Cloning And Expressing Of NPY Gene And SMAD3 Gene,and Their Association With Laying Performance In Muscovy Duck

Muscovy duck is one of the excellent meat poultry,because of its fast growth,high lean meat percentage,special wildfowl taste for meat,great fat liver performance,good adapting to roughage etc,strong disease resistance,high reward of feed.However,compared with domestic ducks,Muscovy duck has existed obviously disadvantage of long breeding cycle,less egg number of annual production,strong broodiness,which caused the low reproductive performance of Muscovy duck and high cost of feeds.so the development of intensive and large-scale feeding of Muscovy ducks has been severely restricted by the factor of low reproductive performance,in order to offer molecular information for breeding of reproductive traits of Muscovy duck,NPY gene and SMAD3 gene were selected as the candidate gene of reproductive performance for Muscovy duck,the CDS region of NPY gene and SMAD3 gene were cloned and analysised by bioinformatics tools,and the transcriptional level of NPY gene and SMAD3 gene in different tissues were also been analysised by q-PCR.Moreover,the polymorphic loci of NPYgene and SMAD3 gene were screened and genotyped.Additionally,the associative study between genotypes and reproductive traits were conducted.The result are as follows:1.The whole-length CDS region of NPY gene was 294 bp,which coded 97 amino acids,through sequencing and homology comparison,the nucleotide sequence of NPY gene shared the highest identy with Anas platyrhynchos(99.7%),and shared the second identy with Meleagris gallopavo(97.3%),the amino acid sequence homology of NPY gene with Anas platyrhynchos and Meleagris gallopavo was 100%,secondary was Gallus gallus(99%).Phylogenetic analysis showed that Tianzhu Muscovy duck had a close relationship with Anas platyrhynchos.The property of NPY protein were acidic and unstable,and NPY protein which all located in extracellular had a signal peptide which occured at the positions of the first to twenty-eighth amino acids.In addition,there were two o-glycosylation sites and abundant phosphorylation sites in NPY protein,which existed a functional domain named PAH,The spatial structure of NPY protein were mainly assembled with a-helix and random coil.The results were expected to provide some references for further study on research of structure and function of NPY gene in Muscovy duck.2.The whole-length CDS region of SMAD3 gene was 966 bp,which coded 321 amino acids.The nucleotide sequence of NPY gene shared the highest identy with Anas platyrhynchos(99.8%),and shared the second identy with Anser cygnoides(99%),except for Haliaeetus leucocephalus,the amino acid sequence homology of SMAD3 gene shared 100% identy with Anas platyrhynchos and other birds,the amino acid sequence homology with Neophocaena asiaeorientalis and other mammal,and zebrafish were more than 96.6%,phylogenetic analysis showed that Tianzhu Muscovy duck had a close relationship with Anas platyrhynchos.The property of SMAD3 protein was acidic and unstable,and SMAD3 protein which mainly located in organelles of cytoplasm hadno signal peptide.Moreover,there were riched of glycosylation and phosphorylation sites in SMAD3 protein,which existed two functional domains named MH1 and MH2.The spatial structure of SMAD3 protein were assembled with random coil.The results were expected to provide some references for further study on research of structure and function of SMAD3 gene in Muscovy duck.3.Q-PCR results showed that the NPY mRNA were widely expressed in all tissues,and the highest expressive level of NPY mRNA was in cerebrum,and the second one were in pancreas and glandular stomach,which were highly significant difference with others tissues(P<0.01),the lowest one was in gizzard.The SMAD3 mRNA were widely distributed in all tissues,and the highest expressive level of were in muscle tissues,and the second one was in uterus,both which were highly significant difference with others tissues(P<0.01),the lowest one was in gizzard.4.Two polymorphic sites named A-411 G,A-540 G were founded in 5’ promoter region of NPY,which existed AA,AG,GG three genotypes.The correlative analysis with reproductive performance indicated that the laying start age for AG genotype was earlier than that of AA and GG genotype,which were extremely significant with AA and GG Genotype(P<0.01),the weight with the first egg of the genotype GG was significantly higher than that of AA genotype(P<0.05).The total egg yield with 300-days-old of age for genotype AG was significant difference with that of genotype AA group(P<0.05).The results suggest that the dominant genotypes of mutat sites of A-411 G and A-540 G of NPY gene might be as the potential molecular markers for reproductive performance in Muscovy duck.5.Fifteen SNPs were detected in exons of SMAD3 gene,The AA genotype of A+6185G and G+6269T,CC genotype of C+24204T and A+27515G sites were extremely significantly or significantly correlated with the the laying start age of Muscovy duck(P<0.01 or P<0.05),the CC genotype of C+24204T,the AG genotype of A+27515G and A+27541G sites were extremely significantly or significantly correlated with the total egg yield for 300-days-old of Muscovy duck(P<0.01 or P<0.05),the weight at the laying start age of genotype CG was significantly higher than that of genotype GG at C+27350G site(P<0.05),the weight at the laying start age of genotype TT was significantly higher than that of genotype CT at C+24295T site(P<0.05),the weight at the laying start age of genotype CC was significantly higher than that of genotype CT at C+27535T site(P<0.05).In summary,the dominant genotypes of those SNPs mutation of SMAD3 gene might be as the potential molecular markers for the reproductive performance of Muscovy ducks,which can be used for marker-assisted selection.