SLC22A3基因负性调控元件的定位

Identification of negative regulatory element in SLC22A3 gene

摘要:

目的：拟寻找和确定人第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，24 h后检测荧光素酶活性（lu－ciferase 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，并对发现的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，pGL3-pro-SLCi7-NO6.3，p-GL3-pro-SLCi7-NO10.2，pGL3-pro-SLCi7-NO6.21，pGL3-pro-SLCi7-NO10.22各组荧光素酶活性较pGL3-Promoter组降低（P<0.05）；而截短型野生重组质粒pGL3-pro-SLCi7-NO6.21-300 bp（P>0.05）和pGL3-pro-SLCi7-NO10.22-300 bp（P >0.05）组荧光素酶活性较pGL3-Promoter组无统计学差异。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基因蛋表达调控方面的研究提供了理论依据。

Abstract:

Objective：To research and identify the specific sequence position of the key negative regulatory element in intron7 of SLC22A3 gene on chromosome 6q26-q27. Methods：Wild type truncated plasmids of intron7 were constructed by gene recombination to stepwise locate the specific sequence position of this negative regulatory element. Specific truncated gene fragments were amplified the by four stages using bacterial artificial chromosome（including human SLC22A3 gene） as templates，and were inserted into luci-ferase report vector to construct recombinant plasmids respectively. Recombinant plasmids and internal reference plasmids were co-transfected into HEK293T cells with ratio of 50∶1，and the luciferase activity was detected after 24 h to determine which inserted frag－ment showed regulatory effect on gene expression；transcription factor binding sites were predicted in element. Results：The luciferase activities of recombinant plasmids of pGL3-pro-SLCi7-NO6，pGL3-pro-SLCi7-NO10，pGL3-pro-SLCi7-NO6.2，pGL3-pro-SLCi7-NO6.3，pGL3-pro-SLCi7-NO10.2，pGL3-pro-SLCi7-NO6.21，pGL3-pro-SLCi7-NO10.22 were decreased compared with those of pGL3-Promoter（P<0.05）；but there was no significant difference in luciferase activity between pGL3-pro-SLCi7-NO6.21-300 bp（P >0.05），pGL3-pro-SLCi7-NO10.22-300 bp（P>0.05）and pGL3-Promoter. Conclusion：The results demonstrated that intron7 of SLC22A3 gene on chromosome 6q26-q27 contains two negative regulatory elements，which might provide theoretical basis for further studies at protein level.