CDNA Cloning Of Gonadotropin-releasing Hormone (GnRH) And Differentially Expression Of The Nesting Related Genes In Muscovy Duck

The objective of this study is to clone and make bioinformatics analysis of the sequence ofGonadotropin-releasing hormone (GnRH) from Muscovy duck. The full length of GnRH was cloned byreverse transcription polymerase chain reaction (RT-PCR) and rapid amplification cDNA ends (RACE), thegene sequence obtained and the putative amino acid sequence were analyzed by bioinformatics software,and the three-dimensional structure of GnRH was predicted by homology modeling.According to the gene sequences of duck's PRL,GH and FSH genes in GenBank and GnRH gene clonedin the study, four pairs of primer based on their conserved regions were designed with duck beta-actin(β-Actin) as an interna control to construct Real-time fluorescent quantitative PCR (RT-FQPCR)assayand Tissue specific expression in nesting and no-nesting ducks were study.The result showed:1. The GnRH cDNA was cloned successfully, the full sequence was474bp that contained one CDS of279bp and encoded a polypeptide of92amino acids (AA). The result of sequence alignment shows that thenucleotide sequence shares95%and94%and the AA sequence shares97%and91%identity to Anasplatyrhynchos (ABU39914.1)and Anser (AAY59003.1), respectively. The protein molecular weight was10306.84kD and the isoelectric point was6.72. Phylogenetic analysis indicates that GnRH belongs to thetype I and the protein structure has a Gonadotropin-releasing hormones signature domain. Atransmembrane structure was speculated and a signal peptide was found in No.24AA. Thethree-dimensional structure of GnRH wasn't predicted.2. The standard curve of every gene established shown fine linear relationship between threshold cycleand template concentration. The correlation coefficients were more than0.995. The amplificationefficiency (E) of each gene was close to1and the difference between each purpose gene and interna controlwas less than5%. The product of each gene was specific to a single peak, it suggest that all the primers canbe used to conduct RT-FQPCR analysis of each gene.3. The GnRH mRNA gene had been found to express higher in hypothalamus, pituitary and ovary.Theexpression quantity in pituitary, hypothalamus, ovary, muscular stomach, pancreas, heart, duodenum and spleen were very significant differences among nesting and no-nesting ducks(P<0.01); the expressionquantity in liver and glandular stomach were significant differences(P<0.05)and in kidney and lung wereno significant differences(P>0.05).4. The PRL mRNA gene had been found to express higher in pituitary, hypothalamus and heart.Theexpression quantity in pituitary, hypothalamus, muscular stomach, kidney, pancreas, liver, heart andduodenum were very significant differences among nesting and no-nesting ducks(P<0.01); the expressionquantity in ovary, glandular stomach, lung and spleen and were no significant differences(P>0.05).5. The GH mRNA gene had been found to express higher in pituitary and heart.The expression quantityin hypothalamus, ovary, muscular stomach, kidney, heart and duodenum were very significant differencesamong nesting and no-nesting ducks(P<0.01); the expression quantity in pituitary was significantdifferences(P<0.05)and in pancreas, liver, glandular stomach, lung and spleen and were no significantdifferences(P>0.05).6. The FSH mRNA gene had been found to express higher in pituitary, heart and spleen.The expressionquantity in pituitary, hypothalamus, ovary, kidney, pancreas, liver, heart, duodenum and lung were verysignificant differences among nesting and no-nesting ducks(P<0.01); the expression quantity in muscularstomach was significant differences(P<0.05)and in glandular stomach and spleen were no significantdifferences(P>0.05).