<i>SENP1</i>启动子G-四链体鉴定及其作用研究 <sup>*</sup>

doi:10.13523/j.cb.1905003

中国生物工程杂志

2020, Vol. 40

Issue (1-2): 92-101 DOI: 10.13523/j.cb.1905003

研究报告

SENP1启动子G-四链体鉴定及其作用研究 ^*

周炎鑫,韩梦,刘娜女,黄伟伟(

)

西北农林科技大学生命科学学院杨陵 712100

Identification and Functional Study of G-quadruplexes in SENP1 Promoter

ZHOU Yan-xing,HAN Meng,LIU Na-nv,HUANG Wei-wei(

)

College of Life Science, Northwest A & F University, Yangling 712100, China

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摘要：

目的:研究G-四链体(G4)对SUMO特异性蛋白酶1(SUMO-specific proteases 1, SENP1)基因的转录调控作用。方法:克隆不同的SENP1启动子片段构建SENP1启动子报告质粒,通过报告基因检测鉴定SENP1启动子核心转录调控区;分析SENP1启动子核心转录调控区序列,并进行G4形成序列预测;合成G4形成序列寡核苷酸,利用圆二色谱分析检测G4形成序列寡核苷酸的拓扑结构;通过G4配体TMPyP4处理和过表达G4解旋酶G4R1结合报告基因检测和Western blot鉴定启动子G4对 SENP1转录表达的调控作用。结果:发现-910 ~+226区域是SENP1启动子的核心转录调控区,序列富含G/C;生信分析发现SENP1启动子核心区存在G4形成序列;圆二色谱分析证实SENP1启动子G4形成序列能够形成G4结构;报告基因检测和Western blot检测发现启动子G4对SENP1转录表达具有抑制作用。结论:SENP1启动子核心转录调控区存在G4结构并对其转录表达具有抑制作用,为揭示SENP1在生理和病理过程中的作用机制提供新的研究思路和试验线索。

关键词： SENP1; G-四链体; TMPyP4; G4R1

Abstract:

Objective: The regulation effect of G-quadruplexes (G4) on the SUMO-specific proteases 1 (SENP1) transcriptional expression was studied. Method: Reporting plasmids of SENP1 promoter were constructed by cloning different SENP1 promoter fragments. The core transcriptional regulatory region of SENP1 promoter was identified by using reporter assay. The sequence of core transcriptional regulatory region of SENP1 promoter was analyzed and G-quadruplex (G4) formation sequence was predicted. Oligonucleotides of G4 formation sequence from SENP1 promoter were synthesized, and their topological structure was detected by circular dichroism analysis. G4 ligand TMPyP4 and G4 helicase G4R1 were used to detect the regulatory effect of promoter G4 on SENP1 transcriptional expression by using reporter assay and Western blot. Result: The -910~+226 region was found to be the core transcriptional regulatory region of SENP1 promoter. Sequence analysis showed the core region of SENP1 promoter is rich in G/C and contains G4 formation sequences. Circular dichroism analysis confirmed that the oligonucleotides of G4 formation sequence from SENP1 promoter form G4 structure. Reporter assay and Western blot showed that promoter G4 inhibited the transcriptional expression of SENP1. Conclusion: G4 exist in the core transcriptional regulatory region of SENP1 promoter and show negative regulation on SENP1 transcriptional expression, which provides new research ideas and experimental clues for revealing the mechanism of SENP1 in physiological and pathological processes.

Key words: SENP1 G-quadruplexe TMPyP4 G4R1

收稿日期: 2019-05-05 出版日期: 2020-03-27

ZTFLH:

Q291

基金资助: * 国家自然科学基金(31300654);陕西省自然科学基金(2014JQ3098)

通讯作者: 黄伟伟 E-mail: whuang0210@163.com

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引用本文:

周炎鑫,韩梦,刘娜女,黄伟伟. SENP1启动子G-四链体鉴定及其作用研究 ^*[J]. 中国生物工程杂志, 2020, 40(1-2): 92-101.

ZHOU Yan-xing,HAN Meng,LIU Na-nv,HUANG Wei-wei. Identification and Functional Study of G-quadruplexes in SENP1 Promoter. China Biotechnology, 2020, 40(1-2): 92-101.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1905003 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I1-2/92

表1 PCR引物序列

图1 SENP1启动子报告质粒及萤光素酶报告基因检测

图2 SENP1启动子序列分析和G4形成序列预测

图3 SENP1启动子G4形成序列及圆二色谱分析

图4 G4配体TMPyP4对SENP1表达的影响

图5 G4解旋酶G4R1对SENP1表达的影响

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