固原鸡生肌调节因子MyoD基因克隆及其组织表达分析
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摘要
为研究MyoD在固原鸡生个体生长发育中的作用,运用荧光定量PCR、TA克隆和核酸测序等技术构建了固原鸡MyoD时序表达谱,并对固原鸡MyoD基因CDS区进行克隆和生物信息学分析。结果表明,MyoD在肌肉组织中表达量极显著高于其他组织(P<0.01),且胸肌中表达量高于腿肌。同时发现MyoD在固原鸡个体发育阶段表达量存在差异性,呈先上升后下降趋势,10日龄达到最高。扩增获得固原鸡MyoD基因CDS区,新发现一个同义突变(126A>C)。亚细胞定位及结构预测显示,MyoD主要分布于细胞核(69.6%),其氨基酸二级结构呈现螺旋-环-螺旋(bHLH)结构。构建的固原鸡与其他11个物种的系统发育树表明,固原鸡与原鸡、火鸡、鹌鹑及绿头鸭之间存在较高的同源性。研究结果为探究固原鸡生长发育机制及分子标记辅助育种提供理论依据。
In order to explore the roles of MyoD relating to growth trait of Guyuan chicken, CDS region of MyoD gene was cloned and expression pattern was analyzed with Real-time PCR, TA cloning and sequencing technology. The results showed that the expression level of MyoD was high in the muscle(P<0.01) and significantly higher in breast muscle than that in leg muscle. In addition, the expression pattern of MyoD was different at different stages and increased firstly then declined, the highest level was at 10 days of age. CDS region of MyoD gene was obtained and one novel synonymous mutations(126A>C) was identified. Subcellular localization of MyoD was mainly in nucleus(69.6%)and the secondary structure of MyoD was a helix loop helix(bHLH). Phylogenetic tree of the eleven species based on MyoD gene coding region analysis showed that Guyuan chicken had a higher homology with Gallus gallus, Meleagris gallopavo, Coturnix coturnix and Anas platyrhynchos. This information could help us understand the molecular mechanism involved in MyoD gene better and was useful for molecular marker-assisted selection in Guyuan chicken.
In order to explore the roles of MyoD relating to growth trait of Guyuan chicken, CDS region of MyoD gene was cloned and expression pattern was analyzed with Real-time PCR, TA cloning and sequencing technology. The results showed that the expression level of MyoD was high in the muscle(P<0.01) and significantly higher in breast muscle than that in leg muscle. In addition, the expression pattern of MyoD was different at different stages and increased firstly then declined, the highest level was at 10 days of age. CDS region of MyoD gene was obtained and one novel synonymous mutations(126A>C) was identified. Subcellular localization of MyoD was mainly in nucleus(69.6%)and the secondary structure of MyoD was a helix loop helix(bHLH). Phylogenetic tree of the eleven species based on MyoD gene coding region analysis showed that Guyuan chicken had a higher homology with Gallus gallus, Meleagris gallopavo, Coturnix coturnix and Anas platyrhynchos. This information could help us understand the molecular mechanism involved in MyoD gene better and was useful for molecular marker-assisted selection in Guyuan chicken.
引文
[1]杨文清,钱爱萍,李自强,等.固原鸡的形成、发展与产业化开发[J].中国家禽,2006,28(6):46-47.
[2]顾亚玲,张娟,杨彦军,等.不同群体固原鸡肌肉氨基酸及脂肪酸含量差异性测定[J].中国家禽,2013,35(24):28-32.
[3]张娟,顾亚玲,刘立刚,等.不同世代不同群体固原鸡鸡肉粗脂肪、粗蛋白的研究[J].中国家禽,2015,37(2):57-59.
[4]LEE H,HABAS R,ABATE-SHEN C. MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis[J]. Science, 2004, 304(5677):1675-1678.
[5]RUDNICKI M A, SCHNEGELSBERG P N, STEAD R H,et al. MyoD or Myf-5 is required for the formation of skeletal muscle[J]. Cell, 1994, 75(7):1351-1359.
[6]CASES S,SMITH S J,ZHENG Y W. Identification of a gene encoding anacyl CoA:Diacylglycerol acyltransferase,a key enzyme in triacylglycerol synthesis[J]. Proceedings of National Academy of Sciences USA, 1998, 95(22):13018-13023.
[7]LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtMethod[J]. Methods, 2001, 25(4):402-408.
[8]KOZAK M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNA[J].Nucleic Acids Research, 1984, 12(2):857-872.
[9]YIN H, PRICE F, RUDNICKI M A. Satellite cells and the muscle stem cell niche[J]. Physiological Reviews, 2013,93(1):23-67.
[10]GOUDENEGE S, PISANI D F, WDZIEKONSKI B, et al. Enhancement of myogenic and muscle repair capacities of human adipose-derived stem cells with forced expression of MyoD[J]. Molecular Therapy the Journal of the American Society of Gene Therapy, 2009, 17(6):1064-1072.
[11]刘伍限,贾斌,石国庆.绵羊FGF5基因编码蛋白的生物信息学分析[J].石河子大学学报(自然科学版),2011,29(3):296-300.
[12]GREGORY D J, WALDBIESER G C, BOSWORTH B G.Cloning and characterization of myogenic regulatory genes in three Ictalurid species[J]. Animal Genetics, 2004, 35(6):425-430.
[13]邓彦飞,石德顺,刘庆友,等.水牛转录因子c-Myc克隆和表达研究[J].中国畜牧杂志,2014,50(7):14-18.
[14]HELLEMANS J, MORTIER G, PAEPE D A, et al. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[J]. Genome Biology, 2007, 8(2):R19.
[15]PFAFFL M W, HORGAN G W, DEMPFLE L. Relative Expression Software Tool(REST?)for group wise comparison and statistical analysis of relative expression results in real-time PCR[J]. Nucleic Acids Research, 2002, 30(9):e36.
[16]PFAFFL M W, TICHOPAD A, PRGOMET C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:Bestkeeper-excelbased tool using pair-wise correlations[J]. Biotechnology Letters, 2004, 26(6):509-515.
[17]俞兆曦.兰州鲇生长性状相关基因的分离、序列特征及组织特异性表达分析[D].兰州:甘肃农业大学,2016.
[18]牛姣艳. Pax7、MyoD和MyoG基因在猪背最长肌中的发育性表达研究[D].晋中:山西农业大学,2015.
[19]祁艳霞,张小辉,庞有志,等.南阳黄牛肌肉发育过程中的MyoD和MEF2基因的表达变化研究[J].河南农业大学学报,2012,46(5):558-561.
[20]朱春红,陶志云,宋迟,等.高邮鸭和金定鸭胸肌早期发育及MyoD1基因表达分析[J].家畜生态学报,2013,34(4):15-18.
[21]MAVES L, WASKIEWICZ A J, PAUL B, et al. Pbx homeodomain proteins direct MyoD activity to promote fastmuscle differentiation[J]. Development, 2007, 134(18):3371-3382.
[2]顾亚玲,张娟,杨彦军,等.不同群体固原鸡肌肉氨基酸及脂肪酸含量差异性测定[J].中国家禽,2013,35(24):28-32.
[3]张娟,顾亚玲,刘立刚,等.不同世代不同群体固原鸡鸡肉粗脂肪、粗蛋白的研究[J].中国家禽,2015,37(2):57-59.
[4]LEE H,HABAS R,ABATE-SHEN C. MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis[J]. Science, 2004, 304(5677):1675-1678.
[5]RUDNICKI M A, SCHNEGELSBERG P N, STEAD R H,et al. MyoD or Myf-5 is required for the formation of skeletal muscle[J]. Cell, 1994, 75(7):1351-1359.
[6]CASES S,SMITH S J,ZHENG Y W. Identification of a gene encoding anacyl CoA:Diacylglycerol acyltransferase,a key enzyme in triacylglycerol synthesis[J]. Proceedings of National Academy of Sciences USA, 1998, 95(22):13018-13023.
[7]LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtMethod[J]. Methods, 2001, 25(4):402-408.
[8]KOZAK M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNA[J].Nucleic Acids Research, 1984, 12(2):857-872.
[9]YIN H, PRICE F, RUDNICKI M A. Satellite cells and the muscle stem cell niche[J]. Physiological Reviews, 2013,93(1):23-67.
[10]GOUDENEGE S, PISANI D F, WDZIEKONSKI B, et al. Enhancement of myogenic and muscle repair capacities of human adipose-derived stem cells with forced expression of MyoD[J]. Molecular Therapy the Journal of the American Society of Gene Therapy, 2009, 17(6):1064-1072.
[11]刘伍限,贾斌,石国庆.绵羊FGF5基因编码蛋白的生物信息学分析[J].石河子大学学报(自然科学版),2011,29(3):296-300.
[12]GREGORY D J, WALDBIESER G C, BOSWORTH B G.Cloning and characterization of myogenic regulatory genes in three Ictalurid species[J]. Animal Genetics, 2004, 35(6):425-430.
[13]邓彦飞,石德顺,刘庆友,等.水牛转录因子c-Myc克隆和表达研究[J].中国畜牧杂志,2014,50(7):14-18.
[14]HELLEMANS J, MORTIER G, PAEPE D A, et al. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[J]. Genome Biology, 2007, 8(2):R19.
[15]PFAFFL M W, HORGAN G W, DEMPFLE L. Relative Expression Software Tool(REST?)for group wise comparison and statistical analysis of relative expression results in real-time PCR[J]. Nucleic Acids Research, 2002, 30(9):e36.
[16]PFAFFL M W, TICHOPAD A, PRGOMET C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:Bestkeeper-excelbased tool using pair-wise correlations[J]. Biotechnology Letters, 2004, 26(6):509-515.
[17]俞兆曦.兰州鲇生长性状相关基因的分离、序列特征及组织特异性表达分析[D].兰州:甘肃农业大学,2016.
[18]牛姣艳. Pax7、MyoD和MyoG基因在猪背最长肌中的发育性表达研究[D].晋中:山西农业大学,2015.
[19]祁艳霞,张小辉,庞有志,等.南阳黄牛肌肉发育过程中的MyoD和MEF2基因的表达变化研究[J].河南农业大学学报,2012,46(5):558-561.
[20]朱春红,陶志云,宋迟,等.高邮鸭和金定鸭胸肌早期发育及MyoD1基因表达分析[J].家畜生态学报,2013,34(4):15-18.
[21]MAVES L, WASKIEWICZ A J, PAUL B, et al. Pbx homeodomain proteins direct MyoD activity to promote fastmuscle differentiation[J]. Development, 2007, 134(18):3371-3382.