Molecular cloning and expression analysis of Foxp1a/b from Nile tilapia(Oreochromis niloticus)

To investigate whether the mRNA expression of Nile tilapia(Oreochromis niloticus) Foxp1a and Foxp1b are involved in the lymphocyte activation and regulated by 17β-estradiol(E2) level in vivo,the coding sequences and mRNA expression patterns of OnFoxp1a and OnFoxp1b were characterized and analyzed by Real-time PCR. OnFoxp1a has an open reading frame 1710 bp encoding 569 aa,while OnFoxp1b with 2040 bp encoding 679 aa.Both of the putative OnFoxp1a/b contain a C2H2 zinc finger domain,a leucine zipper-like domain(V-x(6)-L-x(6)-L-x(6)-L) and a winged-helix/fork head domain,which are typical motifs of Foxp subfamily.Compared with OnFoxp1a,OnFoxp1b shows a higher amino acid identity(77.4% versus 55.0%) to mammalian Foxp1.Phylogenetic analysis reveals that OnFoxp1a/b together with the other known vertebrate Foxp1 form a different clade from Foxp2,Foxp3 and Foxp4.Meanwhile,OnFoxp1a/b separately form a fish-specific clade and OnFoxp1b is further clustered with Foxp1 from the higher vertebrates,which supports that OnFoxp1b has a closer relationship with mammalian Foxp1.The expressions of OnFoxp1a/b were detectable in all examined tissues.The highest mRNA expression of OnFoxp1a was observed in the testis,while OnFoxp1b in the heart.Moreover,the moderate expression of OnFoxp1b was also observed in the immune-related tissues,such as spleen,kidney,gills and intestine.Stimulation of peripheral blood mononuclear cells with PHA(50μg/mL) and PMA(50 ng/mL) led to a significant increase of OnFoxp1b mRNA expression at 6,12 and 24 h,respectively,while no significant changes of OnFoxp1a mRNA expression were observed except stimulation of PHA at 24 h.These results imply that the homologous Foxp1 in lower vertebrate is involved in lymphocyte activation.After 24 h treatment of 6-month male(XY) tilapias with estrogen(17-beta-estradiol,E2),the expressions of OnFoxp1a/b were significantly up-regulated in the intestine and kidney but not in the spleen.Taken together,our results suggest that both of OnFoxp1a/b coded by two different genes are the orthologues of mammals and have potential disproportionate functions.Moreover,the mRNA expression patterns of OnFoxp1a/b are related with the lymphocyte activation and regulated by E2 level in vivo.This study not only provides scientific data for the research on the molecular evolution of Foxp subfamily members in lower vertebrates,but also will promote further study of the fish molecular immune response.