Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (18): 3711-3718.doi: 10.3864/j.issn.0578-1752.2015.18.014

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

cDNA Cloning and Expression of MITF Gene and Its Effect on Melanin Deposition in Silky Fowl

ZHENG Nen-zhu1, XIN Qing-wu1, ZHU Zhi-ming1, MIAO Zhong-wei1, Li Li1, Liu Feng-hui1, HUANG Qin-lou1   

  1. 1 Institute of Animal Science and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013
    2 Station of Animal Science, Licheng District, Putian 351100, Fujian
  • Received:2014-10-30 Online:2015-09-16 Published:2015-09-16

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Abstract: 【Objective】The aim of this paper was to clone microphthalmia-associaated transcription factor(MITF) , explore its expression and relationship with melanin deposition in different tissues of Silky Fowl, for further studying molecular mechanism of melanin deposition among Silky Fowl.【Method】A total of 150 day-old healthy Silky Fowls were selected as the research materials in this experiments. Degenerate primers and Nested PCR were used to isolate MITFgene from the skin of Silky Fowl, and DNAStar, Clustal X, BioEdit and MEGA4.0 software were adopted to analyze biological information of the obtained sequence. Quantitative real-time PCR (qRT-PCR) and ultraviolet spectrophotometry (US) were employed, respectively, to study MITF expression characteristics and melanin deposition in tissues of Silky Fowl, and linear regression estimation was applied to identify the correlation between MITF gene expression and melanin deposition in the tissues of Silky Fowl.【Result】Results showed that the 1 431 bp cDNA sequence of MITF gene was obtained by sequencing and splicing, which contained a complete CDS (25-1 431bp). The CDS domain was composed by 468 amino acids (GenBank accession No. KC879241) with a predicted molecular weight of 52.42kD , an isoelectric point of 6.47, and a conserved basic-helix-loop-helix leucine-zipper (bHLH-zip) protein structure.The homology comparison indicated that the Silky MITF gene shared 100% amino acid homology to Gallus gallus, above 98% to other birds，and about 90% to mammal in CDS domaim. The amino acid phylogenetic tree analysis demonstrated that Silky Fowl MITF had the closest genetic relationship to Gallus gallus, but the farthest to pig and human. The qRT-PCR confirmed that different expressions of MITF gene were detected among tissues of Silky Fowl (P＜0.01), with the highest expression in the skin which was about 2.9 times that of the kidney, 4.4 times that of the gizzard, 11.4 times that of the liver, and 86.2 times that of the muscle. The ultraviolet spectrophotmeter examination showed that the law of melanin depositionwas consistent with thatof MITF expression in tissues, with an overall trend: skin＞kidney＞gizzard ＞liver＞muscular. Correlation analysis diplayed that there was a positive correlation between MITF expression and melanin deposition(P＜0.05).【Conclusion】 MITF gene is highly conservative in the evolution of animal species, the gene expression is specific in tissues, and plays a positive role in the regulation of melanin deposition on Silky Fowl, whose high expression may contribute to the formation of melanin.

Key words: Silky Fowl, melanin, MITF, cloning, gene mRNA expression

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