猪MITF-M的转录调控分析

Transcriptional Regulation Analysis of Pig MITF-M Gene

旨在研究猪MITF-M(microphthalmia associated transcription factor M)转录调控区域活性以及验证重要转录因子。以猪DNA为模板,利用PCR扩增获得MITF-M转录调控区,基因克隆得到5′端缺失重组载体,运用双荧光素酶报告基因试验进行转录活性分析,再通过干扰RNA试验验证重要转录因子。本试验成功克隆得到MITF-M 6 414bp的转录调控区域;构建了9个调控区5′端缺失重组载体;鉴定出MITF-M转录调控区内3个转录活性显著变化区域(P<0.001),分别是位于-1 140～-1 397和-5 429～-6 036bp的正调控区域、-2 470～-3 753bp的负调控区域;发现了转录因子KLF4显著负向调控MITF-M的转录活性(P<0.01)。结果提示,转录因子KLF4抑制MITF-M的转录活性,调控黑色素细胞色素的合成与沉积,从而影响猪毛色的表型。本试验为进一步研究猪MITF-M基因的表达机制奠定了基础,对深入研究猪毛色变化的表达调控机理具有重要意义。

This experiment was conducted to study the transcription regulation region activity of pig MITFM （microphthalmia associated transcription factor M） gene and verify the important transcription factors. PCR amplification with pig DNA as a template was conducted to get the 5r end deletion recombinant vectors were constructed; Three important regulatory regions of MITF＇M were identified （P〈0. 001） ,locating in 1 140 1 397, 5 429 6 036 bp positive regulation regions and 2 470 3 753 negative regulation region; The transcription factor KLF4 significantly negatively regulated the transcription activity of MITF＇M（P〈0. 01）. These results indicated that the KLF4 inhibited the transcriptional activity of MITF＇M and regulated melanin synthesis and deposition in the pigment ceils and affected the pig coat color. These data laid the foundation for further studying studying the expression mechanism of pig MITF＇M, and was of great sig the regulation mechanism of pigs coat color.