| 郭燕 朱东丽 张燕 闫菡 杨铁林.SOX6 基因3'UTR 区野生型和突变型重组质粒的构建与鉴定[J].,2016,16(22):4201-4206 |
| SOX6 基因3'UTR 区野生型和突变型重组质粒的构建与鉴定 |
| Construction and Identification of Recombinant Plasmid with the Wild andMutant-type Including SNP in SOX6 Gene 3'UTR |
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| DOI： |
| 中文关键词: SOX6 基因 单核苷酸多态性 3'非编码区 双荧光素酶报告基因 微小RNA |
| 英文关键词: SOX6 SNP 3'UTR Dual luciferase reporter gene miRNA |
| 基金项目:国家自然科学基金项目（31371278，31471188，81573241）；中国博士后科学基金项目（2015M570819）；
陕西省自然科学基础研究计划（2015JQ3089） |
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| 中文摘要: |
| 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miRNA，为进一步研究此SNP位点的功能及miRNA 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pMIR-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miRNA。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pMIR-REPORT-3'UTR-T 构建成功；经定点突变，成功将pMIR-REPORT-3'UTR-T 质粒转变为pMIR-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miR-190b、miR-190a-5p、miR-451b、miR-4791 与SOX6 基
因3'UTR 的结合区域，其多态的改变可以影响miRNA与mRNA 的结合效率。结论：本研究成功构建了含SNP 位点rs1065024 的
pMIR-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miRNA 与SOX6 基因
3'UTR 区之间的关系研究奠定基础。 |
| 英文摘要: |
| 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. |
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