Molecular Clonging,Expression And SNP Detection On ASIP And TYRP1 In The Candidate Genes Of Goose Plumage Color

Plumage color is an important economic trait in poultry, and is also a genetic marker for the breeding of the varieties (lines), it plays an important role in determining breed purity and phylogenesis. Studies showed that the genetic variation of agouti signaling protein (ASIP) and tyrosinase related protein-1 (TYRP1) genes were associated with the coat (plumage) color of animals, and they have been proposed to be important candidate genes in the genetics of coat (plumage) color. In this study, Zhedong White goose、Wanxi White goose (white feather)、Wanxi White goose (flower feather)、Shitou goose (gray feather) and Shitou goose (white feather) were used as the experimental materials, ASIP gene and TYRP1 gene were selected as candidate genes of the plumage color trait in goose. We cloned the nucleic acid sequence, analyzed the sequence structure and tissue expression profile and screened the SNP sites in the genomic DNA sequence. The main obtained results were as follows:1. The whole cDNA and genomic DNA of goose ASIP gene were cloned by the comparative cloning method. The length of cDNA is 448 bp (GenBank:KP404626), contains a 38 bp 5’-UTR,390 bp ORF and a 20 bp 3’-UTR. The ORF encodes a protein of 130 amino acids with a molecular weight of 14.88 kDa and a isoelectric point of 9.73. The length of goose ASIP genomic DNA is 6176 bp, including three exons and two introns. Multiple sequence alignments and phylogenetic analysis display that the amino acid sequence of ASIP is conserved in vertebrates, especially in the bird species. The semi-quantitative RT-PCR reveals that the expression of goose ASIP mRNA is differentially expressed in the pigment deposition tissues, including eye, flipper, hair follicle, skin of back, as well as in skin of abdomen. Meanwhile, the expression level of ASP gene in skin of abdomen was higher than that in skin of back.45 single nucleotide polymorphisms (SNPs) and 3 insert-deletion (indel) variations were identified in the goose ASIP genomic DNA sequence. For c.3758G>A and C.4599C>T mutations, they could be detected by HhaⅠ and HindⅢ, especially. Both of them generated three kinds of genotypes. The genotyping results indicated that three genotypes appeared in the four goose populations. For c.3758G>A mutation, the allele N was in dominance in Wanxi White goose (white feather), Wanxi White goose (flower feather) and Shitou goose (white feather) populations, the allele D was in dominance in the Shitou goose (gray feather) population. For c.4599C>T mutation, the allele N was in dominance in all detected populations.2. The whole cDNA and genomic DNA of goose TYRP1 gene were cloned by the comparative cloning method. The length of cDNA is 1866bp (GenBank:KR080365), contains a 141 bp 5’-UTR,1611 bp ORF and a 114 bp 3’-UTR. The ORF encodes a protein of 536 amino acids with a molecular weight of 60.66 kDa and a isoelectric point of 5.68. The length of goose ASIP genomic DNA is 10331 bp, including seven exons and six introns. Multiple sequence alignments and phylogenetic analysis revealed that the deduced goose TYRP1 protein had a closed genetic relationship and evolutional distance with that of duck, chicken, quail, turkey and zebra finch. The semi-quantitative RT-PCR analysis displayed that the goose TYRP1 mRNA is highly expressed in the pigment deposition tissues, including eye, skin of back, skin of abdomen and hair follicle, intermediate in flipper, brain, muscle and glandular, absent in heart, liver, spleen and kindey tissues. The expression level of TYRP1 gene in skin of abdomen also was higher than that in skin of back.20 single nucleotide polymorphisms (SNPs) and 3 insert-deletion (indel) variations were identified in the goose TYRP1 genomic DNA sequence. For c.6290A>G mutation, it could be detected by restriction enzyme Hinfl and generated three genotypes. The genotyping results indicated that three genotypes appeared in the four goose populations, and the allele N was in dominance in all detected populations.All the aboved findings will help us understand the funcations of ASIP and TYRP1 genes in geese plumage color.