斑马鱼Notch信号通路配体基因delta-like 4对smad基因的调控作用

斑马鱼Notch信号通路配体基因delta-like 4对smad基因的调控作用

程瑶, 王子睿, 周泽斌, 张鹏, 邱军强, 李伟明, 张庆华

大连海洋大学学报 ›› 2023, Vol. 38 ›› Issue (1) : 76-85.

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大连海洋大学学报 ›› 2023, Vol. 38 ›› Issue (1) : 76-85. DOI: 10.16535/j.cnki.dlhyxb.2022-086

斑马鱼Notch信号通路配体基因delta-like 4对smad基因的调控作用

程瑶, 王子睿, 周泽斌, 张鹏, 邱军强, 李伟明, 张庆华*

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Regulation of smad gene by Notch signal pathway ligand delta-like 4 in zebrafish (Danio rerio)

CHENG Yao, WANG Zirui, ZHOU Zebin, ZHANG Peng, QIU Junqiang, LI Weiming, ZHANG Qinghua*

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摘要

为研究斑马鱼(Danio rerio)的Notch信号通路配体delta-like 4(dll4)对smad基因(smad1和smad7)的调控作用，利用qRT-PCR方法检测受精后2 d(day post fertilization,dpf)的斑马鱼dll4纯合突变体smad家族中smad1和smad7的表达变化，利用生物信息学软件预测smad1和smad7启动子序列的转录结合位点和CpG岛；采用同源重组的方法，构建p3×Flag-CMV-dll4真核表达载体及pGL3-smad1-Luc和pGL3-smad7-Luc报告基因载体，并通过双荧光素酶试验检测dll4对smad1和smad7的调控活性；分别转染两种启动子报告基因载体到HEK293T细胞，共转染启动子报告基因载体与真核表达载体到HEK293T细胞。结果表明：dll4纯合突变体斑马鱼中smad1和smad7表达量均显著下调(P<0.000 1)；两种基因启动子均包含HNF-3、GATA-1和Oct-1等转录因子结合位点，只有smad7启动子存在CpG岛；报告基因pGL3-smad1-Luc和pGL3-smad7-Luc的活性分别为对照组的7.3倍和142.7倍,两种报告基因与p3×Flag-CMV-dll4表达载体共转后，转录活性均升高，分别为对照组的2.8倍和2.2倍，说明p3×Flag-CMV-dll4可以促进pGL3-smad1-Luc和pGL3-smad7-Luc的转录表达活性。研究表明，斑马鱼Notch信号通路配体基因dll4可通过smad基因家族在血管生成中发挥调控作用，为损伤血管的修复和肿瘤血管生成提供特异的生物学靶点。

Abstract

To investigate the regulation of Notch signal pathway ligand delta-like 4 (dll4) on smad genes (smad1 and smad7) in zebrafish (Danio rerio), the expression of smad1 and smad7 genes of Smad family was conducted in 2 dpf(day post fertilization) zebrafish dll4 homozygous mutants using qRT-PCR method. The transcriptional binding sites and CpG islands of smad1 and smad7 promoter sequences were predicted by bioinformatics software. The regulation of dll4 on smad1 and smad7 was verified in HEK293T cells by double luciferase assay. The results showed that the expression levels of smad1 and smad7 in dll4 homozygous mutant zebrafish were significantly down-regulated (P<0.000 1), only smad7 gene promoter had CpG island, and both gene promoters contained transcription factor binding sites such as HNF-3, GATA-1 and Oct-1. The activity of pGL3-smad1-Luc and pGL3-smad7-Luc reporter gene vectors in HEK293T cells was about 7.3 times and 142.7 times higher than that in the control group, respectively. Furthermore, p3×Flag-CMV-dll4 was co-transfected with pGL3-smad1-Luc and pGL3-smad7-Luc into HEK293T cells, and the luciferase activity of p3×Flag-CMV-dll4 was 2.8 and 2.2 times comparing with the control group, respectively. These findings indicate that the ligand dll4 of Notch signaling pathway in zebrafish can play a positive regulatory role in angiogenesis through smad gene family, and provide a specific biological target for the repair of damaged blood vessels and tumor angiogenesis.

关键词

斑马鱼 / Notch 信号通路配体 / delta-like 4 / smad1 / smad7 / 双荧光素酶报告基因

Key words

Danio rerio / Notch signal pathway ligand / delta-like 4 / smad1; smad7 / double luciferase reporter gene

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程瑶, 王子睿, 周泽斌, 张鹏, 邱军强, 李伟明, 张庆华. 斑马鱼Notch信号通路配体基因delta-like 4对smad基因的调控作用[J]. 大连海洋大学学报, 2023, 38(1): 76-85 https://doi.org/10.16535/j.cnki.dlhyxb.2022-086

CHENG Yao, WANG Zirui, ZHOU Zebin, ZHANG Peng, QIU Junqiang, LI Weiming, ZHANG Qinghua. Regulation of smad gene by Notch signal pathway ligand delta-like 4 in zebrafish (Danio rerio)[J]. Journal of Dalian Fisheries University, 2023, 38(1): 76-85 https://doi.org/10.16535/j.cnki.dlhyxb.2022-086

中图分类号： S 917.4 Q 71

基金

国家重点研发计划“蓝色粮仓科技创新”项目(2019YFD0900102)；教育部留学回国人员科研启动基金(D-8002-15-0042)；水产动物疾病与基因编辑育种的平台建设和前沿科学研究项目(A1-3201-19-3013)

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