plasmid was transformed into E. coli. IPTG was used to induce the expression of CSW3 protein. The proteins were isolated and purified with His-tag purification of affinity chromatography. The western-blot analysis showed that the recombinant protein was 33 kDa, which was consistent with the bioinformatics prediction. We then investigated the function of CSW3 protein using protein transfection. The liposomes encapsulating CSW3 protein were injected into the gonad of male half-smooth tongue sole. The experimental group was injected with bioactive CSW3 protein, and the control group was injected with heat-inactivated CSW3 protein. Real time quantitative PCR analysis showed that there were changes in the expression of three sex-related genes including one female-related gene and two male-related genes. The results suggested that the female specific CSW3 protein may cause the up-regulation of the female-related gene foxl2, and the down-regulation of the male-related genes sox9a and amh.