调控民猪ZBED6基因转录元件的筛选与分析
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摘要
ZBED6是锌指蛋白家族的一员,在胎盘哺乳动物中极其保守,可通过对IGF2的调控参与骨骼肌生长。为进一步探究ZBED6基因自身的表达调控机制,本研究以民猪基因组DNA为模板,通过常规PCR扩增ZBED6基因启动子区系列截短片段,构建克隆质粒,通过双酶切和连接反应定向连入pGL3-basic载体,利用PK15细胞和双荧光素酶检测系统测定重组质粒的相对荧光素酶活性;利用在线软件预测启动子区的转录因子结合位点,使用重叠PCR定点缺失转录因子结合位点,构建突变载体并在PK15细胞中检测突变载体的相对荧光素酶活性。结果表明:ZBED6基因启动子区-2 053～-1 777 bp存在多个转录因子结合位点,尤其是-1 808～-1 777 bp,该片段缺失造成启动子活性下降(P<0.01);利用在线软件在该区间预测到3个转录因子HINFP、Adf-1和CREB3,经实验验证后发现这3个转录因子均可调控ZBED6基因的转录,其中Adf-1效果最为明显。据此推测,民猪ZBED6基因的转录调控机制较为复杂,其启动子区存在HINFP、Adf-1和CREB3等多个调控元件的结合位点。
ZBED6, a member of the zinc finger protein family, is extremely conserved in placental mammals. It regulates the growth of skeletal muscle through IGF2. In order to further explore the mechanism of expression and regulation of ZBED6,the genomic DNA of Min pig was used as template to amplify the series truncated fragments of ZBED6 gene promoter by PCR. The cloning plasmid was constructed and linked into pGL3-basic vector by double enzyme digestion and linkage reaction. The relative luciferase activity of the recombinant plasmid was predicted by online software, and the relative luciferase activity of the mutant vector was detected in PK15 cells. The results showed that there were multiple transcription factor binding sites in the promoter region of ZBED6 gene-2 053～-1 777 bp, especially in the region-1 808 ～-1 777 bp. The deletion of this fragment would cause a significant decrease in promoter activity(P<0.01). Three transcription factors, HINFP,Adf-1 and CREB3, were predicted by on-line software. The experiment results showed that these three transcription factors could regulate the transcription of ZBED6 gene, and Adf-1 was the most effective. The conclusion is that the transcription regulation mechanism of ZBED6 gene is relatively complicated, and there are many binding sites including HINFP, Adf-1 and CREB3 in the promoter region.
ZBED6, a member of the zinc finger protein family, is extremely conserved in placental mammals. It regulates the growth of skeletal muscle through IGF2. In order to further explore the mechanism of expression and regulation of ZBED6,the genomic DNA of Min pig was used as template to amplify the series truncated fragments of ZBED6 gene promoter by PCR. The cloning plasmid was constructed and linked into pGL3-basic vector by double enzyme digestion and linkage reaction. The relative luciferase activity of the recombinant plasmid was predicted by online software, and the relative luciferase activity of the mutant vector was detected in PK15 cells. The results showed that there were multiple transcription factor binding sites in the promoter region of ZBED6 gene-2 053～-1 777 bp, especially in the region-1 808 ～-1 777 bp. The deletion of this fragment would cause a significant decrease in promoter activity(P<0.01). Three transcription factors, HINFP,Adf-1 and CREB3, were predicted by on-line software. The experiment results showed that these three transcription factors could regulate the transcription of ZBED6 gene, and Adf-1 was the most effective. The conclusion is that the transcription regulation mechanism of ZBED6 gene is relatively complicated, and there are many binding sites including HINFP, Adf-1 and CREB3 in the promoter region.
引文
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[12]Timmerman C,Suppiah S,Gurudatta B V,et al.The drosophila transcription factor Adf-1(nalyot)regulates dendrite growth by controlling fasii and staufen expression downstream of CaMKIIand neural activity[J].J Neurosci,2013,33(29):11916-11931.
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[2]Van Laere A S,Nguyen M,Braunschweig M,et al.A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig[J].Nature,2003,425(6960):832-836.
[3]Markljung E,Jiang L,Jaffe J D,et al.ZBED6,a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth[J].PLoS Biol,2009,7(12):e1000256.
[4]Younis S,Sch?nke M,Massart J,et al.The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals[J].Proc Natl Acad Sci USA,2018,115(9):E2048-E2057.
[5]Wang X,Jiang L,Wallerman O,et al.ZBED6 negatively regulates insulin production,neuronal differentiation,and cell aggregation in MIN6 cells[J].FASEB J,2018,33(1):88-100.
[6]Huang Y Z,Zhang Z J,He H,et al.Correlation between ZBED6Gene Upstream CpG Island methylation and mRNA expression in cattle[J].Anim Biotechnol,2017,28(2):104-111.
[7]汤方,涂慧珍.真核启动子研究进展[J].林业科技开发,2015,29(2):7-12.
[8]冯琳璎,张利绒.表达相关的转录因子相互作用模式[J].内蒙古大学学报(自然科学版),2018,49(5):1-10.
[9]Andersson L,Andersson G,Hj?lm G,et al.ZBED6:The birth of a new transcription factor in the common ancestor of placental mammals[J].Transcription,2010,1(3):144-148.
[10]Huang Y Z,Sun Y J,Zhan Z Y,et al.Expression,SNP identification,linkage disequilibrium,and haplotype association analysis of the growth suppressor gene ZBED6 in Qinchuan beef cattle[J].Anim Biotechnol,2014,25(1):35-54.
[11]Mitra P,Xie R,Harper J W,et al.HiNF‐P is a bifunctional regulator of cell cycle controlled histone H4 gene transcription[J].J Cell Biochem,2010,101(1):181-191.
[12]Timmerman C,Suppiah S,Gurudatta B V,et al.The drosophila transcription factor Adf-1(nalyot)regulates dendrite growth by controlling fasii and staufen expression downstream of CaMKIIand neural activity[J].J Neurosci,2013,33(29):11916-11931.
[13]祁艳梅.猪CREB家族基因的克隆及表达研究的初步探讨[D].武汉:华中农业大学,2009:1-2.