鸭MyoG基因启动子的生物信息学分析及其甲基化频率对基因表达的影响
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摘要
采用RT–PCR技术扩增和克隆鸭Myo G基因启动子,并对其启动子序列进行生物信息学分析,采用Sequenom Mass Array技术检测Cp G岛在鸭肌肉组织中的甲基化水平,用q RT–PCR检测Myo G基因的表达量。结果表明,扩增得到鸭Myo G基因启动子序列2 730 bp,对启动子序列预测后,发现存在2个Cp G岛,其中Cp G岛(–2 536~–1 997 bp)存在5个转录因子结合位点和多个真核生物结构元件。甲基化检测结果表明:在鸭的个体和组织水平上,启动子甲基化率均未聚类在一起;Cp G位点甲基化频率存在个体差异,22%Cp G位点的甲基化频率与Myo G的m RNA表达量呈负相关(P>0.05),78%Cp G位点的甲基化频率呈正相关(P>0.05),其中,腿肌甲基化位点Cp G_1、Cp G_26.27.28.29的甲基化频率与Myo G基因表达水平均呈显著相关(P<0.05)。Myo G基因在鸭与在哺乳动物中的转录调控机制存在差异。试验中发现多个影响鸭Myo G基因转录的潜在甲基化位点,其中Cp G_1与Cp G_26.27.28.29能通过DNA甲基化修饰影响Myo G基因在鸭腿肌中的转录。本研究结果可为鸭Myo G基因转录调控提供参考依据。
Promoter sequence of gene Myo G in duck was amplified and cloned using RT–PCR, and its bioinformatic info was analyzed as well; the methylation level in Cp G island(from –2 536 to –1 997 bp) of promoter Myo G in muscle tissues was detected using the Sequenom Mass Array technique, meanwhile, its expression level was detected using q RT–PCR. The results showed that the amplified Myo G promoter sequences was 2 730 bp which contained 2 Cp G islands in promoter region. The Cp G island(from –2 536 to –1 997 bp) included 5 binding sites of transcription factor and several eukaryotes structure components. The methylation data showed that methylation status from different individuals and tissues did not clustered together. Methylation frequency in Cp G loci varied with individuals, and the methylation frequency on 22% of locus were negatively correlated with Myo G expression levels(P>0.05), whereas, on 78% of locus were positively correlated(P>0.05) with that, among of them, they reached significant difference level on locus of Cp G 1, Cp G 26.27.28.29 from leg muscles(P<0.05). The mechanism of regulating transcription for Myo G was different from mammals. Among the potential locus influencing Myo G transcription, Cp G_1 and Cp G_26.27.28.29 might affect Myo G transcription in skeletal muscle tissues of duck leg through DNA methylation. This study might list foundation data for studying the regulatory mechanisms of Myo G transcription in duck.
Promoter sequence of gene Myo G in duck was amplified and cloned using RT–PCR, and its bioinformatic info was analyzed as well; the methylation level in Cp G island(from –2 536 to –1 997 bp) of promoter Myo G in muscle tissues was detected using the Sequenom Mass Array technique, meanwhile, its expression level was detected using q RT–PCR. The results showed that the amplified Myo G promoter sequences was 2 730 bp which contained 2 Cp G islands in promoter region. The Cp G island(from –2 536 to –1 997 bp) included 5 binding sites of transcription factor and several eukaryotes structure components. The methylation data showed that methylation status from different individuals and tissues did not clustered together. Methylation frequency in Cp G loci varied with individuals, and the methylation frequency on 22% of locus were negatively correlated with Myo G expression levels(P>0.05), whereas, on 78% of locus were positively correlated(P>0.05) with that, among of them, they reached significant difference level on locus of Cp G 1, Cp G 26.27.28.29 from leg muscles(P<0.05). The mechanism of regulating transcription for Myo G was different from mammals. Among the potential locus influencing Myo G transcription, Cp G_1 and Cp G_26.27.28.29 might affect Myo G transcription in skeletal muscle tissues of duck leg through DNA methylation. This study might list foundation data for studying the regulatory mechanisms of Myo G transcription in duck.
引文
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[11]张晓卉.民猪Myo G基因的克隆、表达与启动子核心区的定位[D].哈尔滨:东北农业大学,2012.
[12]宋兴超,荣敏,邢秀梅,等.甘肃马鹿肌细胞生成素(Myo G)基因启动子区序列克隆与分析[J].特产研究,2009,31(2):12–16.DOI:10.3969/j.issn.1001–4721.2009.02.004.
[13]赵忠海,李辉,易恒洁,等.Myo G基因多态性对鸭屠宰性状的影响[J].基因组学与应用生物学,2015,34(6):1189–1194.
[14]孙朕,孙英民,郑素月,等.家禽血液基因组DNA的快速提取[J].安徽农业科学,2010,38(16):8449–8452.DOI:10.3969/j.issn.0517–6611.2010.16.063.
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[17]张小辉,祁艳霞.真核生物启动子TATA–box·GC–box和CAAT–box的分析[J].安徽农业科学,2008,36(4):1380–1381.
[18]KIM J.Methylation–sensitive binding of transcription factor YY1 to an insulator sequence within the paternally expressed imprinted gene,Peg3[J].Human Molecular Genetics,2003,12(3):233–245.DOI:10.1093/hmg/12.3.233.
[19]肖剑,王红梅,马继登,等.猪皮下和内脏脂肪组织中G蛋白偶联受体120(GPR120)第2内含子Cp G岛甲基化促进其mR NA的转录[J].农业生物技术学报,2014,22(8):992–1000.
[20]EL–OSTA A,KANTHARIDIS P,ZALCBERG J R,et al.Precipitous release of methyl–CpG binding protein 2 and histone deacetylase 1 from the methylated human multidrug resistance gene(MDR1)on activation[J].Mol Cell Biol,2002,22(6):1844–1857.DOI:10.1128/mcb.22.6.1844–1857.2002.
[21]FRAGA M F,BALLESTAR E,MONTOYA G,et al.The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties[J].Nucleic Acids Res,2003,31(6):1765–1774.DOI:10.1093/nar/gkg249.
[22]张广华.子宫内膜癌细胞孕激素受体基因表现遗传学调控的研究–DNA甲基化及表达沉默相关的染色质成分变化[D].天津:天津医科大学,2010.10.7666/d.y1749766.
[23]李明桂.牛睾丸组织b–Boule基因表达的甲基化调控研究[D].南京:南京农业大学,2012.10.7666/d.Y2361886.
[24]李长雷,郑聪颖,刘军,等.山羊克隆胎儿不同组织中H19基因Cp G岛甲基化模式及表达量检测[J].生物工程学报,2010,26(5):582–587.
[25]康倩.Aroclor1254对小鼠生殖细胞印记基因表达与甲基化的影响[D].长春:吉林农业大学,2013.
[2]WYSZY?SKA–KOKO J,PIERZCHA?A M,FLISIKOWSKI K,et al.Polymorphisms in coding and regulatory regions of the porcine MYF6 and Myo G genes and expression of the MYF6 gene in m.longissimus dorsi versus productive traits in pigs[J].J Appl Genet,2006,47(2):131–138.DOI:10.1007/BF0319 4612.
[3]BECKER C,HAGMANN J,MüLLER J,et al.Spontaneous epigenetic variation in the Arabidopsis thaliana methylome[J].Nature,2011,480(7376):245–249.DOI:10.1038/nature10555.
[4]NABESHIMA Y,HANAOKA K,HAYASAKA M,et al.Myogenin gene disruption results in perinatal lethality because of severe muscle defect[J].Nature,1993,364(6437):532–535.DOI:10.1038/364532a0.
[5]WEINTRAUB H,DAVIS R,TAPSCOTT S,et al.The Myo D gene family:nodal point during specification of the muscle cell lineage[J].Science,1991,251(4995):761–766.DOI:10.1126/science.1846704.
[6]王珊,陈宏,蔡欣.牛MyoG基因启动子的克隆与序列分析[J].西北农林科技大学学报(自然科学版),2006,34(8):12–16.DOI:10.3321/j.issn:1671–9387.2006.08.003.
[7]王秋华,曹允考,李树峰,等.牛Myo G基因启动子的克隆及功能的初步分析[J].畜牧兽医学报,2012,43(1):37–43.
[8]李硕,郝斐,梁浩,等.牛肌细胞生成素基因(Myo G)启动子的活性及组织特异性分析[J].农业生物技术学报,2014,22(1):87–93.DOI:10.3969/j.issn.1674–7968.2014.01.011.
[9]WANG X,LU M,FENG L H,et al.Effects of CMV enhancer on activity and specificity of bovine Myo G Gene Promoter[J].Journal of Northeast Agricultural University(English Edition),2013,20(4):34–38.
[10]刘铮铸,巩元芳,张文香,等.山羊MyoG基因启动子区序列分析与结构预测[J].畜牧兽医学报,2012,43(1):29–36.
[11]张晓卉.民猪Myo G基因的克隆、表达与启动子核心区的定位[D].哈尔滨:东北农业大学,2012.
[12]宋兴超,荣敏,邢秀梅,等.甘肃马鹿肌细胞生成素(Myo G)基因启动子区序列克隆与分析[J].特产研究,2009,31(2):12–16.DOI:10.3969/j.issn.1001–4721.2009.02.004.
[13]赵忠海,李辉,易恒洁,等.Myo G基因多态性对鸭屠宰性状的影响[J].基因组学与应用生物学,2015,34(6):1189–1194.
[14]孙朕,孙英民,郑素月,等.家禽血液基因组DNA的快速提取[J].安徽农业科学,2010,38(16):8449–8452.DOI:10.3969/j.issn.0517–6611.2010.16.063.
[15]刘贺贺.IGF–1对鸭胚肌肉发育影响及其分子机理研究[D].成都:四川农业大学,2012.
[16]NELSON B H.IL–2,regulatory T cells,and tolerance[J].J Immunol,2004,172(7):3983–3988.DOI:10.4049/jimmunol.172.7.3983.
[17]张小辉,祁艳霞.真核生物启动子TATA–box·GC–box和CAAT–box的分析[J].安徽农业科学,2008,36(4):1380–1381.
[18]KIM J.Methylation–sensitive binding of transcription factor YY1 to an insulator sequence within the paternally expressed imprinted gene,Peg3[J].Human Molecular Genetics,2003,12(3):233–245.DOI:10.1093/hmg/12.3.233.
[19]肖剑,王红梅,马继登,等.猪皮下和内脏脂肪组织中G蛋白偶联受体120(GPR120)第2内含子Cp G岛甲基化促进其mR NA的转录[J].农业生物技术学报,2014,22(8):992–1000.
[20]EL–OSTA A,KANTHARIDIS P,ZALCBERG J R,et al.Precipitous release of methyl–CpG binding protein 2 and histone deacetylase 1 from the methylated human multidrug resistance gene(MDR1)on activation[J].Mol Cell Biol,2002,22(6):1844–1857.DOI:10.1128/mcb.22.6.1844–1857.2002.
[21]FRAGA M F,BALLESTAR E,MONTOYA G,et al.The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties[J].Nucleic Acids Res,2003,31(6):1765–1774.DOI:10.1093/nar/gkg249.
[22]张广华.子宫内膜癌细胞孕激素受体基因表现遗传学调控的研究–DNA甲基化及表达沉默相关的染色质成分变化[D].天津:天津医科大学,2010.10.7666/d.y1749766.
[23]李明桂.牛睾丸组织b–Boule基因表达的甲基化调控研究[D].南京:南京农业大学,2012.10.7666/d.Y2361886.
[24]李长雷,郑聪颖,刘军,等.山羊克隆胎儿不同组织中H19基因Cp G岛甲基化模式及表达量检测[J].生物工程学报,2010,26(5):582–587.
[25]康倩.Aroclor1254对小鼠生殖细胞印记基因表达与甲基化的影响[D].长春:吉林农业大学,2013.