牦牛MDHI基因的克隆及组织表达分析
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摘要
苹果酸脱氢酶具有氧化还原活性,主要控制动物机体的能量代谢。旨在克隆牦牛MDHI基因,结合生物信息学和组织表达分析,为研究牦牛MDHI基因的理化性质及功能奠定基础。结果显示,该基因的cDNA序列长1 169 bp,CDS区全长1 005 bp,编码334个氨基酸,编码的蛋白为亲水性稳定蛋白;属于保守结构域家族;有两个跨膜区,分别是6-22位和34-54位;存在26个磷酸化位点,其中有11个Ser磷酸化位点、11个Thr磷酸化位点、4个Tyr磷酸化位点;二级结构主要由α-螺旋(152个,占45.51%),无规卷曲(110个,占32.93%)组成;三级结构与猪细胞质苹果酸脱氢酶、α-酮丙二酸和四氢NAD的三元复合物的晶体结构具有98.50%的相似性;亚细胞定位分析得到在内质网中分布最多,在高尔基体和细胞核中分布最少;同源性及系统进化树分析表明牦牛该基因与普通牛的同源性最高,推测牦牛MDHI基因在进化水平上较为保守;组织表达分析表明该基因在臀脂中表达量最高,在臀肌中表达量较低,说明该基因参与机体脂肪沉积的调控。成功克隆了MDHI基因CDS区、并进行了生物信息学及组织表达分析。
Malate dehydrogenase has redox activity and plays a key role in energy metabolism of animal organisms. This study aims to clone the yak MDHI gene,combined with bioinformatics analysis and tissue expression analysis,which may lay a foundation for studying the physical and chemical properties and functions of the yak MDHI gene. The results showed that the cDNA sequence of this gene was 1169 bp,the CDS region was 1 005 bp in length and it encoded 334 amino acids. The encoded protein was a hydrophilic and stable,which belonged to the conserved domain family. There were two transmembrane areas(located in 6-22 and 34-54 amino acids respectively)and 26 phosphorylation sites(11 Ser phosphorylation sites,11 Thr phosphorylation sites and 4 Tyr phosphorylation sites). The secondary structure was mainly composed of α-helix(152,accounting for 45.51%),random coil(110,accounting for 32.93%). The tertiary structure had a similarity of 98.50% with crystal structure of the ternary complex of cytoplasmic malate dehydrogenase,α-Ketomalonic acid and tetrahydro NAD in porcine. Subcellular localization analysis revealed that most of them were distributed in the endoplasmic reticulum,least in the Golgi and nucleus. Furthermore,homology and phylogenetic tree analysis indicated that its homology was the highest with common cattle,which can be speculate that the yak MDHI gene maybe highly conserved at the evolution level. Tissue expression analysis showed that the gene was expressed the highest in gluteal fat but lower in gluteal muscle,indicating that the gene may function in fat deposition. In sum,we cloned MDHI gene successfully and performed bioinformatics and tissue expression analysis.
Malate dehydrogenase has redox activity and plays a key role in energy metabolism of animal organisms. This study aims to clone the yak MDHI gene,combined with bioinformatics analysis and tissue expression analysis,which may lay a foundation for studying the physical and chemical properties and functions of the yak MDHI gene. The results showed that the cDNA sequence of this gene was 1169 bp,the CDS region was 1 005 bp in length and it encoded 334 amino acids. The encoded protein was a hydrophilic and stable,which belonged to the conserved domain family. There were two transmembrane areas(located in 6-22 and 34-54 amino acids respectively)and 26 phosphorylation sites(11 Ser phosphorylation sites,11 Thr phosphorylation sites and 4 Tyr phosphorylation sites). The secondary structure was mainly composed of α-helix(152,accounting for 45.51%),random coil(110,accounting for 32.93%). The tertiary structure had a similarity of 98.50% with crystal structure of the ternary complex of cytoplasmic malate dehydrogenase,α-Ketomalonic acid and tetrahydro NAD in porcine. Subcellular localization analysis revealed that most of them were distributed in the endoplasmic reticulum,least in the Golgi and nucleus. Furthermore,homology and phylogenetic tree analysis indicated that its homology was the highest with common cattle,which can be speculate that the yak MDHI gene maybe highly conserved at the evolution level. Tissue expression analysis showed that the gene was expressed the highest in gluteal fat but lower in gluteal muscle,indicating that the gene may function in fat deposition. In sum,we cloned MDHI gene successfully and performed bioinformatics and tissue expression analysis.
引文
[1]钟金城.牦牛遗传与育种[M].成都:四川科学技术出版社,1996.
[2]Wiener G, Han JL, Long RJ. The Yak[M]. 2nd ed. Bangkok,Thailand:Regional Office for Asia and the Pacific of the Food and Agriculture Organization of the United Nations, 2003.
[3]高志英.我国牦牛业和牦牛科研的进展[J].中国牦牛, 1985(3):1-3.
[4]Lee ML, Woo KS. Enzyme polymorphism of Apis cerana Fabr. In Korea[J].Honeybee Science, 1991, 12(2):58-60.
[5]李绍文,孟玉萍,张宗炳,等.意蜂和中蜂四种同工酶的研究[J].昆虫学报, 1988, 31(1):15-31.
[6]RozalskiRJ,SakuraiH,TsuehidaK.Esteraseandmalate dehydrogenase lsozymes analysis in the population of honeybees,Apis cerana japon:ea and Apis mellifrea[J].Japanese Journal of Entomology, 1996, 64(4):910-917.
[7]Guan HY, Tang ZQ, Li H. Correlation analysis between singlenucleotide polymorphism of malate dehydrogenase gene 5’-flanking region and growth and body composition traits in chicken[J]. Acta Genetics Sinica, 2006, 33(6):501-506.
[8]季海峰.中国科学技术协会首届青年学术年会论文集[C].农科分册, 1992.
[9]郑玉才,苏永杰,文勇立,等.牦牛肌红蛋白的基因克隆测序、分离纯化、含量及其与乳酸脱氢酶和苹果酸脱氢酶活力的关系[J].畜牧兽医学报, 2007, 38(7):646-650.
[10]Lima Queiroz A, Zhang B, Comstock DE, et al. miR-126-5p targets malate dehydrogenase 1 in non-small cell lung carcinomas[J].Biochem Biophys Res Commun, 2018, 499(2):314-320.
[11]Zhang B, Tornmalm J, Widengren J, et al. Characterization of the role of the malate dehydrogenases to lung tumor cell survival[J].Journal of Cancer, 2017, 8(11):2088-2096.
[12]HanseEA,RuanC,KachmanM,etal.Cytosolicmalate dehydrogenase activity helps support glycolysis in actively proliferating cells and cancer[J]. Oncogene, 2017, 36(27):3915-3924.
[13]依姆兰(Muhammad Imran).棉花苹果酸脱氢酶基因家族的分子鉴定及其特征[D].北京:清华大学, 2017.
[14]王西成,钱亚明,吴伟民,等. 6-BA对葡萄果实中有机酸积累及相关基因表达的影响[J].华北农学报, 2017, 32(5):149-153.
[15]李海贤,马艳玲,曾荣.丁香酚对金黄色葡萄球菌MDH和SDH酶活力的影响[J].食品科技, 2018, 43(3):213-217.
[16]张亨,刘盈盈,陈云,等.戈壁异常球菌Dgl5蛋白生物学功能研究[J].生物技术通报, 2018, 34(3):177-184.
[17]陶勇,任善茂,倪黎纲,等.苏姜猪血清HSL、MDH活性变化规律的研究[J].西南农业学报, 2017, 30(7):1672-1674.
[18]宋庆文.不同营养水平下荣昌猪和DLY猪脂肪(酸)代谢关键酶活性的发育性变化研究[D].呼和浩特:内蒙古农业大学,2007.
[19]Gibal AMJ, Young ME, Taegtmeyer H. Anaplerosis of the citrie acid cyele:role in energy metabolism of heart and skeletal muscle[J]. Aeta Physiolo Scand, 2000, 168:657-665.
[20]祝仁铸,尹逊河,王元虎,等.猪肌肉组织MDH和LPL基因表达与肌内脂肪含量和脂肪酸组成关系的研究[J].畜牧兽医学报, 2013, 44(8):1182-1188.
[21]杨芳.陆川猪与杜洛克猪正反交F1代肉质性状、肌内脂肪酶活性和相关基因的研究[D].南宁:广西大学, 2017.
[22]王美玲,陈仲建,吕林,等.不同形态锰对肉仔鸡脂肪代谢关键酶活性及其基因表达的影响[J].中国农业科学, 2011, 44(18):3850-3858.
[23]Kim EY, Kim WK, Kim JH et al. Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity[J]. J Lipid Res, 2012, 53(9):1864-1876.
[24]Picard B, Gagaoua M, Al-Jammas M, et al. Beef tenderness and intramuscular fat proteomic biomarkers:muscle type effect[J].Peer J, 2018, 6:e 4891.
[2]Wiener G, Han JL, Long RJ. The Yak[M]. 2nd ed. Bangkok,Thailand:Regional Office for Asia and the Pacific of the Food and Agriculture Organization of the United Nations, 2003.
[3]高志英.我国牦牛业和牦牛科研的进展[J].中国牦牛, 1985(3):1-3.
[4]Lee ML, Woo KS. Enzyme polymorphism of Apis cerana Fabr. In Korea[J].Honeybee Science, 1991, 12(2):58-60.
[5]李绍文,孟玉萍,张宗炳,等.意蜂和中蜂四种同工酶的研究[J].昆虫学报, 1988, 31(1):15-31.
[6]RozalskiRJ,SakuraiH,TsuehidaK.Esteraseandmalate dehydrogenase lsozymes analysis in the population of honeybees,Apis cerana japon:ea and Apis mellifrea[J].Japanese Journal of Entomology, 1996, 64(4):910-917.
[7]Guan HY, Tang ZQ, Li H. Correlation analysis between singlenucleotide polymorphism of malate dehydrogenase gene 5’-flanking region and growth and body composition traits in chicken[J]. Acta Genetics Sinica, 2006, 33(6):501-506.
[8]季海峰.中国科学技术协会首届青年学术年会论文集[C].农科分册, 1992.
[9]郑玉才,苏永杰,文勇立,等.牦牛肌红蛋白的基因克隆测序、分离纯化、含量及其与乳酸脱氢酶和苹果酸脱氢酶活力的关系[J].畜牧兽医学报, 2007, 38(7):646-650.
[10]Lima Queiroz A, Zhang B, Comstock DE, et al. miR-126-5p targets malate dehydrogenase 1 in non-small cell lung carcinomas[J].Biochem Biophys Res Commun, 2018, 499(2):314-320.
[11]Zhang B, Tornmalm J, Widengren J, et al. Characterization of the role of the malate dehydrogenases to lung tumor cell survival[J].Journal of Cancer, 2017, 8(11):2088-2096.
[12]HanseEA,RuanC,KachmanM,etal.Cytosolicmalate dehydrogenase activity helps support glycolysis in actively proliferating cells and cancer[J]. Oncogene, 2017, 36(27):3915-3924.
[13]依姆兰(Muhammad Imran).棉花苹果酸脱氢酶基因家族的分子鉴定及其特征[D].北京:清华大学, 2017.
[14]王西成,钱亚明,吴伟民,等. 6-BA对葡萄果实中有机酸积累及相关基因表达的影响[J].华北农学报, 2017, 32(5):149-153.
[15]李海贤,马艳玲,曾荣.丁香酚对金黄色葡萄球菌MDH和SDH酶活力的影响[J].食品科技, 2018, 43(3):213-217.
[16]张亨,刘盈盈,陈云,等.戈壁异常球菌Dgl5蛋白生物学功能研究[J].生物技术通报, 2018, 34(3):177-184.
[17]陶勇,任善茂,倪黎纲,等.苏姜猪血清HSL、MDH活性变化规律的研究[J].西南农业学报, 2017, 30(7):1672-1674.
[18]宋庆文.不同营养水平下荣昌猪和DLY猪脂肪(酸)代谢关键酶活性的发育性变化研究[D].呼和浩特:内蒙古农业大学,2007.
[19]Gibal AMJ, Young ME, Taegtmeyer H. Anaplerosis of the citrie acid cyele:role in energy metabolism of heart and skeletal muscle[J]. Aeta Physiolo Scand, 2000, 168:657-665.
[20]祝仁铸,尹逊河,王元虎,等.猪肌肉组织MDH和LPL基因表达与肌内脂肪含量和脂肪酸组成关系的研究[J].畜牧兽医学报, 2013, 44(8):1182-1188.
[21]杨芳.陆川猪与杜洛克猪正反交F1代肉质性状、肌内脂肪酶活性和相关基因的研究[D].南宁:广西大学, 2017.
[22]王美玲,陈仲建,吕林,等.不同形态锰对肉仔鸡脂肪代谢关键酶活性及其基因表达的影响[J].中国农业科学, 2011, 44(18):3850-3858.
[23]Kim EY, Kim WK, Kim JH et al. Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity[J]. J Lipid Res, 2012, 53(9):1864-1876.
[24]Picard B, Gagaoua M, Al-Jammas M, et al. Beef tenderness and intramuscular fat proteomic biomarkers:muscle type effect[J].Peer J, 2018, 6:e 4891.