人多巴胺D2受体基因内含子区51103T/C多态性对基因转录活性的影响  

Effect of rs2075652 Polymorphism in DRD2 Third Intron Region on Transcription Activity in Human

作　　者：段朝霞[1] 张洁元[1] 陈魁君[1] 李晓霞[1] 王建民[1] 李兵仓[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所第六研究室,创伤,烧伤,复合伤国家重点实验室,重庆400042

出　　处：《解放军医药杂志》2014年第11期30-33,42,共5页Medical & Pharmaceutical Journal of Chinese People’s Liberation Army

基　　金：国家自然科学基金资助项目(81101422)

摘　　要：目的探讨人多巴胺D2受体(DRD2)基因第3内含子区51 103 bp位点单核苷酸多态性(rs2075652)对DRD2基因转录活性的影响,进一步揭示DRD2基因第3内含子区的单核苷酸多态性影响创伤后应激障碍发病风险的分子机制。方法以人全血基因组DNA为模板,扩增DRD2基因第3内含子区序列(399 bp),51 103 bp处分别为T或C,与报告基因载体pmir Glo相连接,构建重组荧光素酶表达载体,经限制性内切酶酶切及测序得以鉴定,然后分别与pRL-CMV共转染人胚肾癌细胞株HEK293,检测细胞中荧光素酶的相对表达量。结果双酶切及测序结果证实成功构建重组质粒pmir Glo-T/pmir Glo-C,荧光素酶检测结果显示,当第3内含子区51 103 bp位点为C时荧光素酶活性显著高于为T时(P<0.01)。结论成功构建了pmir Glo-T/pmir Glo-C报告基因重组质粒,DRD2基因第3内含子区rs2075652位点由T突变为C后,可能增强基因转录活性,该结果为进一步研究DRD2基因内含子区的功能奠定了基础。Objective To investigate the effect of single nucleotide polymorphism（ SNP） in 51103 bp（ rs2075652） of Dopamine receptor D2（ DRD2） gene of the third intron region on transcription activity and to further study the effect of gene mechanism on onset risk of post-traumatic stress disorder（ PTSD） in human. Methods The third intron region sequences of DRD2 gene T or C at site 51103 bp were amplified using whole blood genomic DNA as template,the obtained fragments of 399 bp（ the transcription start site is marked as ＋ 1） were inserted into pmir Glo respectively. The recombinant vector or pmir Glo-Basic vector was co-transfected together with pRL-CMV vector containing renal luciferase reporter gene into primarily cultured HEK293 after identifying with restriction enzyme double digestion and sequencing,and the corresponding expression value of firefly luciferase was detected. Results The correct construction of recombinant expression vector pmir Glo-DRD2-T / pmir Glo-DRD2-C was confirmed by restriction enzyme double digestion and sequencing analysis. The luciferase detection showed that expression of firefly luciferase,when the site of 51103 bp was C,was significantly higher than that when the site of 51 103 was T（ P〈0. 01）. Conclusion Luciferase reporter vectors pmir Glo-T / pmir Glo-C can be successfully constructed. The transcription activity of DRD2 gene may be enhanced when the DRD2 gene of the third intron region 51 103 bp site changes from C to T,and these results may be the basis for further study of the function of DRD2 gene intron region.

关 键 词：多巴胺受体D2 内含子区 荧光素酶活性 基因多态性 

分 类 号：R349.6[医药卫生—基础医学]