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| 斑马鱼notch1a对pka报告基因转录调控作用 |
| Transcriptional regulation of notch1a on pka in zebrafish |
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| DOI：10.3969/j.issn.1006-7108.2021.04.007 |
| 中文关键词: 斑马鱼 骨质疏松 notch1a pka 双荧光素酶报告基因 |
| 英文关键词:zebrafish osteoporosis notch1a pka luciferase |
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| 中文摘要: |
| 目的 通过双荧光素酶报告基因研究斑马鱼notch1a对pka的转录调控作用。方法 利用NCBI数据库获得斑马鱼notch1a基因序列，克隆notch1a基因的胞内段NICD（Notch intracellular domain），构建pCMV-N1aICD表达载体，利用蛋白质印迹法检测N1aICD蛋白的表达水平；利用加州大学圣克鲁兹分校基因组浏览器数据库获得斑马鱼pka基因的启动子序列，构建pGL3-pka报告基因载体，然后在HEK293T细胞系中证实构建的报告基因载体具有活性；将重组荧光素酶报告载体和N1aICD表达载体共转染HEK293T细胞，用双荧光素酶检测试剂盒测定荧光素酶活性，验证notch1a胞内段对pka基因转录水平的调控作用。结果 经测序验证克隆成功后，蛋白质印迹法检测N1aICD蛋白能够正常表达，且蛋白大小与预测结果一致。双荧光素酶报告基因显示，pGL3-pka组活性为pGL3空载组的3.25倍。转染pGL3-pka和pCMV-N1aICD组活性为转染pGL3-pka和pCMV空载对照组的0.31倍。结论 斑马鱼notch1a对pka启动子具有转录抑制作用。在骨形成和骨重建中，Notch通路可能通过调控pka来参与成骨与破骨细胞的分化。 |
| 英文摘要: |
| Objective To investigate the transcriptional regulation of zebrafish notch1a on pka by luciferase assay. Methods In this study, we used NCBI database to obtain the sequence of notch1a. Then, we cloned and constructed pCMV-N1aICD vector. Next, we detected the expression of pCMV-N1aICD vector by Western blotting. The University of California Santa Cruz Genome Browse Database was used to predict the promoter sequence of pka. We cloned the pka promoter and constructed dual luciferase reporter vectors. And then we used the dual luciferase reporter assay system detecting the activity of pGL3-pka in HEK293T cell line. We had cotransfected HEK293T cells with pGL3-pka and pCMV-N1aICD, and we verified the intracellular regulation of notch1a on the transcriptional level of pka. Results The results showed that we verified the constructed plasmids by sequencing. The N1aICD proteins were detected by Western blot with normal expression, and the protein size was consistent with the predicted results. The dual luciferase reporter assay system showed that the activity of pGL3-pka group was 3.25 times that of the pGL3 group. The activity of the cotransfected pGL3-pka and pCMV-N1aICD group was 0.31 times lower than that of the cotransfected pGL3-pka and pCMV group. Conclusion It was confirmed that notch1a of zebrafish has transcriptional inhibition on pka promoter. In the process of bone formation and reconstruction, Notch pathway may be involved in the differentiation of osteoblasts and osteoclasts by regulating pka. |
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