猪MITF-M的转录调控分析

doi:10.11843/j.issn.0366-6964.2018.11.006

摘要/Abstract

摘要：

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 6 414 bp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～-6 036 bp的正调控区域、-2 470～-3 753 bp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。

Abstract:

This experiment was conducted to study the transcription regulation region activity of pig MITF-M (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 transcription regulation region of pig MITF-M, and 5'-end deletion recombinant vector were obtained by gene cloning. The transcriptional activity of MITF-M was tested via dual luciferase reporter gene experiment, and RNA interference were carried out to verify important transcription factors. The results showed that a 6 414 bp transcription regulation region was cloned; Nine 5'-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 cells and affected the pig coat color. These data laid the foundation for further studying the expression mechanism of pig MITF-M, and was of great significance for studying the regulation mechanism of pigs coat color.

中图分类号:

S828.2

张廷焕, 柴捷, 陈磊, 龙熙. 猪MITF-M的转录调控分析[J]. 畜牧兽医学报, 2018, 49(11): 2349-2358.

ZHANG Ting-huan, CHAI Jie, CHEN Lei, LONG Xi. Transcriptional Regulation Analysis of Pig MITF-M Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(11): 2349-2358.

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