SOX6 基因3'UTR 区野生型和突变型重组质粒的构建与鉴定

目的:构建含单核苷酸多态性(SNP)位点rs1065024的SOX6基因3'UTR双荧光素酶报告基因载体,并用生物信息学软件预测与rs1065024位点区域相结合的mi RNA,为进一步研究此SNP位点的功能及mi RNA与SOX6基因3'UTR区之间的关系奠定基础。方法:提取人全血基因组DNA,以基因组DNA为模板,通过PCR扩增含SNP位点在内的SOX6基因3'UTR片段,经过胶回收纯化后,将回收的目的片段插入双荧光素酶报告基因载体p MIR-REPORT中,再经DH5a转化扩增,挑单克隆进行菌落PCR并进行质粒提取,对质粒进行双酶切鉴定,最后进行DNA测序鉴定。针对SNP进行定点突变,构建出野生型和突变型重组质粒,并用生物信息学软件预测出与SNP位点相结合的mi RNA。结果:经单菌落质粒测序验证显示带有T碱基的SOX6基因3'UTR重组质粒p MIR-REPORT-3'UTR-T构建成功;经定点突变,成功将p MIR-REPORT-3'UTR-T质粒转变为p MIR-REPORT-3'UTR-C,经比对未引入任何其他突变;生物信息学预测显示,rs1065024位点位于mi R-190b、mi R-190a-5p、mi R-451b、mi R-4791与SOX6基因3'UTR的结合区域,其多态的改变可以影响mi RNA与m RNA的结合效率。结论:本研究成功构建了含SNP位点rs1065024的p MIR-REPORT-SOX6-3'UTR野生型和突变型重组质粒,为今后SOX6基因3'UTR的SNP位点的功能及mi RNA与SOX6基因3'UTR区之间的关系研究奠定基础。

Construction and Identification of Recombinant Plasmid with the Wild and Mutant-type Including SNP in SOX6 Gene 3'UTR

Objective:To construct luciferase reporter gene vectors containing SOX6 gene 3''UTR with SNP (rs1065024) and to predict miRNA binding site with this SNP by using the bioinformatics software, which could lay foundation for investigating the function of this SNP.Methods:First, genomic DNA was extracted fromhuman whole blood.A 840 bp fragment including the SNP (rs1065024) of the SOX6 3''UTR were amplified from genomic DNA. The PCR product was inserted into the reporter gene vector pMIR-REPORT after the target band extracting from the agrose, and then transformed into DH5a cells for further propagation under the selection of appropriate antibiotics. Then, we chose the single bacteria to conduct the bacteria PCR, and the recombinant plasmid was extracted from bacterial pellet and verified by double enzymatic restriction analysis and DNA sequencing. Finally, pMIR-REPORT-3''UTR-T vector was used as PCR template to construct the pMIR-REPORT-3''UTR-C by site-specific mutagenesis. pMIR-REPORT-3''UTR-C vectors were transferred into DH5a and verified by double-enzyme digestions and DNA sequencing. We predicted miRNA binding site with SNP of SOX6 3''UTR by using the bioinformatics software.Results:Two recombinant plasmids were successfully constructed and the sequences of them were the same as expected. Furthermore, the bioinformatics analysis suggested that rs1065024 T allele may bind with miR-190a-5p, miR-190b, miR-451b and miR-4791.Conclusion:We constructed reporter plasmids with the mutant and wildtype of SOX6 (840 bp of 3''UTR) linked to luciferase (pMIR-REPORT-SOX6-wild and pMIR-REPORT-SOX6-mut 5005T/C) successfully, It will be further studied in related SNP function analysis.