Cloning And Expression Of AMH In Yellow River Carp And Two Loaches

In mammals, a multitude of studies have shown that anti-Müllerian hormone (AMH), also knownas the Mullerian inhibiting substance (MIS), belonging to the beta transforming growth factor (TGF-β)superfamily member, apart from inducing Müllerian duct regression during male sexual differentiation,exerts inhibitory effects on male and female gonadal steroidogenesis and differentiation. However, in lowervertebrates like teleost fish, the function of AMH has been far less explored. As a first step to unravel itspotential role in reproduction in teleost fish, we isolated and characterised the AMH gene in the yellowriver carps and two species of loach, and analysed its gene expression and those of transcripts.In this article, degenerate primers was designed according to the sequences in conserved regionof AMH genes and PCR was used to amplify cDNA in the yellow river carps and two species of loach, onebands of455bp appeared steadily in PCR product electrophoresis results; After sequencing the yellow rivercarps and two species of loach are AMH genes sequence. According to these results, the full-length cDNAof AMH in yellow river carps and AMH in two species of loach are achieved by combination3′RACE andthe method of PCR. Sequence analysis revealed the full-length of yellow river carps AMH is a2000bpcDNA through putting together with these two fragments, containing the340bp3′-untranslated region(including polyA) and1676bp open reading frame which encode a AMH protein of538amino acid. Andmisgurnus anguillicaudatu AMH is a1331bp cDNA through putting together with these two fragments,containing the328bp3′-untranslated region (including polyA) which encode a AMH protein of345aminoacid. paramisgurnus dabryanus AMH is a1327bp cDNA through putting together with these twofragments, containing the341bp3′-untranslated region (including polyA) which encode a AMH protein of328amino acid.Yellow river carps and two species of loach AMH contained a TGF-beta domain at theC-terminus which has eight cysteine residues.Sequence analysis revealed that the yellow river carps AMHwas clustered with AMHs of teleosts, and shared highest homologies with homologues from Squaliuspyrenaicus and Squalius alburnoides (81.4and76.1%respectively). Sequence analysis exhibited theidentity rate of amino acid of AMH among two species of loach are both above88.7%. The result indicated yellow river carps and two species of loach with cyprinid fish the AMH gene is extremely conservative inthe progress of evolution.Two species of loach tissue and embryo RT-PCR analysis results show that: MaAMH wasexpressed in all the six tissues but it was higher expressed than any others in ovary. In different stagesof developing embryo, MaAMH level of expression in yolk-sac absorption stage is highest, but lowerexpression in other stage. PdAMH was expressed in all the six tissues but it was higher expressed thanany others in heart.