AIM; To investigate the effect of G/A mutation in rs5418 site of solute carrier family 2, facilitated glucose transporter,member 4 protein(SLC2A4) promoter region on gene expression. METHODS: The core promoter re-gion of SLC2A4 gene was amplified by PCR. Mutant and wild - type recombinant expression vectors containing promoter of SLC2A4 gene were constructed by recombinant gene technique and the strategy of site - directed mutagenesis. Recombinant vectors were transfected into HEK293T cells by lipofectamine and the expression activity of the reporter gene in the recom-binant expression vectors with different alleles was detected by a dual - luciferase reporter assay system. RESULTS: The 716 - bp SLC2A4 promoter was amplified and the recombinant expression vectors pGL3 - SLC2A4 -prom(A) and pGL3 -SLC2A4 - prom( G) were successfully constructed. The luciferase reporter vector containing SLC2A4 promoter with rs5418 -A alleles produced significantly higher relative luciferase activity (19.49 ±4.41) than that with rs5418 -G allele (13. 04 ±4.45; P <0. 05). CONCLUSION: The G→A variation of rs5418 site in SLC2A4 promoter region increases the ex-pression of SLC2A4 gene, thereby affecting the gene function.%目的:研究葡萄糖转运蛋白4(SLC2A4)基因启动子区rs5418多态位点G→A变异对基因表达的影响.方法:PCR法扩增SLC2A4基因核心启动子区序列.采用基因重组、定点突变等技术,构建rs5418位点含有不同等位基因的SLC2A4基因启动子重组表达载体.脂质体转染法将重组质粒转入HEK293T细胞,采用双萤光素酶报告系统,观察携带不同等位基因的重组质粒中下游报告基因的表达活性.结果:PCR扩增获得长度为716 bp的SLC2A4基因核心启动子序列,成功构建pGL3-SLC2A4-prom(A)和pGL3-SLC2A4-prom(G)重组表达载体.双萤光素酶报告基因活性检测结果显示,携带A等位基因的载体启动下游报告基因表达的相对活性(19.49±4 41)比携带G等位基因的载体(13.04±4.45)强,两者存在显著差异(P<0.05).结论:SLC2A4基因启动子区rs5418位点的G→A突变能显著增强启动子活性,从而影响SLC2A4基因表达活性.
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