Molecular Cloning And Sequence Analysis Of The Peking Duck Prolactin Receptor Gene And Estrogen Receptor α, β Gene

As the specific receptor of PRL, PRLR was involved in a series of physiological process. The prolactin receptor (PRLR) was selected as a candidate gene for broodiness because it is thought that a major gene involved in susceptibility to broodiness resides on the chicken Z chromosome manifest as sex linkage of the trait. The estrogen receptor was also attractive as a candidate gene for broodiness.Up to now, sequence information for duck prolactin receptor and estrogen receptor are not yet reported.In this study, sequence of prolactin receptor and estrogen receptor were cloned from Peking duck. Expression of PRLR mRNA and ESR mRNA were detected using semi-quantitative RT-PCR. It will be the base for molecular breeding of Peking duck. Progresses of this research are following:1. Prolactin receptor (PRLR) cDNA of Peking duck was cloned using RT-PCR method (GenBank accession no. EF205054). The dPRLR cDNA contains 2439bp including the ATG start codon and TAA stop that encodes 832 amino acid residues. The putative receptor protein had 2 strong hydrophobic stretches: one was the signal sequence in N-terminal region and the other, the single transmembrane region of 24 amino acid residues in the central portion. Excluding the signal sequence, the mature dPRLR contained 808 amino acid residues with a theoretical molecular mass of 91.5 kDa. The mature protein could be divided into three domains, extracellular domain (ECD), transmembrane domain (TM) and intracellular domain (ICD). The ECD contained two homologous repeat units with 66.7% amino acids sequence identity to each other. The membrane distal unit and proximal unit consisted of 204 and 212 amino acids, respectively. Each unit was similar to the singular ECD of the mammalian PRLRs. The characteristic extracellular cysteines and the WSXWS motif of the cytokine receptor were conserved in both repeat units. The ICD of dPRLR consisted of 368 amino acids at the C-terminus, and contain two conserved region—Box1, Box2. The all characters of duck were similar to chicken and mammalians, which implied the dPRLR, had same mechanism with other species. Sequence similarity analysis shows that the dPRLR shares 44.8%~ 96.5% identities with other species at deduced amino acid level; the dPRLR shares 29.6%~96.1% identities with other species at nucleic acid level, share highest identity with goose PRLR at both level. In a phylogenetic analysis, the duck PRLR was found to cluster with the PRLR of goose, turkey, chicken and pigeon. Semi-quantitative RT-PCR indicated that dPRLR mRNA was expressed in all eight tissues. Expression In the kidney was highest in varietals tissues.2. Partial cDNA of Peking duck estrogen receptorα(dESRα) and estrogen receptorβ(dESRβ) were obtained using RT-PCR method. The dESRαcDNA contains 1593bp including the start codon (ATG) that encodes 531 amino acid residues (GenBank accession no. EF205052). The dESRβcDNA contains 1085bp including the start codon (ATG) that encodes 361 amino acid residues (GenBank accession no. EF621308). Sequence analysis shows that both dESRαand d ESRβcould be divided into five domains (A-E), which are conserved in the nuclear receptor super family. Sequence similarity analysis shows that the A/B domain of dESRαshares 11.5% identities with A/B domain of dESRβat deduced amino acid level, the C domain shares 90% between dESRαand dESRβ, the D domain shares 30%, the E domain shares 35%. The amino acid sequences in the C domain were highly conserved between ESRαand ESRβ, that implied both ESRαand ESRβhave similar target genes. PredictNLS computer program was use for predicting nuclear localization signal (NLS). Results indicated that ESRαcontain two NLS, one in C domain and another in D domain; ESRβcontains one NLS locating in C domain. Semi-quantitative RT-PCR indicates that both ESRαand ESRβmRNA expression in female were higher than in male. In the kidney, ESRαmRNA expression was lower than that of ESRβmRNA. But in both male and female duck gonads, ESRαmRNA expression was higher than that of ESRβmRNA.3. A DNA fragment of Peking duck prolactin receptor (PRLR) spanning 1325bp was obtained by PCR method (GenBank accession no. EF502053). The fragment contains an intron of 635bp in length. Sequence analysis showed that this fragment consisted of partial of exon9 (57bp), intro9 (635bp), and partial of exon10 (633bp). It will provide a chance of finding polymorphism loci of duck prolactin receptor.