团头鲂SREBP-1基因的克隆及其表达分析
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摘要
为阐明团头鲂SREBP-1的功能,采用RT-PCR技术克隆获得SREBP-1基因的cDNA序列,利用荧光定量PCR(qRT-PCR)检测SREBP-1基因在不同发育时期及不同组织中的表达情况。生物信息学分析发现,SREBP-1基因cDNA的编码序列(coding sequence,CDS)为3 426bp(GenBank登录号:MH633449),开放阅读框(ORF)编码1 141个氨基酸,与大西洋鲑、斑马鱼、草鱼、鲤、金线鲃SREBP-1的氨基酸序列相似性较高(74%～99%)。保守结构域分析显示,团头鲂SREBP-1蛋白具有SREBPs家族典型的"碱性螺旋-环-螺旋-亮氨酸拉链"(bHLH-Zip)结构域。表达分析结果显示,SREBP-1基因在团头鲂胚胎发育过程中的表达呈现先下降后增高的趋势,在眼囊期最低,至出膜期达到最高。在团头鲂幼鱼中,SREBP-1在脑组织中的表达量最高,其次是眼、肝脏、心脏、肌肉、脂肪组织、肾脏,鳃中表达量最低。在成鱼中,SREBP-1在脑组织表达量最高,其次是性腺组织、肌肉、肝脏、眼,肾脏、脾脏和心脏中的表达量较低。以上结果表明,SREBP-1基因在团头鲂幼鱼和成鱼的不同组织中的表达模式存在差异。
This study was conducted to investigate the function of the SREBP-1 gene in Megalobrama amblycephala.The SREBP-1 gene was cloned by RT-PCR,and its expression were analyzed using qRT-PCR.Bioinformatics analysis showed that the cDNA coding sequence(CDS)of SREBP-1 was3 426 bp(GenBank Accession number:MH633449),with an open reading frame(ORF)encoding 1 141 amino acids.The M.amblycephala SREBP-1 protein shared high similarity(74%～99%)with Salmo salar,Danio rerio,Ctenopharyngodon idella,Cyprinus carpio and Sinocyclocheilus grahami.Conserved domain analysis showed that the SREBP-1 protein has the bHLH-Zip domains,which belong to the SREBPs family.Expression analysis showed that the SREBP-1 expression leveled was the lowest at the optic vesicle stage and highest at the hatching stage during embryonic development of M.amblycephala.For juvenile M.amblycephala,the SREBP-1 gene expressed highest in the brain,followed by the eye,liver,heart,fat tissue,kidney and gill.For adult M.amblycephala,the SREBP-1 gene expressed highest in the brain,followed by the gonad,muscle,liver,eye,while expressed relatively low in the kidney,spleen and heart.The tissue expression pattern of the SREBP 1 gene was different between the juvenile and adult M.amblycephala.
This study was conducted to investigate the function of the SREBP-1 gene in Megalobrama amblycephala.The SREBP-1 gene was cloned by RT-PCR,and its expression were analyzed using qRT-PCR.Bioinformatics analysis showed that the cDNA coding sequence(CDS)of SREBP-1 was3 426 bp(GenBank Accession number:MH633449),with an open reading frame(ORF)encoding 1 141 amino acids.The M.amblycephala SREBP-1 protein shared high similarity(74%～99%)with Salmo salar,Danio rerio,Ctenopharyngodon idella,Cyprinus carpio and Sinocyclocheilus grahami.Conserved domain analysis showed that the SREBP-1 protein has the bHLH-Zip domains,which belong to the SREBPs family.Expression analysis showed that the SREBP-1 expression leveled was the lowest at the optic vesicle stage and highest at the hatching stage during embryonic development of M.amblycephala.For juvenile M.amblycephala,the SREBP-1 gene expressed highest in the brain,followed by the eye,liver,heart,fat tissue,kidney and gill.For adult M.amblycephala,the SREBP-1 gene expressed highest in the brain,followed by the gonad,muscle,liver,eye,while expressed relatively low in the kidney,spleen and heart.The tissue expression pattern of the SREBP 1 gene was different between the juvenile and adult M.amblycephala.
引文
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[18]聂涛.猪SREBPs和CREB-1基因的克隆、染色体定位及其功能研究[D].武汉:华中农业大学,2005.
[19]杨云超.山羊SREBP1-1a、SREBP1-1c基因的鉴定及功能初步研究[D].杨凌:西北农林科技大学,2016.
[20]付常振,昝林森,王虹,等.秦川牛SREBP1基因重组腺病毒载体的构建与病毒包装[J].畜牧兽医学报,2013,44(8):1323-1329.
[21]廖碧云.水牛SCAP和SREBP-1基因的克隆、表达规律和多态性检测研究[D].南宁:广西大学,2015.
[22]LIU H L,ZHANG H K,ZHENG H P.Regulatory roles of sterol regulatory element-binding protein(SREBP)on lipid metabolism in marine invertebrate Chlamys nobilis[J].Aquaculture,2018,493:251-257.
[23]JONES S.An overview of the basic helix-loop-helix proteins[J].Genome biology,2004,5(6):226.https://doi.org/10.1186/gb-2004-5-6-226.
[24]何登菊,姚俊杰,赵云龙,等.瓯江彩鲤胚胎发育过程中脂、蛋白及碳水化合物水平的变化[J].动物学杂志,2011,46(2):102-107.
[25]牟洪民,马珊,姚俊杰,等.普安银鲫胚胎发育早期的总脂与脂肪酸变化[J].西南农业学报,2012,25(5):1925-1928.
[26]蒋左玉,熊铧龙,姚俊杰,等.普安银鲫胚胎发育中脂质代谢酶基因的表达及葡萄糖的影响[J].水产科学,2017,36(3):359-363.
[27]ASSAF S,HAZAR D,PITEL F,et al.Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2(SREBP-2),chromosomal localization,and tissue expression of chicken SREBP-1and-2genes[J].Poultry science,2003,82(1):54-61.
[2]SHIMANO H,SATO R.SREBP-regulated lipid metabolism:convergent physiology-divergent pathophysiology[J].Nature reviews endocrinology,2017,13(12):710-730.
[3]DEBOSE-BOYD R A,YE J.SREBPs in lipid metabolism,insulin signaling,and beyond[J].Trends in biochemical sciences,2018,43(5):358-368.
[4]EBERLE D,HEGARTY B,BOSSARD P,et al.SREBP transcription factors:master regulators of lipid homeostasis[J].Biochimie,2004,86(11):839-848.
[5]JEON T,OSBOME T F.SREBPs:metabolic integrators in physiology and metabolism[J].Trends in endocrinology and metabolism,2012,23(2):65-72.
[6]BROWN M S,GOLDSTEIN J L.The SREBP pathway:regulation of cholesterol metabolism by proteolysisof a membranebound transcription factor[J].Cell,1997,89(3):331-340.
[7]WANG Y,CHEN K,YAO Q,et al.Phylogenetic analysis of zebrafish basic helix-loop-helix transcription factors[J].Journal of molecular evolution,2009,68(6):629-640.
[8]MINGHETTI M,LEAVER M J,TOUCHER D R.Transcriptional control mechanisms of genes of lipid and fatty acid metabolism in the Atlantic salmon(Salmo salar L.)established cell line,SHK-1[J].Biochimica et biophysica acta(BBA)-molecular and cell biology of lipids,2011,1811(3):194-202.
[9]刘芳.黄斑蓝子鱼固醇调节元件结合蛋白-1基因克隆及表达研究[D].汕头:汕头大学,2011.
[10]孙君君,卢荣华,杨峰,等.草鱼SREBP-1基因的克隆及糖对其在肝脏中表达的影响[J].水产学报,2014,38(8):1057-1067.
[11]张伟,姬明丽,郭前进,等.红鳍东方鲀SREBP-1和SREBP-2基因克隆及表达分析[J].华北农学报,2016,31(4):74-79.
[12]王卫民.团头鲂养殖产业现状[J].科学养鱼,2009(4):44-45.
[13]汤晓丽,邓立彬,林加日,等.固醇调节元件结合蛋白1及其靶基因网络[J].遗传,2013,35(5):607-615.
[14]XIAO X,SONG B L.SREBP:a novel therapeutic target[J].Acta biochimica et biophysica sinica,2013,45(1):2-10.
[15]SOYAL S M,NOFZIGER C,DOSSENA S,et al.Targeting SREBPs for treatment of the metabolic syndrome[J].Trends in pharmacological sciences,2015,36(6):406-416.
[16]SHIMOMURA I,SHIMANO H,HORTON J D,et al.Differential expression of exons 1aand 1cin mRNAs for sterol regulatory element binding protein-1in human and mouse organs and cultured cells[J].The journal of clinical investigation,1997,99(5):838-845.
[17]KHESHT F A,HASSANABADI A.Effects of sterol regulatory element-binding protein(SREBP)in chickens[J].Lipids in health and disease,2012,11:20.https://doi.org/10.1186/1476-511X-11-20.
[18]聂涛.猪SREBPs和CREB-1基因的克隆、染色体定位及其功能研究[D].武汉:华中农业大学,2005.
[19]杨云超.山羊SREBP1-1a、SREBP1-1c基因的鉴定及功能初步研究[D].杨凌:西北农林科技大学,2016.
[20]付常振,昝林森,王虹,等.秦川牛SREBP1基因重组腺病毒载体的构建与病毒包装[J].畜牧兽医学报,2013,44(8):1323-1329.
[21]廖碧云.水牛SCAP和SREBP-1基因的克隆、表达规律和多态性检测研究[D].南宁:广西大学,2015.
[22]LIU H L,ZHANG H K,ZHENG H P.Regulatory roles of sterol regulatory element-binding protein(SREBP)on lipid metabolism in marine invertebrate Chlamys nobilis[J].Aquaculture,2018,493:251-257.
[23]JONES S.An overview of the basic helix-loop-helix proteins[J].Genome biology,2004,5(6):226.https://doi.org/10.1186/gb-2004-5-6-226.
[24]何登菊,姚俊杰,赵云龙,等.瓯江彩鲤胚胎发育过程中脂、蛋白及碳水化合物水平的变化[J].动物学杂志,2011,46(2):102-107.
[25]牟洪民,马珊,姚俊杰,等.普安银鲫胚胎发育早期的总脂与脂肪酸变化[J].西南农业学报,2012,25(5):1925-1928.
[26]蒋左玉,熊铧龙,姚俊杰,等.普安银鲫胚胎发育中脂质代谢酶基因的表达及葡萄糖的影响[J].水产科学,2017,36(3):359-363.
[27]ASSAF S,HAZAR D,PITEL F,et al.Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2(SREBP-2),chromosomal localization,and tissue expression of chicken SREBP-1and-2genes[J].Poultry science,2003,82(1):54-61.