生物技术通报 ›› 2021, Vol. 37 ›› Issue (12): 160-168.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0335
包林珠1,2(), 时灿1,2, 卢玲儿1,2, 徐行1,2, 周泽斌1,2, 任建峰1,2, 李伟明3, 张庆华1,2()
收稿日期:
2021-03-18
出版日期:
2021-12-26
发布日期:
2022-01-19
作者简介:
包林珠,女,硕士研究生,研究方向:分子生物学;E-mail: 基金资助:
BAO Lin-zhu1,2(), SHI Can1,2, LU Ling-er1,2, XU Xing1,2, ZHOU Ze-bin1,2, REN Jian-feng1,2, LI Wei-ming3, ZHANG Qing-hua1,2()
Received:
2021-03-18
Published:
2021-12-26
Online:
2022-01-19
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。
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.
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 |
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 | |
tp53-F2 | CGGGGTACCCCCATGATGTGGGGACACAA | 报告基因载体构建 Construction of reporter gene vector |
tp53-R2 | CCGCTCGAGTCAAACGATCCCGGCAAGTA | |
mapk1-F1 | ATGGCGACAGCTGCGGTTTC | CDS序列扩增 Amplification of CDS sequences |
mapk1-R1 | CTATGGTCTGTAGCCTGGCT | |
mapk1-F2 | CCGGAATTCATGGCGACAGCTGCGGTTTC | 表达载体构建 Construction of expression vector |
mapk1-F2 | CCGCTCGAGCTATGGTCTGTAGCCTGGCT |
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 | |
tp53-F2 | CGGGGTACCCCCATGATGTGGGGACACAA | 报告基因载体构建 Construction of reporter gene vector |
tp53-R2 | CCGCTCGAGTCAAACGATCCCGGCAAGTA | |
mapk1-F1 | ATGGCGACAGCTGCGGTTTC | CDS序列扩增 Amplification of CDS sequences |
mapk1-R1 | CTATGGTCTGTAGCCTGGCT | |
mapk1-F2 | CCGGAATTCATGGCGACAGCTGCGGTTTC | 表达载体构建 Construction of expression vector |
mapk1-F2 | CCGCTCGAGCTATGGTCTGTAGCCTGGCT |
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 |
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 |
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 |
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)
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 |
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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