Study On The Melanogenic ASIP And AGRP Gene Of Duck

Muscovy duck, GaoYou duck and Cherry Valley duck (the British strain of Pekin-duck) were collected to study the effects of nondestructive samples and destructive samples on total RNA extraction in order to provide a new method for further molecular biology researches in hair follicle tissues of the poultry.Furthermore, we cloned parts of nucleotide sequences of Agouti signaling protein(ASIP) and Agouti-related protein (AGRP) genes. Semi-quantitative RT-PCR method was used to study expression patterns of ASIP and AGRP genes in various tissues in Muscovy duck. The major findings were as follows:1.The Agouti signaling protein (ASIP) CDS fragments (308bp) of Muscovy duck, GaoYou duck and the British strain of Pekin-duck were amplified from the extracted total RNA of nondestructive collected hair follicle by using RT-PCR, cloned and sequenced. The results showed that the obtained sequences of GaoYou duck were same as those of the British strain of Pekin-duck. When compared with the chicken and quail corresponding CDS sequences of ASIP gene in GenBank, the homologies of the nucleotide sequences and the 102 amino acid sequences of ASIP gene in Muscovy duck, GaoYou duck and the British strain of Pekin-duck were higher than 92.53% and 92.16%, respectively. These results proved that the ASIP gene in birds was considerably conserved. It also showed the feasibility of nondestructive sampling,providing a new reference for sampling methods of avian hair follicles.2.Primers were designed for amplification of AGRP gene according to the known conserved region sequences of chicken, quail, and anas platyrhynchos. The fragments of ASIP gene in Muscovy duck, GaoYou duck and the British strain of Pekin-duck were obtained by PCR amplification. The nucleotide sequences of Muscovy duck, GaoYou duck and the British strain of Pekin-duck were 1213bp, 1195bp, 1196bp, respectively, containing two introns, exon 2, incomplete exon 1 and exon 3. Homologue analysis suggested that the homologies between ducks and species of Galliformes were high, especially in coding sequence and amino acid sequence. However, the duck sequence of AGRP intron 2 was 200bp less than that of Galliformes species.3.Semi-quantitative RT-PCR was used to detect the expression of ASIP and AGRP gene in hair follicles, skin, heart, liver, spleen, lung, kidney, skeletal muscle, ovary (♀), testis(♂), hypothalamus, pituitary, adrenal gland, uropygial gland, fat, totally 15 tissues of Muscovy duck. The results showed that ASIP gene expressed in the skin (hair follicle and skin), central nervous system (hypothalamus) and other non-skin tissues of avian. The AGRP gene was expressed in all tissues,displaying high expression levels in hypothalamus and adrenal gland.The ASIP gene expression in hair follicle was not different from that in the skin (P>0.05), while the relative ASIP gene expression level in the hypothalamus was significantly higher than that of the skin (P<0.01). There were significant difference among the expressions of AGRP gene in the hypothalamus, hair follicle and skin (P<0.01), with the highest expression level in hypothalamu, followed by hair follicle, and finally the skin.