Full-length CDNA Cloning Of Gonadotropin-releasing Hormone Receptor (GnRHR) Gene, Temporal And Spatial Expression And Association Studying On Laying Performance In Muscovy Duck

In order to investigate the mechanism of nesting behavior in Muscovy duck, Gonadotropin-releasinghormone receptor (GnRHR) gene as a key factor to reproductive ability was cloned and analyzed. Reversetranscription polymerase chain reaction (RT-PCR) and rapid amplification cDNA ends (RACE) were usedto clone the full length cDNA of Gonadotropin-releasing hormone receptor (GnRHR) gene. The sequenceof gene and protein encoded were analysed by using a variety of bioinformatics softwares, it was well toprovide a theoretical basis to carry out the study of the molecular mechanisms of gene function.Specific primers were designed in the conserved region that based on the cloned GnRHR cDNAsequence and GnRH gene sequence of Muscovy duck.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene having been as reference gene, the differentially expressions were observated in differentorganizations, in different periods of the Muscovy duck’s two genes, through the establishment of theReal-time PCR detection method. And the expression trend was researchde on.Using the covering method, the DNA of GnRHR gene was obtained on the basis of the obtainedfull-length cDNA sequence, By PCR-SSCP and PCR-RFLP method to detect the GnRHR genepolymorphism, and analysis of the correlation between the polymorphism and laying performance.The test results were as follows:1. The GnRHR gene of Muscovy pituitary was successfully cloned and the full-length cDNA of the geneis1680bp. Among them, the full-length of amino acid coding region (CDS) is1248bp, encoding415aminoacids. By different species sequence alignment and homology analysis, the result shows that gene nucleicacid sequence with Gallus gallus and Jungle fowl homology, are90%and88%, respectively; the aminoacid sequence homology with Gallus gallus is88%. The phylogenetic tree analysis showed that GnRHRgene of Muscovy pituitary belongs to type II and the protein structure has a G-protein coupled receptorsfamily1domain. It has seven transmembrane structures and belongs to the G protein-coupled receptorfamily. That protein has α-helix of48.43%, β-sheet of6.02%, and random coil structure of45.54%insecondary structure. Three-dimensional structure was predicted after Homology modeling.2. The expression level of GnRHR was showed higher in hypothalamus, pituitary and gonads. The threetissues was significantly higher than the muscular stomach, pancreas, heart, duodenum, spleen, liver andglandular stomach of expressionsignificantly (P <0.05).The differentially expressions were studied on inthe HPG axis of Muscovy duck for GnRHR and GnRH gene.The results show that for GnRHR more expression of female’s pituitary is found in laying period and is significant differences to otherperiods(P<0.05). For GnRH more expression of pituitary is found in laying and sexual maturity period, butis not significant differences for each other(P＞0.05).But in male more expression is found in sexualmaturity period.The expressions of hypothalamus and gonad are similar to the pituitary’s.3. Three introns had been found to sequence the GnRHR gene of Muscovy duck; the nucleotidemutations of the one loci were detected in the fragment GR2, and it was CT mutations. The genotypes ofthe fragment was detected and analysed, there were three genotypes of AA, AB and BB in the fragmentGR1. And significant different genotypes has been significantly associated with age at first egg,the highestclutch days, the average clutch days and300-day-old egg number(P＜0.05).