SLC22A3基因intron7对基因转录的负性调控作用研究

Negative regulation role of SLC22A3 gene intron7 in gene transcription

摘要:

目的：探讨人SLC22A3基因intron7序列及其SNPrs2048327（A/G）位点对基因转录的调控作用。方法：以包含人SLC22A3基因全长序列的细菌人工染色体（bacterial artificial chromosome，BAC）为模板，PCR扩增hSLC22A3 intron7，克隆入荧光素酶报告基因载体pGL3-Basic和pGL3-Promoter，得到重组质粒pGL3-Basic-SLCi7wt、pGL3-Promoter-SLCi7wt和pGL3-Promoter-SLCi7mut；重组质粒与内参质粒pRL-SV40共转染入人胚肾细胞HEK293T，24 h后检测荧光素酶活性。结果：野生型重组质粒pGL3-Basic-SLCi7wt荧光素酶活性与pGL3-Basic无统计学差异（P=0.986）；野生型重组质粒pGL3-Promoter-SLCi7wt荧光素酶活性（3.514±0.096） 相较于pGL3-Promoter（6.286±0.370）降低（P=0.000）；突变型重组质粒pGL3-Promoter-SLCi7mut荧光素酶活性（2.089±0.332）相对于pGL3-Promoter亦有所下降（P=0.000），且较野生型重组质粒pGL3-Promoter-SLCi7wt下调（P=0.000）。结论：人SLC22A3基因intron7序列具有负性调节活性，能下调荧光素酶报告基因的表达水平；定位于该序列上的SNP rs2048327（A/G）位点A→G的改变，可能增强该内含子所具有的负性调节作用。

Abstract:

Objective：To explore the regulatory effect of the human SLC22A3 gene intron7 and SNPrs2048327（A/G） on gene tran－scription. Methods：The hSLC22A3 intron7，amplified from the bacterial artificial chromosome（BAC） including whole human SLC22A3 gene，was cloned into pGL3-Basic and pGL3-Promoter respectively to construct the recombinant plasmid pGL3-Basic-SLCi7wt，pGL3-Promoter-SLCi7wt and pGL3-Promoter-SLCi7mut. The recombinants with internal reference plasmid pRL-SV40 were co-trans－fected into HEK293T cells，then the luciferase activity was detected. Results：There was no significant difference in luciferase activity between pGL3-Basic-SLCi7wt and pGL3-Basic（P=0.986）. Luciferase activity of pGL3-Basic-SLCi7wt was lower than that of pGL3-Promoter（（3.514 ± 0.096） vs. （6.286 ± 0.370）；P=0.000）. Luciferase activity of pGL3-Promoter-SLCi7mut was lower than that of pGL3-Promoter and pGL3-Promoter-SLCi7wt（P=0.000）. Conclusions：The human SLC22A3 gene intron7 has negative regulatory activity，which could significantly down-regulate the expression of luciferase reporter gene. The mutation（A→G） of SNPrs2048327（A/G） located in the hSLC22A3 intron7 may enhance the negative regulatory activity of this intron.