Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (17): 3433-3442.doi: 10.3864/j.issn.0578-1752.2016.17.016

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

Influences of Pax6 PAI Subdomain on MITF, TYR, TYRP1 and TYRP2 in Melanocytes

NIE Rui-qiang1, YANG Yu-jing1, XIE Jian-shan1,2, FAN Rui-wen1, XU Dong-mei1, YU Xiu-ju1, DUAN Zhi-cheng1, DONG Chang-sheng1   

  1. 1College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
    2School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001
  • Received:2016-01-26 Online:2016-09-01 Published:2016-09-01

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Abstract: 【Objective】 Highly conserved PAX family has important effects on the differentiation of melanocytes and production of melanin. It has mainly binding sites with target gene in PD domain that is included by all of PAX family, on the other hand, the PAI subdomain, which is located in amino terminal of PD domain, has most important effects on PD domain which bound with target gene. Many reports show that Pax6 has the most important effects on the differentiation of retinal pigment epithelial cells. This experiment studies the function of PD domain and PAI subdomain of PAX family by analysing the function of Pax6 PAI subdomain.【Method】The structure of Pax6 PD domain was analyzed by Psipred. The target gene binding sites of Pax6 PD domain was analyzed by NCBI. The binding sites of Pax6 PD domain to the promoter of MITF, TYR, TYRP1, and TYRP2 were analyzed by Jasper. The coding sequences of Pax6 PAI subdomain was amplified by PCR and the Pax6 PAI subdomain 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 promoter driving the expression of green fluorescent protein (GFP). The plasmid vector was confirmed by sequencing. Then, the mouse melanocytes were transfected with the vector using Liposome 2000. Three methods were used in the result test, they were quantitative real-time PCR, western blot and melanin content measurement. 【Result】The target gene binding sites of Pax6 PD domain was mainly distributed in PAI subdomain which is located in amino terminal of PD domain. There was a binding site of Pax6 PD domain at -695 base of MITF promoter; Two binding sites of Pax6 PD domain at -873 base and -1133 base of TYR promoter; One binding site of Pax6 PD domain at -629 base of TYRP1 promoter; And one binding site of Pax6 PD domain at -655 base of TYRP2 promoter. The RT-PCR and western blot results showed that the four target genes and melanin content were significantly increased. MITF mRNA was significantly increased by 2.05 times (P＜0.01), TYR mRNA was increased by 2.09 times, TYRP1 mRNA was increased by 2.93 times(P＜0.05), TYRP2 mRNA was increased by 3.62 times (P＜0.01). Compared with the control group, MITF protein was significantly increased by 1.7 times (P＜0.01), TYR protein was increased to 2 times (P＜0.05), TYRP1 protein was increased by 1.9 times(P＜0.01), TYRP2 protein was increased to 1.37 times. Meanwhile, the melanin content was significantly increased by 1.33 times (P＜0.001).【Conclusion】Results of the study demonstrated that the Pax6 PAI subdomain still promoted the expression of MITF, TYR, TYRP1, and TYRP2, while increased the production of melanin of melanocytes.

Key words: PAI subdomain, Pax6, melanin

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[1] NIE Rui-qiang, YANG Yu-jing, XIE Jian-shan, FAN Rui-wen, GAO Wen-jun, DONG Chang-sheng. The Influences of Over-Expressing Pax610Neu on MITF and TYR in Melanocytes [J]. Scientia Agricultura Sinica, 2016, 49(11): 2214-2221.
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