Abstract:We present molecular cloning and tissue expression analysis of three estrogen receptor (ER) subtypes, vbERα, vbERβ1 and vbERβ2, from liver of the cyprinid fish Varicorhinus barbatulus through reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The sequence alignment and phylogenetic analysis reconfirmed the evolutionary relationship of V. barbatulus within the family Cypriniformes. Directional constraints for subtype-specific substitution of critical amino acids were observed in the E2 binding region. For amino acid substitution, vbERβ exhibited a M517L change in the ligand-dependent transactivation region. The tissue distributions were investigated using RT-PCR with subtype-distinguishable primers. Both vbERα and vbERβ1 were most highly expressed in liver, while vbERβ2 was higher in intestine. Here we demonstrate that the identification and cloning of ER subtypes using PCR is feasible in wildlife in that the temporal and spatial observations are consistent with those from phylogeny analysis and crystal structural investigation by others.