Regulation of Gene mapk1 in Danio rerio on Gene tp53

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

Abstract

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

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.

Figures/Tables 13

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

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

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

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

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

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

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

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

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

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）

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

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

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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