SLC22A3基因负性调控元件的定位

发布时间：2018-01-17 17:08

本文关键词：SLC22A3基因负性调控元件的定位　出处：《重庆医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的拟寻找人第6号染色体q26-q27区域的SLC22A3基因intron7序列中具有重要负性调控功能的核心调控元件的具体序列位置。方法用基因重组的方法,分别构建intron7截短型野生重组质粒,分段查找该负性调控元件的具体序列位置。以包含人SLC22A3基因全长序列的细菌人工染色体为模板,PCR扩增所需截短型片段,分别插入荧光素酶报告基因载体pGL3-Promoter,将重组质粒与内参质粒pRL-SV40以50:1的比例共转染HEK293T细胞,24h后检测荧光素酶活性(Luciferase activity),通过对比重组质粒和pGL3-Promoter荧光素酶活性,判断插入片段是否具有调控作用,随后通过实时定量PCR对Luciferase基因mRNA水平进行检测,并且验证其调控元件作用与其位置的相关性,最后对发现元件进行转录因子结合位点预测。结果截短型野生重组质粒pGL3-pro-SLCi7-NO6,pGL3-pro-SLCi7-NO10,pGL3-pro-SLCi7-NO6.2,p GL3-pro-SLCi7-NO6.3,p-GL3-pro-SLCi7-NO10.2,pGL3-pro-SLCi7-NO6.21,p GL3-pro-SLCi7-NO10.22各组荧光素酶活性较pGL3-Promoter组降低(P0.05);而截短型野生重组质粒pGL3-pro-SLCi7-NO6.21-300 bp(P0.05)和p GL3-pro-SLCi7-NO10.22-300 bp(P0.05)组荧光素酶活性较pGL3-Promoter组无统计学差异,实时定量PCR验证mRNA水平与蛋白表达水平相一致,且调控元件发挥作用与其所处位置无相关。同时,通过生物软件预测发现与负性调控元件结合的四个转录因子。结论人类第6号染色体q26-q27区域的SLC22A3基因intron7序列中存在2个负性调控元件,为今后进一步对SLC22A3基因表达调控方面的研究提供了新的理论依据。
[Abstract]:Objective to search for the specific position of the core regulatory elements in the intron7 sequence of the SLC22A3 gene in the q26-q27 region of human chromosome 6, which have important negative regulatory functions. The method of restructuring. Intron7 truncated wild recombinant plasmids were constructed, and the specific sequence location of the negative regulatory element was found. The artificial chromosome of bacteria containing the full-length sequence of human SLC22A3 gene was used as template. The truncated PCR fragments were inserted into the luciferase reporter gene vector pGL3-Promoter respectively. The recombinant plasmid pRL-SV40 was co-transfected into HEK293T cells at the ratio of 50: 1. Luciferase activity and luciferase activity of luciferase activity were detected 24 hours later. The recombinant plasmid and pGL3-Promoter luciferase activity were compared. Then the mRNA level of Luciferase gene was detected by real-time quantitative PCR, and the correlation between the role of its regulatory element and its position was verified. Finally, transcription factor binding sites were predicted. Results truncated wild recombinant plasmid pGL3-pro-SLCi7-NO6 + pGL3-pro-SLCi7-NO10. PGL3-pro-SLCi7-NO6.2pGL3-pro-SLCi7-NO6.3pGL3-pro-SLCi7-NO10.2. PGL3-pro-SLCi7-NO6.21. The luciferase activity of P GL3-pro-SLCi7-NO10.22 group was lower than that of pGL3-Promoter group. The truncated wild recombinant plasmid pGL3-pro-SLCi7-NO6.21-300 bpn. P0. 05). There was no significant difference in luciferase activity between the two groups compared with the pGL3-Promoter group. Real-time quantitative PCR was used to verify that the level of mRNA was consistent with the level of protein expression, and the role of regulatory elements was not related to their position. Four transcription factors combined with negative regulatory elements were found by biosoftware prediction. Conclusion there are two SLC22A3 gene intron7 sequences in the q26-q27 region of human chromosome 6. Negative regulatory elements. It provides a new theoretical basis for further research on the regulation of SLC22A3 gene expression.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R541.4

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