| With people’s attention to "green food" and "black food",black-bone chicken has become the first choice of green and healthy food,which makes the market demand of black-bone chicken,which is a trinity of meat,egg and medicine,higher and higher.Melanin in black-bone chicken is considered as the material basis for its medicinal effect.In order to explore the distribution and deposition law and molecular regulation mechanism of melanin in Guyuan black-bone chicken,the morphology and distribution of melanin and melanocytes in Guyuan black-bone chicken tissues were observed by HE staining,transmission electron microscopy and immunohistochemical staining.Four-week-old Guyuan black-bone chickens were fed with five different dietary tyrosine levels,the levels of which were the control group(0%),0.2,0.4,0.6 and 0.8%.They were killed at the age of 12 weeks,and the contents of immunoglobulin,melanin,tyrosinase and melanocyte stimulating hormone in each group were determined by enzyme-linked immunosorbent assay.Fluorescence quantitative PCR was used to detect the expression differences of four genes related to melanin synthesis in Guyuan black-bone chicken tissues at different tyrosine levels,including tyrosinase gene(TYR),tyrosinase-related protein 1(TYRP1),microphthalmia-related transcription factor(MITF)and cAMP response element binding protein 1(CREB1),and to explore the gene expression of melanin synthesis.The results showed that melanin granules were observed in the skin,lung,small intestine,chest muscle and trachea of Guyuan black-bone chicken,but not in the liver.The melanin cells were found in the skin,trachea and small intestine of Guyuan black-bone chicken,and the melanin cells in the skin were mainly distributed around blood vessels.The typical characteristics of melanin cells in Guyuan Black Chicken are that there are round melanosomes with high electron density in the cytoplasm.The process of melanosome maturation is divided into four stages,Ⅰ,Ⅱ,Ⅲ and Ⅳ.Most of the melanosomes in trachea and small intestine are in stage Ⅳ of maturation.Immunohistochemical staining showed that TYR had a positive reaction in lung tissue,TYRP1 had a positive reaction in muscle and liver tissue,and MITF and CREB1 had positive results in skin,liver,small intestine and chest muscle tissue.The statistical results showed that dietary tyrosine level affected the contents of immunoglobulin A,melanin,tyrosinase and melanocyte-stimulating hormone.When the tyrosine level was 0.8%,the difference in the contents of melanin,tyrosinase and melanocyte-stimulating hormone between the control group and the treatment group was most significant(p<0.05).When the tyrosine level was 0.6%,the difference in immunoglobulin A content between the control group and the treatment group was significant(p<0.05).However,the addition of dietary tyrosine had no significant effect on the contents of immunoglobulin M and G,but the difference was not significant with the control group(P>0.05).The results of mRNA expression levels of melanin-related genes at different dietary tyrosine levels showed that there was a linear correlation between MITF and TYR gene expressions and melanin content.The study on the distribution of melanin in Guyuan black-bone chicken showed that there were melanin granules in lung,skin,trachea,small intestine and chest muscle of Guyuan black-bone chicken,but no melanin granules were observed in liver.The content of melanin,tyrosinase and MSH in Guyuan black-bone chicken can be significantly increased when the dietary tyrosine level is 0.4-0.8%.TYR and MITF genes can be used as major candidate genes for melanin production in Guyuan black-bone chicken.The research results enrich the biological content of melanocytes and the regulation and change law of melanin deposition,and also provide technical and theoretical basis for human skin whitening and research and development of black food. |
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