山羊PMEL基因启动子活性及转录调控元件分析

doi:10.11843/j.issn.0366-6964.2017.05.006

摘要/Abstract

摘要：

旨在筛选调控山羊毛色基因PMEL的启动子活性区域及转录因子，为探究该基因的表达调控机制提供理论依据，并为彩色山羊的育种和改良提供思路。以山羊基因组DNA为模板，PCR扩增PMEL基因不同长度的启动子缺失片段，定向克隆至pGL3-basic载体，将重组质粒转染到293T和A375细胞，通过双荧光素酶检测系统测定相对荧光素酶活性值；利用生物信息学方法对PMEL基因核心启动子区的转录因子结合位点进行预测，随后利用重叠延伸PCR分别对pGL3-327质粒上预测的转录因子结合位点进行点突变并构建突变载体，利用双荧光素酶检测系统进行活性验证。结果显示，本研究成功构建了7个不同长度的启动子片段，其中6个片段具有明显的启动子活性。经过双荧光素酶活性检测发现山羊PMEL基因-251/+76区域为核心启动子区域。通过不同长度的启动子片段的活性比较发现，-251/-62区域的缺失造成启动子活性从最高到消失，表明该区域对山羊PMEL基因转录调控有重要影响，生物信息学分析发现该区域存在5个转录因子结合位点，利用点突变构建了5个突变载体，经过双荧光素酶检测发现5个突变载体的活性均显著下降。提示这5个转录因子是山羊PMEL基因转录的正调控元件。本研究确定了山羊PMEL基因启动子核心区域为-251/+76，NF-1（-206/-197）、Sp1（-186/-174）、Sp1（-151/-139）、CREB（-91/-82）和Sp1（-82/-71）结合位点为山羊PMEL基因转录的正调控元件。

关键词: PMEL基因, 启动子, 瞬时表达, 点突变, 转录调控元件

Abstract:

The research was designed to screen the promoter activity region and transcription factors of goat PMEL gene, and to provide a theoretical basis for exploring the mechanism of expression regulation of PMEL gene and provide ideas for breeding and improvement of colored goats. Using goat genomic DNA as template, PCR was used to amplify PMEL gene promoter sequence of different length fragments, and the fragment was cloned into pGL3-basic vector. The recombinant plasmid was transfected into 293T and A375 cells, the relative luciferase activity was measured by dual luciferase assay system, and the transcription factor binding sites in the core promoter region were predicted by bioinformatics. The transcription factor binding sites on the pGL3-327 plasmid were mutated by overlap-extension PCR, and the activity of mutants was verified.The results showed that 7 promoter fragments of different lengths were constructed successfully, and 6 of them had obvious promoter activity. -251/+76 region was detected by dual luciferase activity as the core promoter region. By comparing the activity of PMEL gene promoter sequence of different length fragments, the deletion of -251/-62 region made the promoter activity from the highest to none, which indicated that the region had important influence on the transcriptional regulation of goat PMEL gene. Bioinformatics analysis found that the -251/-62 region had 5 transcription factor binding sites, the 5 mutation vectors were constructed by point mutation, their activity were significantly decreased by double luciferase assay. It is suggested that these 5 transcription factors were the positive regulatory elements in goat PMEL gene transcription.The results indicate that the core promoter of goat PMEL gene is -251/+76 region, NF-1 (-206/-197), Sp1 (-186/-174), Sp1 (-151/-139), CREB (-91/-82) and Sp1 (-82/-71) binding sites are positive regulatory elements of goat PMEL gene.

Key words: PMEL gene, promoter, transient expression, point mutation, transcriptional regulatory elements

中图分类号:

S827
S813.3

李丽莎, 彭永东, 郑晓宁, 李祥龙. 山羊PMEL基因启动子活性及转录调控元件分析[J]. 畜牧兽医学报, 2017, 48(5): 826-835.

LI Li-sha, PENG Yong-dong, ZHENG Xiao-ning, LI Xiang-long. Analysis of the Promoter Activity and Transcriptional Regulatory Elements of Goat PMEL Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(5): 826-835.

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