猪SEPW1基因的转录调控分析
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摘要
本研究旨在对猪SEPW1基因的潜在启动子区进行克隆及转录活性分析,获得其核心启动子区域,并进一步分析转录因子SP1对SEPW1基因转录活性的影响,为探索SEPW1基因在猪肉质性状方面的功能奠定基础。利用实时荧光定量PCR检测SEPW1基因在大白猪各组织中的表达量,构建空间表达谱;通过PCR技术克隆得到6个逐级缺失的SEPW1基因启动子片段,构建6个双荧光素酶报告载体,通过检测各载体的双荧光素酶活性获得SEPW1基因的核心启动子区域;对核心启动子区进行生物信息学分析,发现潜在的SP1转录因子结合位点;通过过表达、抑制表达、定点突变及凝胶迁移试验(EMSA)确认SP1转录因子结合位点的存在及其对SEPW1基因转录活性的影响。结果显示,SEPW1基因在所检测的4月龄大白猪12个组织中均有表达,其中在腓肠肌及心脏中的表达量较高。双荧光素酶活性显示,猪SEPW1基因5′侧翼区-443～-231 bp为其核心启动子区,且-378～-306 bp存在1个潜在的SP1结合位点。过表达和抑制表达SP1基因结果显示,转录因子SP1能够促进SEPW1基因的转录;定点突变及EMSA试验确认,转录因子SP1可直接与SEPW1基因启动子区的SP1结合位点(-348～-339 bp)相结合。综合以上结果表明,转录因子SP1可直接靶向SEPW1基因的启动子区并促进SEPW1基因的转录。
This study was aimed to clone the potential promoter region of porcine SEPW1 gene and analyze its transcriptional activity,obtain its core promoter region,and analyze the effect of transcription factor SP1 on the transcriptional activity of SEPW1 gene for further studying the function of porcine SEPW1 gene in meat traits.The expression level of SEPW1 gene in Large White pigs tissues was detected by Real-time quantitative PCR,and the spatial expression profile was constructed.6 SEPW1 gene promoter deletion fragments were cloned by PCR,and 6 dual luciferase reporter vectors were constructed,the core promoter region of SEPW1 gene was obtained by detecting the dual luciferase activity of each vector;The potential transcription factor SP1 binding site was predicted through bioinformatics analysis of the core promoter region.The transcription factor SP1 binding site and its effect on the transcriptional activity of SEPW1 gene were confirmed by over-expression,down-expression,site-directed mutation and EMSA.The results showed that SEPW1 gene was widely expressed in 12 tissues of 4-month-old Large White pigs,while the expression levels were higher in castrocnemius and heart.The results of dual luciferase activity indicated that there was the core promoter region of SEPW1 gene 5′-flanking region at-443 to-231 bp,and there was a potential SP1 binding site at-378 to-306 bp.The results of over-expression and down-expression of SP1 gene indicated that transcription factor SP1 promoted the transcription of SEPW1 gene.Site-directed mutagenesis and EMSA assay confirmed that transcription factor SP1 could directly bind to SP1 binding sites(-348 to-339 bp) in the promoter region of SEPW1 gene.The results suggested that transcription factor SP1 could directly target the promoter region of SEPW1 gene and promote SEPW1 gene transcription.
This study was aimed to clone the potential promoter region of porcine SEPW1 gene and analyze its transcriptional activity,obtain its core promoter region,and analyze the effect of transcription factor SP1 on the transcriptional activity of SEPW1 gene for further studying the function of porcine SEPW1 gene in meat traits.The expression level of SEPW1 gene in Large White pigs tissues was detected by Real-time quantitative PCR,and the spatial expression profile was constructed.6 SEPW1 gene promoter deletion fragments were cloned by PCR,and 6 dual luciferase reporter vectors were constructed,the core promoter region of SEPW1 gene was obtained by detecting the dual luciferase activity of each vector;The potential transcription factor SP1 binding site was predicted through bioinformatics analysis of the core promoter region.The transcription factor SP1 binding site and its effect on the transcriptional activity of SEPW1 gene were confirmed by over-expression,down-expression,site-directed mutation and EMSA.The results showed that SEPW1 gene was widely expressed in 12 tissues of 4-month-old Large White pigs,while the expression levels were higher in castrocnemius and heart.The results of dual luciferase activity indicated that there was the core promoter region of SEPW1 gene 5′-flanking region at-443 to-231 bp,and there was a potential SP1 binding site at-378 to-306 bp.The results of over-expression and down-expression of SP1 gene indicated that transcription factor SP1 promoted the transcription of SEPW1 gene.Site-directed mutagenesis and EMSA assay confirmed that transcription factor SP1 could directly bind to SP1 binding sites(-348 to-339 bp) in the promoter region of SEPW1 gene.The results suggested that transcription factor SP1 could directly target the promoter region of SEPW1 gene and promote SEPW1 gene transcription.
引文
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[14] DENG B,ZHANG F,CHEN K,et al.MyoD promotes porcine PPARgamma gene expression through an E-box and a MyoD-binding site in the PPARgamma promoter region[J].Cell and Tissue Research,2016,365(2):381-391.
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[17] BRAHMACHARY M,SCHONBACH C,YANG L,et al.Computational promoter analysis of mouse,rat and human antimicrobial peptide-coding genes[J].BMC Bioinformatics,2006,7(Suppl5):S8.
[18] LEE B J,PARK S I,PARK J M,et al.Molecular biology of selenium and its role in human health[J].Molecules and Cells,1996,6(5):509-520.
[19] REDERSTORFF M,KROL A,LESCURE A.Understanding the importance of selenium and selenoproteins in muscle function[J].Cellular and Molecular Life Sciences,2006,63(1):52-59.
[20] VENDELAND S C,BEILSTEIN M A,CHEN C L,et al.Purification and properties of selenoprotein W from rat muscle[J].The Journal of Biological Chemistry,1993,268(23):17103-17107.
[21] 王晓龙,宋晓东,宋月峰,等.SelW mRNA下调对肌细胞状态影响的FCM评估[J].吉林农业大学学报,2007,29(2):152-154.WANG X L,SONG X D,SONG Y F,et al.Assessment of cell conditions by FCM after SelW mRNA down-regulation[J].Journal of Jilin Agricultural University,2007,29(2):152-154.(in Chinese)
[22] PEDERSEN N D,WHANGER P D,WESWIG P H,et al.Selenium binding proteins in tissues of normal and selenium responsive myopathic lambs[J].Bioinorganic Chemistry,1973,2(1):33-45.
[2] ZHU Y G,ELIZABETH A H,ZHAO F J,et al.Selenium in higher plants:Understanding mechanisms for biofortification and phytoremediation[J].Trends in Plant Science,2009,14(8):436-442.
[3] FAIRWEATHER-TAIT S J,BAO Y,BROADLEY M R,et al.Selenium in human health and disease[J].Antioxidants & Redox Signaling,2011,14(7):1337-1383.
[4] 卢建新,张仲欣,任丽影.富硒食品加工现状与发展分析[J].农产品加工,2014,21:45-51.LU J X,ZHANG Z X,REN L Y.Processing status and development of selenium-accumlating food[J].Academic Periodica of Farm Products Processing,2014,21:45-51.(in Chinese)
[5] 王晓龙,许凯,秦藕菊,等.SelW基因转录后沉默对小鼠骨骼肌细胞内GSH及GPx影响研究[J].畜牧兽医学报,2009,40(7):1013-1018.WANG X L,XU K,QIN O J,et al.The influence of SelW on GSH and GPx after post-transcriptional gene silencing in mouse skeletal muscle cell[J].Acta Veterinaria et Zootechnica Sinica,2009,40(7):1013-1018.(in Chinese)
[6] 杨旭,董文宾.富硒食品的研究进展[J].食品安全质量检测学报,2017,8(6):2091-2097.YANG X,DONG W B.Research progress of Se-enriched food[J].Journal of Food Safety & Quality,2017,8(6):2091-2097.(in Chinese)
[7] 胡万明.富硒食品的研究意义与进展[J].食品安全导刊,2018,12:42.HU W M.Research significance and progress of selenium enriched food[J].China Food Safety Magazine,2018,12:42.(in Chinese)
[8] JEONG D,KIM T,CHUNG Y,et al.Selenoprotein W is a glutathione-dependent antioxidant in vivo[J].FEBS Letter,2002,517(1-3):225-228.
[9] GU Q P,SUN Y,REAM L W,et al.Selenoprotein W accumulates primarily in primate skeletal muscle,heart,brain and tongue[J].Molecular and Cellular Biochemistry,2000,204(1-2):49-56.
[10] PRINZ W A,ASLUND F,HOLMGREN A,et al.The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm[J].The Journal of Biological Chemistry,1997,272(25):15661-15667.
[11] BELLINGHAM J,GREGORY-EVANS K,FOX M F,et al.Gene structure and tissue expression of human selenoprotein W,SEPW1,and identification of a retroprocessed pseudogene,SEPW1P[J].Biochimica et Biophysica Acta,2003,1627(2-3):140-146.
[12] LI J G,ZHOU J C,ZHAO H,et al.Enhanced water-holding capacity of meat was associated with increased Sepw1 gene expression in pigs fed selenium-enriched yeast[J].Meat Science,2011,87(2):95-100.
[13] LOFLIN J,LOPEZ N,WHANGER P D,et al.Selenoprotein W during development and oxidative stress[J].Journal of Inorganic Biochemistry,2006,100(10):1679-1684.
[14] DENG B,ZHANG F,CHEN K,et al.MyoD promotes porcine PPARgamma gene expression through an E-box and a MyoD-binding site in the PPARgamma promoter region[J].Cell and Tissue Research,2016,365(2):381-391.
[15] YEH J Y,BEILSTEIN M A,ANDREWS J S,et al.Tissue distribution and influence of selenium status on levels of selenoprotein W[J].FASEB Journal,1995,9(5):392-396.
[16] GU Q,SUN Y,REAM L,et al.Selenoprotein W accumulates primarily in primate skeletal muscle,heart,brain and tongue[J].Molecular and Cellular Biochemistry,2000,204(1-2):49-56.
[17] BRAHMACHARY M,SCHONBACH C,YANG L,et al.Computational promoter analysis of mouse,rat and human antimicrobial peptide-coding genes[J].BMC Bioinformatics,2006,7(Suppl5):S8.
[18] LEE B J,PARK S I,PARK J M,et al.Molecular biology of selenium and its role in human health[J].Molecules and Cells,1996,6(5):509-520.
[19] REDERSTORFF M,KROL A,LESCURE A.Understanding the importance of selenium and selenoproteins in muscle function[J].Cellular and Molecular Life Sciences,2006,63(1):52-59.
[20] VENDELAND S C,BEILSTEIN M A,CHEN C L,et al.Purification and properties of selenoprotein W from rat muscle[J].The Journal of Biological Chemistry,1993,268(23):17103-17107.
[21] 王晓龙,宋晓东,宋月峰,等.SelW mRNA下调对肌细胞状态影响的FCM评估[J].吉林农业大学学报,2007,29(2):152-154.WANG X L,SONG X D,SONG Y F,et al.Assessment of cell conditions by FCM after SelW mRNA down-regulation[J].Journal of Jilin Agricultural University,2007,29(2):152-154.(in Chinese)
[22] PEDERSEN N D,WHANGER P D,WESWIG P H,et al.Selenium binding proteins in tissues of normal and selenium responsive myopathic lambs[J].Bioinorganic Chemistry,1973,2(1):33-45.
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