山羊PMEL基因启动子活性及转录调控元件分析

发布时间：2018-02-13 04:23

本文关键词： PMEL基因启动子瞬时表达点突变转录调控元件　出处：《畜牧兽医学报》2017年05期 　论文类型：期刊论文

【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。
[Abstract]:The aim of this study was to screen the promoter active regions and transcription factors of goat coat color gene PMEL, and to provide theoretical basis for exploring the mechanism of expression and regulation of goat coat color gene. The genomic DNA of goat was used as template to amplify the promoter deletion fragments of different length of PMEL gene, and then cloned into pGL3-basic vector. The recombinant plasmid was transfected into 293T and A375 cells, and the recombinant plasmid was transfected into 293T and A375 cells. The relative luciferase activity was measured by double luciferase detection system, and the transcription factor binding sites in the core promoter of PMEL gene were predicted by bioinformatics. Then the point mutation of the predicted transcription factor binding site on the pGL3-327 plasmid was carried out by overlapping extended PCR and the mutant vector was constructed. The activity was verified by double luciferase detection system. In this study, seven promoter fragments of different lengths were successfully constructed. Six of the fragments showed significant promoter activity. The goat PMEL gene -251 / 76 region was found to be the core promoter region by double luciferase activity test, and the activity of different length promoter fragments was found to be -251 / -62 region. The loss of promoter activity caused the promoter activity to peak to disappear, The results indicated that this region had an important effect on the transcriptional regulation of goat PMEL gene. Bioinformatics analysis showed that there were five transcription factor binding sites in the region and five mutants were constructed by point mutation. After double luciferase detection, it was found that the activity of the five mutant vectors was significantly decreased, which suggested that the five transcription factors were positive regulatory elements of goat PMEL gene transcription. The core region of goat PMEL gene promoter was -251 / 2. The binding sites of 76-NF-1T -206 / -197 / Sp1C1-186-174C / Sp1-151- 151- 139C ~ (-82) and Sp1+ -82 / -71) are positive regulatory elements for the transcription of goat PMEL gene.
【作者单位】：河北科技师范学院;河北农业大学;
【基金】：河北省高校创新团队领军人才培育计划项目(LJRC004) 河北省应用基础研究计划重点基础研究项目(15962901D) 河北省高等学校青年拔尖人才计划项目(BJ2016026) 秦皇岛市科技支撑计划项目(201502A058)
【分类号】：Q78;S827

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