獭兔 MITF不同可变剪接体对黑色素沉积的影响

小眼畸形相关转录因子(microphthalmia-associated transcription factor, MITF)在哺乳动物皮毛的黑色素形成中发挥了重要作用,而兔MITF基因功能及其不同可变剪接体对黑色素沉积的影响还未见报道。基于此,根据NCBI预测结合文献报道,设计MITF编码区特异性引物,采用RT-PCR结合TA克隆测序的方法,鉴定兔 MITF基因的6种可变剪接体： MITF X1、 MITF X2、 MITF X3、 MITF X4、 MITF X5和 MITF X6。通过进化树分析,发现6个可变剪切体归为一个分支,与人和猩猩的进化距离最近。进一步构建MITF不同剪接体的过表达载体,转染到家兔黑色素细胞中,发现MITF蛋白亚细胞定位于细胞核中;通过荧光定量检测发现MITF X1~X4 过表达显著增加GPNMB的mRNA表达量（P<0.05）,MITF X1~X3过表达显著增加TYR的mRNA的表达量（P<0.05）,MITF X2过表达显著增加 PMEL-17的mRNA表达量（P< 0.05）,其余MITF剪接体的过表达对下游基因表达量的影响不显著（P>0.05）;通过黑色素含量测定,发现MITF X1~X6 剪接体显著增加黑色素细胞中总黑色素和真黑色素的含量（P<0.05）,提示MITF N端的剪接变化并不影响黑色素沉积功能,但不同剪接体的下游信号通路存在差异。

Effect of Alternative Spliceosomes of MITF on Melanin Deposition in Rex Rabbit

Microphthalmia-associated transcription factor (MITF) plays an important role in the formation of melanin in furmammal, but the function of rabbit MITF gene and effects of its different alternative spliceosomes on melanin deposition have not been reported at present. According to the NCBI prediction and relative reports, the MITF coding region-specific primers were designed and six alternative spliceosomes were identified by RT-PCR and TA cloning, MITF X1, MITF X2, MITF X3, MITF X4, MITF X5 and MITF X6. Through phylogenetic tree analysis, we found that the alternative spliceosomes were grouped into one branch, which was closest to the evolution of humans and orangutans.By overexpression of MITF spliceosomes in rabbit melanocytes, the subcellular localization of MITF protein was in the nucleus. Further, the qRT-PCR detection revealed that the mRNA expression of GPNMB increased significantly after overexpression of MITF X1-X4(P<0.05), the expression of TYR mRNA significantly increased after overexpression of MITF X1-X3(P<0.05)and the mRNA expression of PMEL-17 significantly increased after overexpression of MITF X2 (P< 0.05). The overexpression of other MITF spliceosomes had no significant effect on the expression of downstream gene expression(P>0.05). Through the measurement of melanin content, MITF X1-X6 spliceosome significantly increased the contents of total melanin and true melanin in melanocytes (P< 0.05). It is suggested that the N-terminal spliceosomes of MITF does not affect the melanin deposition function, but the downstream signal pathways of different spliceosomes are different.