Molecular Cloning, Expression And Sequence Analysis Of Two Activin-β Subunit Genes In Japanese Flouder (Paralichthys Olivaceus)

Activin is a member of the transforming growth factor-β（TGF-β）family andconsisting of two β subunits. And Activin is a glycoprotein hormones that plays a vitalrole in hypothalamus-pituitary-gonad axis （HPG）. Activins regulate pituitarygonadotropin secretion and ovarian functions, such as steroid hormone productionand oocyte maturation.Japanese flounder（Paralichthys olivaceus）is an importantcommercial fish, and has many strong points such as fast-growing in aquaculture. Inthis study, the full-length of the ActivinβAand βBsubunit was cloned from Japaneseflounder. Then the expression file in different tissues and early developmental stageswas demonstrated. The promoters of ActivinβAand βBin Japanese flounder werecloned by genome walking. The mature Peptide sequences were inserted into thePlasmid pET32a（+）to construet the recombined vectors which supply a firm supportfor further study ofActivin biological functions in the future.（1） In this study, based on the cloning and RACE techniques, the full-lengthcDNA of ActivinβAand βBwere obtained. Using bioinformatic softwares andquantitative real-time PCR to study each of them, respectively.A. The full-length P. olivaceus ActivinβAcDNA was1633bp, composed of30bp of5′UTR,1365bp of ORF, and238bp of3′UTR, and it could encode454aaresidues with an estimated molecular mass of49.9kD. This propeptide included asignal peptide of20aa residues. qPCR analysis indicated that ActivinβAwasexpressed in all the ten tissues of adult P. olivaceus, with a highest level in the wholebrain, gill and ovary.B. The full-length P. olivaceus ActivinβBcDNA was1783bp, composed of121bp of5′UTR,1173bp of ORF, and489bp of3′UTR, and it could encode390aaresidues with an estimated molecular mass of43.8kD. This propeptide included a signal peptide of22aa residues. qPCR analysis indicated that ActivinβBwasexpressed in all the ten tissues of adult P. olivaceus, with a highest level in the ovary.Bioinformatic softwares predication indicated ActivinβAand βBof P. olivaceuswere secreted protein. The ActivinβAamino acids sequence alignment among P.olivaceus and other teleosts were remarkedly similar, indicating the highlyconservation of teleost ActivinβAin evolution.（2） The promoters of ActivinβAand βBin P. olivaceus were cloned bygenome walking. The promoters of these two genes were2.7kb and2.4kb in length,respectively. The bounding sites of transcriptional regulation elements were predictedusing bioinformatics method. The result showed that the TATA box of Activin βBwasexisted at31bp in the upstream region of the transcription initiation site, while thisstructure was not found in Activin βA. Numbers of binding sites for transcriptionfactors Sp1, Oct-1, C/EBP, CREB, GATA-1, c-Jun, HNF-3, HNF-1, USF were foundin the two promoters. In addtion, multiple transcription binding sites related toendocrine factors Pit-1, ER, PR, GR, RAR, RXR were also existed. However, MyoD、myogenin and SRY binding sites were only found in Activin βA. In conclusion, Thebioinformatic analysis suggested that basic and hormone-inducible expressionregulation patterns exist both in Activin βAand βB, However, the regulationmechanisms were not completely consistent between the two genes.（3）The P. olivaceusActivinβAand βBpeptide（without signal peptide）wasexpressed in the form of fusion protein using pET32a as expression vectors and BL21（DE3）pLysS as expression host which supply a support for further study ofActivinbiological functions in the future.