中国农业科学2015，Vol.48Issue(18)：3711-3718,8.DOI:10.3864/j.issn.0578-1752.2015.18.014
白绒乌鸡MITF基因的cDNA克隆、表达及其对黑色素沉积的影响
cDNA Cloning and Expression of MITF Gene and Its Effect on Melanin Deposition in Silky Fowl
摘要
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 MITF gene from the skin of Silky Fowl, and DNA Star,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 (Pkidney>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.关键词
白绒乌鸡/黑色素/小眼畸形相关转录因子/克隆/基因表达Key words
Silky Fowl/melanin/MITF/cloning/gene mRNA expression引用本文复制引用
郑嫩珠,辛清武,朱志明,缪中纬,李丽,刘凤辉,黄勤楼..白绒乌鸡MITF基因的cDNA克隆、表达及其对黑色素沉积的影响[J].中国农业科学,2015,48(18):3711-3718,8.基金项目
福建省科技计划项目(2010R1025-6)、福建省农科院创新团队项目(CXTD-2-1314)和福建省农科院基金(MYQJ2014-2) （2010R1025-6）
