斑马鱼（Danio rerio）mapk1基因对tp53基因调控研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0335

摘要/Abstract

摘要：

旨在研究斑马鱼（Danio rerio）mapk1基因对tp53基因的调控作用。通过生信网站分析斑马鱼tp53启动子序列与mapk1基因CDS序列,构建斑马鱼tp53启动子荧光素酶报告基因质粒pGL3-tp53-Luc和 mapk1表达质粒pCMV-Tag2B-mapk1。利用Luciferase实验验证tp53启动子报告基因的活性,以及mapk1基因对tp53基因的活性影响。结果显示,斑马鱼tp53基因启动子区无CpG岛位点,包含 Oct-1（TATGTAAAGC）、Sp-1（AGATCCCGCC）、c-Jun（CTGACGTCAC）、CREB（CTGACGTCAC）、CRE-BP1（CTGACGTCAC）、NF-kappa B1（AGGGGAATCC）和RAR-alpha1（TTGAACTTTT）共7个转录因子结合位点;斑马鱼与人和小鼠MAPK1氨基酸的一致性分别为91.33%和90.79%。pGL3-tp53-Luc在哺乳动物细胞系和鱼类细胞系中均具有很高的活性,分别是对照组的17倍和9倍。在HEK293T细胞中过表达pCMV-Tag2B-mapk1 质粒后pGL3-tp53-Luc的luciferase活性是对照组的3倍左右。实验验证了斑马鱼mapk1基因可促进tp53基因的表达。

关键词: 斑马鱼, tp53, mapk1, 报告基因

Abstract:

The objective is to investigate the regulation of zebrafish（Danio rerio）mapk1 gene on tp53 gene. The tp53 promoter sequence and mapk1 gene CDS sequence of zebrafish were analyzed by bioinformatics websites,and then the luciferase reporter gene plasmid pGL3-tp53-Luc and mapk1 expression plasmid pCMV-Tag2B-mapk1 were constructed. Finally,luciferase assay was used to verify the activity of tp53 promoter reporter gene and the effect of mapk1 gene on the activity of tp53 gene. The results showed that there was no CpG island site in the promoter region of tp53 gene in zebrafish,and there were 7 transcription factors binding sites,including Oct-1（TATGTAAAGC）,Sp-1（AGATCCCGCC）,c-Jun（CTGACGTCAC）,CREB（CTGACGTCAC）,CRE-BP1（CTGACGTCAC）,NF-KappaB1（AGGGGAATCC）,and RAR-alpha1（TTGAACTTTT）. The amino acid consistency of MAPL1 between zebrafish and human and mouse was 91.33% and 90.79%,respectively. The tp53 promoter fragment sequence had high activity in both mammalian cell lines and fish cell lines,which were 17 times and 9 times that of the control group,respectively. After overexpression of pCMV-Tag2B-mapk1 plasmid in the HEK293T cells,the luciferase activity of pGL3-tp53-Luc was about 3 times that of the control group. This experiment verifies that zebrafish mapk1 may promote the expression of tp53.

Key words: Danio rerio, tp53, mapk1, reporter gene

包林珠, 时灿, 卢玲儿, 徐行, 周泽斌, 任建峰, 李伟明, 张庆华. 斑马鱼（Danio rerio）mapk1基因对tp53基因调控研究[J]. 生物技术通报, 2021, 37(12): 160-168.

BAO Lin-zhu, SHI Can, LU Ling-er, XU Xing, ZHOU Ze-bin, REN Jian-feng, LI Wei-ming, ZHANG Qing-hua. Regulation of Gene mapk1 in Danio rerio on Gene tp53[J]. Biotechnology Bulletin, 2021, 37(12): 160-168.

图/表 13

表1 PCR试剂及体系

Table 1 PCR reagents and systems

体系System	体积Volume
上游引物Forward primer	0.5 μL
下游引物Reverse primer	0.5 μL
DNA模板DNA template	10 ng
高保真酶Prime STAR^® Max DNA polymerase	12.5 μL
灭菌水ddH₂O	Up to 25 μL

表2 实验所用的引物序列

Table 2 Primer sequences used in the experiment

引物Primer	序列Sequence（5'-3'）	用途Usage
tp53-F1	CCCATGATGTGGGGACACAA	启动子序列扩增 Amplification of promoter sequences
tp53-R1	TCAAACGATCCCGGCAAGTA	启动子序列扩增 Amplification of promoter sequences
tp53-F2	CGGGGTACCCCCATGATGTGGGGACACAA	报告基因载体构建 Construction of reporter gene vector
tp53-R2	CCGCTCGAGTCAAACGATCCCGGCAAGTA	报告基因载体构建 Construction of reporter gene vector
mapk1-F1	ATGGCGACAGCTGCGGTTTC	CDS序列扩增 Amplification of CDS sequences
mapk1-R1	CTATGGTCTGTAGCCTGGCT	CDS序列扩增 Amplification of CDS sequences
mapk1-F2	CCGGAATTCATGGCGACAGCTGCGGTTTC	表达载体构建 Construction of expression vector
mapk1-F2	CCGCTCGAGCTATGGTCTGTAGCCTGGCT	表达载体构建 Construction of expression vector

图1 pGL3-Enhancer质粒（A）与 pGL3-tp53-Luc重组质粒（B）示意图

Fig. 1 Schematic diagram of pGL3-Enhancer plasmid（A）and pGL3-tp53-Luc recombinant plasmid（B）

表3 检测tp53启动子报告基因活性的转染体系

Table 3 Transfection system for detecting tp53 promoter reporter gene activity

组别Group	pGL3-Enhancer/ng	pGL3-tp53-Luc/ng	phRL-T/ng	Opti-MEM/μL	Fu GENE HD/μL
pGL3-Enhancer	200	0	10	50	0.63
pGL3-tp53-Luc	0	200	10	50	0.63

图2 pCMV-Tag2B质粒（A）与pCMV-Tag2B-mapk1重组质粒（B）示意图

Fig. 2 Schematic diagram of pCMV-Tag2B plasmid（A）and pCMV-Tag2B-mapk1 recombinant plasmid（B）

表4 转染复合物配制表

Table 4 Formulation table of transfected compound

组别Group	pCMV-Tag2B-mapk1/ng	pCMV-Tag2B/ng	pGL3-tp53-Luc/ng	phRL-TK/ng	Opti-MEM/μL	Fu GENE HD/μL
对照Control	0	200	100	10	50	0.93
共转 Co-transfect	200	0	100	10	50	0.93

图3 斑马鱼tp53启动子特征序列分析黑色下划线的碱基为启动子区上下游引物;红色碱基为相对应的转录因子结合位点;7个蓝色碱基TATATAA为TATA-box;单独蓝色碱基A为转录起始位点;3个绿色碱基ATG为起始密码子

Fig. 3 Analysis of the characteristic sequence of the tp53 promoter in zebrafish The bases underlined in black are the upstream and downstream primers of the promoter region. The red bases are corresponding transcription factor binding sites. The seven blue bases TATATAA is TATA-box. Single blue base A is the transcription start site（TSS）. The three green bases,ATG,are the starting codons

图4 Meth primer-Design 软件预测 tp53 基因启动子区 CpG岛

Fig. 4 Prediction of CpG island in tp53 promoter region by Meth Primer-Design software

图5 pGL3-tp53-Luc双酶切检验

Fig. 5 Double enzyme restriction assay of pGL3-tp53-Luc

图6 pGL3-tp53-Luc活性 A:HEK293T 细胞;B:ZF4 细胞。1:pGL3-Enhancer,2:pGL3-tp53-Luc;误差用 mean ± SE 表示（n=3,** P<0.01,***P<0.001）

Fig. 6 Activity of pGL3-tp53-Luc A:HEK293T cells. B:ZF4 cells. 1:pGL3-Enhancer. 2:pGL3-tp53-Luc.The error is expressed as mean ± SE（n=3,** P<0.01,***P<0.001）

表5 斑马鱼与人和小鼠mapk1基因氨基酸序列对比

Table 5 Comparison of amino acid sequences of mapk1 gene between zebrafish and human and mouse

物种 Species	序列来源 Sequence source	一致性 Consistency/%
人 Homo spaiens	NP_002736.3	91.33
小鼠Mus musculus	NP_001033752.1	90.79

图7 mapk1 CDS片段扩增（A）和 pCMV-Tag2B-mapk1双酶切检验（B）

Fig. 7 mapk1 CDS fragment amplification（A）and pCMV-Tag2B-mapk1 double enzyme restriction assay（B）

图8 pCMV-Tag2B-mapk1和pGL3-tp53-Luc共同转染 HEK293T 细胞后荧光素酶相对表达量 1:pCMV-Tag2B;2:共转;误差用 mean ± SE 表示（n=3,*** P<0.001）

Fig. 8 Relative expression of luciferase in the HEK293T cells co-transfected with pCMV-Tag2B-mapk1 and pGL3-tp53-Luc plasmids 1:pCMV-Tag2B. 2:Co-transfect. The error is expressed as mean ± SE（n=3,***P<0.001）

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