绵羊MITF-M在黑素细胞中过表达后的功能分析

doi:10.3864/j.issn.0578-1752.2016.21.015

Abstract

Abstract: 【Objective】 Microphthalmia associated transcription factor M (MITF-M) plays an important role in the melanin synthesis in animal’s skin and hair. The aim of this paper was to clone microphthalmia-associated transcription factor M, to investigate whether the over-expression of oar MITF-M regulated TYR, TYRP-1 and TYRP-2 expression in melanocytes of sheep, and to explore its influence on the formation of melanin. 【Method】The CDS region in oar MITF-M gene was cloned from melanocytes of sheep by primers and PCR, to build a sheep MITF-M cloning vector and eukaryotic expression vector. Over-expression of oar MITF-M was conducted in melanocytes of 5^th generation sheep through the cell transfection technique and the transfer efficiency was observed by fluorescence microscope. The contents of melanin in melanocytes was detected by spectrophotometer, and the levels of MITF, TYR, TYRP-1 and TYRP-2 were detected using Real-time PCR and the proteins of MITF and TYR were detected using Western blot. 【Result】 Results showed that the 1 242 bp cDNA sequence of oar MITF-M gene was obtained by sequencing and splicing. Eukaryotic expression vector was successfully constructed with a startup report gene of green fluorescent protein and specificity TYRP-2 gene promoter. Under the fluorescence microscope, green fluorescence could be identified in melanocytes of sheep. The contents of melanin in melanocytes were increased by 1.15 times (P＜0.05). Real-time PCR results showed that MITF mRNA was significantly increased (P＜0.001) which indicated the oar MITF-M high transfection efficiency. TYR mRNA was significantly increased (P＜0.001). TYRP-1 mRNA was significantly increased by 5.06 times (P＜0.05). TYRP-2 mRNA was not significantly increased by 1.49 times. MITF protein in MITF-M group was 1.65 times more than in empty vector group (P＜0.001) and TYR protein in MITF-M group was 2.38 times more than in empty vector group (P＜0.001), which was consistent with the real-time PCR results. 【Conclusion】 The 1 242 bp length CDS region of oar MITF-M gene was got by PCR, TA cloning and nucleic acid sequencing technology. Results of the study suggested that oar MITF-M increased the production of TYR, TYRP-1 and had little influence on TYRP-2. Therefore, the oar MITF-M may mediate the alteration of coat color through regulating the expression of TYR and TYRP-1.

Key words: microphthalmia associated transcription factor (MITF), TYR, TYRP-1, TYRP-2

YANG Yu-jing, ZHANG DAN-Jin, NIE Rui-qiang, XIE Jian-shan, FAN Rui-wen, GAO Wen-jun, DONG Chang-sheng. The Function Analysis of Over-Expression of Oar MITF-M in Melanocytes[J].Scientia Agricultura Sinica, 2016, 49(21): 4214-4221.

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