山羊PRNP基因启动子转录活性研究

发布时间：2018-05-19 17:04

本文选题：山羊 + PRNP基因　；参考：《中国农业科学》2016年10期

【摘要】：【目的】分析山羊PRNP基因启动子活性区域,旨在筛选调节朊蛋白表达水平的关键区域或转录因子,为阐明山羊PRNP基因的表达调控提供理论依据,并为从遗传学角度降低朊蛋白病的发生提供思路;【方法】以山羊PRNP基因序列(Gen Bank登录号:EU870890)为模板,设计特异性引物,扩增山羊PRNP基因5′侧翼区片段,并将扩增片段克隆至p EASY-T3载体,鉴定为阳性的克隆进行测序;利用生物信息学方法和在线工具进行启动子区域和转录因子结合位点的预测;利用缺失突变技术扩增启动子区不同长度的片段11个,并克隆至p EASY-T3载体后,鉴定为阳性的质粒和pGL3-Basic载体分别用限制性内切酶Mlu I和Bgl II进行酶切,并回收酶切产物;利用T4连接酶进行目的片段与pGL3-Basic连接,鉴定为阳性的荧光素酶报告基因重组质粒进行测序,并提取无内毒素质粒,用脂质体转染法瞬时转染至SH-SY5Y细胞,转染48h后,利用双荧光素酶检测试剂盒进行各缺失突变重组质粒在细胞内的启动活性检测;【结果】成功克隆了山羊PRNP基因5′侧翼区片段,长度为2 332 bp,且该片段含有预测的启动子活性区域、保守的motifs和多个转录因子的结合位点;成功克隆了11个含有不同长度启动子的片段,并与荧光素酶报告基因连接,并构建了目的片段与荧光素酶报告基因的重组质粒;转染时脂质体与DNA的比例为1㑳0.5,萤火虫荧光素酶载体与海肾荧光素酶比例为50㑳1;山羊PRNP基因5′侧翼区存在着核心启动子,启动子活性最强的区域为-519—+82 bp,且在-220—+59 bp这一区域存在着正调控元件,外显子1对启动子活性中起重要的调控作用;4个motifs可能为正调控元件结合位点;在强启动子活性区存在10个Sp1结合位点,2个AP-2 alpha结合位点和1个AP-1结合位点;山羊PRNP基因motif 3和motif 4分别预测为转录因子Foxp3和COE1的结合位点。【结论】确定了山羊PRNP基因启动子的核心区域(-519—+82bp),外显子1对启动子活性起重要的调控作用。
[Abstract]:[objective] to analyze the active region of goat PRNP gene promoter in order to screen the key regions or transcription factors regulating the expression of PRNP gene, and to provide theoretical basis for elucidating the regulation of goat PRNP gene expression. [methods] Goat PRNP gene 5'flanking region was amplified using goat PRNP gene sequence Gen Bank accession number: EU870890 as template, and specific primers were designed to amplify the 5'flanking region of goat PRNP gene. The amplified fragments were cloned into p EASY-T3 vector and identified as positive clones for sequencing. The promoter regions and transcription factor binding sites were predicted by bioinformatics and online tools. Eleven fragments of different length of promoter region were amplified by deletion mutation technique and cloned into p EASY-T3 vector. The positive plasmid and pGL3-Basic vector were digested with restriction endonuclease Mlu I and Bgl II, respectively, and the products were recovered. The target fragment was ligated with pGL3-Basic by T4 ligase. The recombinant plasmid of luciferase reporter gene identified as positive was sequenced, and the non-endotoxin plasmid was extracted. The recombinant plasmid was transiently transfected into SH-SY5Y cells by liposome transfection and transfected into SH-SY5Y cells for 48 h. Double luciferase assay kit was used to detect the priming activity of the deletion mutant recombinant plasmid. [results] the 5'flanking region of goat PRNP gene was cloned successfully. The length of the fragment was 2 332 BP, and the fragment contained predicted promoter active region, conserved motifs and binding sites of multiple transcription factors. 11 fragments with different length promoters were cloned and linked to luciferase reporter gene. The recombinant plasmid of the target fragment and luciferase reporter gene was constructed, the ratio of liposome to DNA was 1: 0. 5, the ratio of luciferase vector to luciferase was 50? 1, and there was a core promoter in 5 'flanking region of goat PRNP gene. The most active region of promoter is -519-82 BP, and there are positive regulatory elements in the region of -220-59 BP, exon 1 plays an important role in the activity of promoter, and four motifs may be positive regulatory element binding sites. There were 10 Sp1 binding sites, 2 AP-2 alpha binding sites and 1 AP-1 binding site in the active region of strong promoter. Goat PRNP gene motif 3 and motif 4 were predicted to be binding sites of transcription factor Foxp3 and COE1 respectively. [conclusion] the core region of goat PRNP gene promoter is identified, and exon 1 plays an important role in the regulation of promoter activity.
【作者单位】：河北农业大学动物科技学院;
【基金】：国家自然科学基金项目(31201775) 河北省首批青年拔尖人才支持计划河北农业大学中青年骨干教师境外研修项目
【分类号】：S827

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