glut9基因启动子区痛风易感snp位点功能的分析-analysis of snp site function of gout susceptibility in glut 9 gene promoter region.docxVIP

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glut9基因启动子区痛风易感snp位点功能的分析-analysis of snp site function of gout susceptibility in glut 9 gene promoter region.docx

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glut9基因启动子区痛风易感snp位点功能的分析-analysis of snp site function of gout susceptibility in glut 9 gene promoter region

AbstractObjectives: Previous studies have shown that the G to C substitution of rlocated in the putative promoter region of glucose transporter 9 (GLUT9), is significantly associated with gout susceptibility in Han Chinese males. Here, we investigate the potential mechanisms underlying association between this polymorphism and gout risk.Methods: Specific fragments incorporating the GLUT9 rpolymorphism were obtained by polymerase chain reaction (PCR), and cloned into a luciferase reporter vector to construct the wild(pGL3-basic-GLUT9/G) and mutant(pGL3-basic-GLUT9/C) recombinant. The recombinant were transient transfected into 293T cells to express. Differences in promoter activities were tested by dual-luciferase activity assay. Electrophoretic mobility shift assay (EMSA) was also performed to determine differences in transcription factor binding activities between G and C alleles.Results: By sequenced the recombinant contained both the sequence of pGL3-basic and that of rof human GLUT9 gene promoter. The segment inserted was in the correct direction. Dual-luciferase activity assays showed significant reductions in transcriptional efficiency with the C compared with G allele (P ??0.05). Furthermore, EMSA demonstrated that the C and G alleles have different affinities for specific transcription factors, and suggests that the G to C substitution of rcauses IRF-1 binding site loss.Conclusions: The rpolymorphism in the GLUT9 promoter effectively influences GLUT9 expression, and possibly IRF-1 plays an important role in the procession. It provides the knowledge to investigate the pathology of gout in Han Chinese males.Postgraduate: Zhu Xuelian（Endocrine and metabolic diseases） Directed by Professor: Li ChangguiKey words: Gout；GLUT9；r录引言 1第 1 章材料和方法21.1 主要仪器 21.2 主要试剂 21.3 实验方法 31.3.1 野生型和突变型载体构建 31.3.2 细胞培养与转染 61.3.3 双荧光素酶报告系统检测基因突变对启动子激活能力的影响 71.3.4 EMSA 实验7第 2 章实验结果112.1 载体构建 11重组质粒 pGL3-basic-GLUT9/G 的双酶切