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

摘要/Abstract

摘要： 【目的】小眼畸形相关转录因子-M型（microphthalmia associated transcription factor M，MITF-M）在动物皮肤和毛发的黑色素合成途径中发挥重要作用，克隆绵羊小眼畸形相关转录因子-M型基因序列，研究在绵羊黑素细胞中过表达绵羊MITF-M是否影响TYR、TYRP-1和TYRP-2的表达，从而探究对黑色素的生成的影响。【方法】使用试验室冻存的第5代绵羊黑素细胞，通过PCR方法用引物以绵羊黑素细胞cDNA为模板克隆MITF-M基因cDNA序列，构建绵羊MITF-M克隆载体和真核表达载体；通过细胞转染技术在细胞水平过量表达绵羊MITF-M；转染后使用荧光显微镜观察细胞转染效率，采用分光光度计对绵羊黑素细胞中黑色素含量进行测定，并进行Real-time PCR试验检测转染后细胞MITF、TYR、TYRP-1和TYRP-2基因在mRNA水平表达量的变化，Western blot试验检测转染后细胞MITF、TYR蛋白水平的变化。【结果】经测序和拼接，最终获得长度为1 242 bp的绵羊 MITF-M基因的cDNA 序列；成功构建真核表达载体，载体上连有一个启动报告基因绿色荧光蛋白和特异性TYRP-2基因启动子；细胞转染后，在荧光显微镜下可观察到黑素细胞带有绿色荧光说明转染效率明显；分光光度计检测显示，转染后绵羊黑素细胞中黑色素量增加1.15倍（P＜0.05）；荧光定量检测结果显示，绵羊黑素细胞中MITF mRNA表达量增加极显著（P＜0.001），表明绵羊MITF-M转染效率显著，TYR mRNA表达量增加极显著（P＜0.001），TYRP-1 mRNA表达量升高至5.06倍（P＜0.05），TYRP-2 mRNA表达量升高至1.49倍，变化不明显。蛋白免疫印迹结果显示，绵羊黑素细胞中MITF-M转染组MITF蛋白表达量是空载体组的1.65倍（P＜0.001），转染组TYR蛋白表达量是空载体组的2.38倍（P＜0.001），这与荧光定量检测结果一致。【结论】通过PCR和克隆技术及核酸测序技术获得了绵羊MITF-M基因全长1 242 bp的CDS区，过表达绵羊MITF-M会使黑素细胞TYR、TYRP-1的表达量增加，对TYRP-2的表达量变化不明显，从而揭示绵羊MITF-M可以通过调节TYR和TYRP-1的表达量控制绵羊黑素细胞中黑色素的生成。

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

杨玉静，张丹瑾，聂瑞强，谢建山，范瑞文，高文俊，董常生. 绵羊MITF-M在黑素细胞中过表达后的功能分析[J]. 中国农业科学, 2016, 49(21): 4214-4221.

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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