Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (11): 2214-2221.doi: 10.3864/j.issn.0578-1752.2016.11.017

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

The Influences of Over-Expressing Pax610Neu on MITF and TYR in Melanocytes

NIE Rui-qiang1, YANG Yu-jing1, XIE Jian-shan1,2, FAN Rui-wen1, GAO Wen-jun1, DONG Chang-sheng1   

  1. 1 College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
    2 School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001
  • Received:2015-10-28 Online:2016-06-01 Published:2016-06-01

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Abstract: 【Objective】 MITF and β-catenin are important regulatory genes of the melanin system. Pax6 regulates the production of melanin indirectly, through regulates MITF and TYR. In order to explore the function of Pax610Neu and the mechanism of Pax6 with its target genes, we explored the function of Pax610Neu, which only included 102 N-terminalanimo acids of the paird domain. 【Method】 According to the sequence of Pax610Neu in NCBI, the primers of Pax610Neu were designed and synthesized by BGI. The coding sequences of Pax610Neu were PCR amplified and then confirmed by sequencing. Sal I and Xba I restriction sites were selected by analyzing the sequences of Pax610Neu and the mammalian expression vector. The Pax610Neu was cloned into the T-Vector, meanwhile, confirmed by sequencing. The fragment was then subcloned into a mammalian expression vector, resulting in a construction that contained a specific mouse tyrp2 promoter driving the expression of the green fluorescent protein (GFP) and the target fragment with Sal I and Xba I restriction sites. The plasmid vector was confirmed by sequencing. The expression vector was amplified by competent cells and obtained without endotoxin by the plasmid midiprep system. Then, the sheep melanocytes were transfected with the vector using Liposome 2000. There were three methods used in the results test which were quantitatively real-time PCR, western blot, and melanin content measurement. 【Result】 The results showed that the MITF mRNA, MITF and TYR protein, melanin content were significantly increased. MITF mRNA was significantly increased to 3.2 times (P＜0.05) and TYR mRNA was increased to 1.31 times, compared with control group, MITF protein was significantly increased to 8.24 times (P＜0.001) and TYR protein was significantly increased to 2.09 times (P＜0.001). Meanwhile, the melanin content was significantly increased to 1.22 times (P＜0.05). 【Conclusion】 We demonstrated that the Pax610Neu still interacts with MITF, and then regulated TYR indirectly, meanwhile changing the production of melanin of melanocytes.

Key words: Pax610Neu, Pax6, MITF, TYR, melanin

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