李虹仪,张茂.调节猪Smad3 表达的miRNA 鉴定[J].广东农业科学,2014,41(23):123-126 |
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调节猪Smad3 表达的miRNA 鉴定 |
Identification of miRNA regulated Porcine Smad3 expression |
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DOI: |
中文关键词: Smad3 miR-23a 荧光素酶 |
英文关键词: Smad3 miR-23a luciferase |
基金项目:福建省自然科学基金(2014J05043) |
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中文摘要: |
生物信息学预测猪Smad3 为miR-23a 的靶基因,为了研究猪Smad3 与miR-23a 之间的调节关系,人工合成miR-23a 双链序列及Smad3 基因3忆UTR 中mR-23a 的结合位点序列,分别与带荧光的慢病毒质粒pshRNAcopGFPLentivector 及萤光素酶质粒pPGL3-control 连接,构建重组质粒LV-shRNA-miR-23a 和pPGL3-control-Smad3-3忆UTR。将两重组质粒共转CHO 细胞,以慢病毒空质粒与pPGL3-control-Smad3-3’UTR 共转作为对照,48 h后收集细胞裂解液检测荧光素酶活性变化,提取细胞总RNA 实时荧光定量检测荧光素酶基因mRNA 表达变化。结果显示,试验成功构建了重组质粒LV-shRNA-miR-23a 和pPGL3-control-Smad3-3’UTR。miR-23a 能够显著抑制荧光素酶基因mRNA 表达(P<0.05),并能通过抑制Smad3 结合位点序列抑制其上游荧光素酶的活性(P<0.05)。初步证明,猪Smad3 与miR-23a 有直接的靶向关系。 |
英文摘要: |
Porcine Smad3 was predicted to be the target gene of miR-23a by online websites. In order to verify the relationship between Smad3 and miR-23a, double stranded miR-23a sequences and its biding site to the 3’untranslated region of Smad3 were synthetized, and conjugated to pshRNA-copGFP Lentivector and pPGL3-control respectively, to construct recombinant plasmids of LV-shRNA-miR-23a and pPGL3-control-Smad3-3’UTR. Then the CHO cell line was cotransfected with these two recombinant plasmids, taking cotransfected with pshRNA-copGFP Lentivector and pPGL3- control-Smad3-3’UTR as negative control. Cells were collected 48 h after transfection, then the luciferase activity was measured using a dual luciferase reporter assay system and the expression of Smad3 mRNA was measured by Quantitative Real-time PCR. The results showed that two recombinant plasmids were successfully constructed. Expression of Smad3 mRNA as well as the luciferase activity were suppressed by miR-23a (P<0.05), which proved that Porcine Smad3 was the target gene of miR-23a. |
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