草鱼脂酰辅酶A合成酶4基因全长cDNA分子克隆及表达调控
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摘要
本试验采用反转录PCR(RT-PCR)技术克隆草鱼脂酰辅酶A合成酶4(ACSL4)基因全长cDNA,利用实时荧光定量PCR技术研究ACSL4基因在草鱼大脑、心脏、脾脏、肝胰脏、肌肉、肾脏、肠系膜脂肪、前肠、中肠、后肠10种组织中的表达情况,并对投喂不同淀粉源饲料以及饥饿再投喂0、3、6、12和24 h后草鱼肝胰脏中ACSL4基因表达情况进行研究。结果显示:草鱼ACSL4基因cDNA全长2 418 bp,其开放阅读框2 121 bp,共编码706个氨基酸;草鱼ACSL4蛋白的氨基酸序列包含1个跨膜结构域、1个脂肪酸绑定基序和1个ATP/AMP绑定基序。草鱼ACSL4 mRNA在大脑和脾脏中相对表达量较高,显著高于其他组织(P <0.05),在肌肉中相对表达量最低;投喂不同淀粉源饲料对草鱼肝胰脏中ACSL4 mRNA相对表达量无显著影响(P>0.05);饥饿再投喂12 h后,草鱼肝胰脏中ACSL4 mRNA相对表达量达到最高值,显著高于其他时间段(P<0.05),24 h后恢复至最初水平。本试验从草鱼10种混合组织中克隆了ACSL4基因全长cDNA,其所编码的氨基酸序列的主要功能性位点脂肪酸绑定基序和ATP/AMP绑定基序在不同物种间高度保守;草鱼ACSL4基因主要在大脑和脾脏中表达;饲料中淀粉源对草鱼肝胰脏中ACSL4基因的表达无显著影响;在饥饿再投喂12 h后,草鱼肝胰脏中ACSL4 mRNA相对表达量达到最高值,随后显著下降至最初水平。
The full-length cDNA of long chain acyl coenzyme A synthetase 4( ACSL4) gene was cloned from grass carp by reverse-transcription PCR( RT-PCR) technique. Tissue distribution of ACSL4 gene in the brain,heart,spleen,hepatopancreas,muscle,kidney,adipose tissues,anterior intestine,mid-intestine and posterior intestine of grass carp was analyzed by real-time quantitative PCR technique. Meanwhile,the ACSL4 gene expression in the hepatopancreas of grass carp fed with diets containing different starch sources was studied. In addition,the ACSL4 gene expression in the hepatopancreas of grass carp at 0,3,6,12 and 24 h after starvation and refeeding was also investigated. The results showed as follows: the full-length cDNA of ACSL4 gene was2 418 bp with a 2 121 bp open reading frame( ORF) encoding 706 amino acids; the ACSL4 protein contained a trans-membrane domain,a ATP/AMP signature motif and a fatty acyl coenzyme A synthetase( FACS) signature motif. The relative expression level of ACSL4 mRNA in brain and spleen were higher,which were significantly higher than those in the other tissues( P<0.05),and the lowest in muscle. There were no significant differences of ACSL4 mRNA relative expression level in the hepatopancreas of grass carp after fed with diets containing different starch sources( P>0.05). However,the highest expression level of ACSL4 mRNA in the hepatopancreas of grass carp was detected after 12 h of starvation and refeeding,which was significantly higher than that at the other time( P<0.05),and then it returned to its beginning level after 24 h of starvation and refeeding. In summary,we have cloned the full-length cDNA of ACSL4 gene from grass carp,and the main functional sites ATP/AMP signature motif and FACS signature motif of amino acid sequences are both highly conserved in different species. Brain and spleen are the main ACSL4 gene expressing tissues in grass carp. Moreover,the ACSL4 gene expression in hepatopancreas of grass carp is not significantly affected by different dietary starch sources,and the highest relative expression level of ACSL4 mRNA in the hepatopancreas of grass carp is detected after 12 h of starvation and refeeding,and then it decreases significantly to its beginning level.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2442-2450]
The full-length cDNA of long chain acyl coenzyme A synthetase 4( ACSL4) gene was cloned from grass carp by reverse-transcription PCR( RT-PCR) technique. Tissue distribution of ACSL4 gene in the brain,heart,spleen,hepatopancreas,muscle,kidney,adipose tissues,anterior intestine,mid-intestine and posterior intestine of grass carp was analyzed by real-time quantitative PCR technique. Meanwhile,the ACSL4 gene expression in the hepatopancreas of grass carp fed with diets containing different starch sources was studied. In addition,the ACSL4 gene expression in the hepatopancreas of grass carp at 0,3,6,12 and 24 h after starvation and refeeding was also investigated. The results showed as follows: the full-length cDNA of ACSL4 gene was2 418 bp with a 2 121 bp open reading frame( ORF) encoding 706 amino acids; the ACSL4 protein contained a trans-membrane domain,a ATP/AMP signature motif and a fatty acyl coenzyme A synthetase( FACS) signature motif. The relative expression level of ACSL4 mRNA in brain and spleen were higher,which were significantly higher than those in the other tissues( P<0.05),and the lowest in muscle. There were no significant differences of ACSL4 mRNA relative expression level in the hepatopancreas of grass carp after fed with diets containing different starch sources( P>0.05). However,the highest expression level of ACSL4 mRNA in the hepatopancreas of grass carp was detected after 12 h of starvation and refeeding,which was significantly higher than that at the other time( P<0.05),and then it returned to its beginning level after 24 h of starvation and refeeding. In summary,we have cloned the full-length cDNA of ACSL4 gene from grass carp,and the main functional sites ATP/AMP signature motif and FACS signature motif of amino acid sequences are both highly conserved in different species. Brain and spleen are the main ACSL4 gene expressing tissues in grass carp. Moreover,the ACSL4 gene expression in hepatopancreas of grass carp is not significantly affected by different dietary starch sources,and the highest relative expression level of ACSL4 mRNA in the hepatopancreas of grass carp is detected after 12 h of starvation and refeeding,and then it decreases significantly to its beginning level.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2442-2450]
引文
[1] PICCINI M,VITELLI F,BRUTTINI M,et al.FACL4,a new gene encoding long-chain acyl-CoA synthetase4,is deleted in a family w ith Alport syndrome,elliptocytosis,and mental retardation[J]. Genomics,1998,47(3):350-358.
[2] WATKINS P A,MAIGUEL D,JIA Z Z,et al. Evidence for 26 distinct acyl-coenzyme a synthetase genes in the human genome[J]. Journal of Lipid Research,2007,48(12):2736-2750.
[3] SOUPENE E,KUYPERS F A.Mammalian long-chain acyl-CoA synthetases[J]. Experimental Biology and M edicine,2008,233(5):507-521.
[4]于莉莉,谭小力,侯文胜.大豆长链脂酰辅酶A合成酶基因GmLACS在酵母中的表达[J].大豆科学,2011,30(5):719-722.
[5] WU M H,LIU H Y,CHEN W,et al.Hepatic expression of long-chain acyl-CoA synthetase 3 is upregulated in hyperlipidemic hamsters[J]. Lipids,2009,44(11):989-998.
[6] LOPES-MARQUES M,CUNHA I,REIS-HENRIQUES M A,et al. Diversity and history of the longchain acyl-CoA synthetase(Acsl)gene family in vertebrates[J]. BM C Evolutionary Biology,2013,13:271.
[7] KANG M J,FUJINO T,SASANO H,et al. A novel arachidonate-preferring acyl-CoA synthetase is present in steroidogenic cells of the rat adrenal,ovary,and testis[J]. Proceedings of the National Academy of Sciences of the United States of America,1997,94(7):2880-2884.
[8] LEWIN T M,VAN HORN C G,KRISANS S K,et al. Rat liver acyl-CoA synthetase 4 is a peripheralmembrane protein located in tw o distinct subcellular organelles,peroxisomes,and mitochondrial-associated membrane[J].Archives of Biochemistry and Biophysics,2002,404(2):263-270.
[9] KOTRONEN A,YKI-JARVINEN H,AMINOFF A,et al.Genetic variation in the ADIPOR2 gene is associated w ith liver fat content and its surrogate markers in three independent cohorts[J].European Journal of Endocrinology,2009,160(4):593-602.
[10] QUINLIVAN V H,FARBER S A. Lipid uptake,metabolism,and transport in the larval zebrafish[J].Frontiers in Endocrinology,2017,8:319
[11] CHENG H L,CHEN S,XU J H,et al.Molecular cloning and nutrient regulation analysis of long chain acyl-CoA synthetase 1 gene in grass carp,Ctenopharyngodon idella L[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and M olecular Biology,2017,204:61-68.
[12]严媛,程汉良,许建和,等.草鱼乙酰辅酶A羧化酶β基因全长cDNA分子克隆与表达分析[J].动物营养学报,2018,30(5):1827-1836.
[13] WEIMAR J D,DIRUSSO C C,DELIO R,et al.Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids. Amino acid residues w ithin the ATP/AM P signature motif of Escherichia coli FadD are required for enzyme activity and fatty acid transport[J]. The Journal of Biological Chemistry,2002,277(33):29369-29376.
[14]郭丽娟.野猪、家猪ACSL4基因的克隆、表达及多态性研究[D].硕士学位论文.哈尔滨:东北农业大学,2008.
[15]王继文,潘志雄,吕佳,等.鹅ACSL4基因克隆及其与肝脂质沉积的关系研究[J].中国畜牧杂志,2012,48(5):6-10.
[16] GOLEJ D L,ASKARI B,KRAMER F,et al. Longchain acyl-CoA synthetase 4 modulates prostaglandin E2release from human arterial smooth muscle cells[J]. Journal of Lipid Research,2011,52(4):782-793.
[17] WU X Y,LI Y R,WANG J H,et al.Long chain fatty acyl-CoA synthetase 4 is a biomarker for and mediator of hormone resistance in human breast cancer[J].PLoS One,2013,8(10):e77060.
[18] MALOBERTI P,CASTILLA R,CASTILLO F,et al.Silencing the expression of mitochondrial acyl-CoA thioesteraseⅠand acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis[J]. The FEBS Journal,2005,272(7):1804-1814.
[19]田丽霞,刘永坚,冯健,等.不同种类淀粉对草鱼生长、肠系膜脂肪沉积和鱼体组成的影响[J].水产学报,2002,26(3):247-251.
[20]郭文英.α-淀粉水平及不同淀粉类型对西伯利亚鲟幼鱼摄食生长和能量收支的影响[D].硕士学位论文.石家庄:河北师范大学,2010.
[21]叶元土,蔡春芳.鱼类营养与饲料配制[M].北京:化学工业出版社,2013:117-118.
[22]张宝龙.饲料中不同水平蛋白、糖、脂肪对鲤鱼生长及相关代谢调控的影响[D].硕士学位论文.天津:天津农学院,2015.
[23] LIN J H,CUI Y B,HUNG S S O,et al.Effect of feeding strategy and carbohydrate source on carbohydrate utilization by w hite sturgeon(Acipenser transmontanus)and hybrid tilapia(Oreochromis niloticus×O.aureus)[J].Aquaculture,1997,148(2/3):201-211.
[24]冯姣.抗性淀粉与OKGM降解产物对齐口裂腹鱼营养成分及脂质代谢相关基因表达的影响[D].硕士学位论文.雅安:四川农业大学,2015.
[25]夏晓杰.多糖对齐口裂腹鱼肌肉品质、脂质代谢及HSP70基因表达的影响[D].硕士学位论文.雅安:四川农业大学,2015.
[26]田娟,涂玮,曾令兵,等.饥饿和再投喂期间尼罗罗非鱼生长、血清生化指标和肝胰脏生长激素、类胰岛素生长因子-Ⅰ和胰岛素mRNA表达丰度的变化[J].水产学报,2012,36(6):900-907.
[27]覃川杰,邵婷,杨洁萍,等.饥饿胁迫对瓦氏黄颡鱼脂肪代谢的影响[J].水生生物学报,2015,39(1):58-65.
[2] WATKINS P A,MAIGUEL D,JIA Z Z,et al. Evidence for 26 distinct acyl-coenzyme a synthetase genes in the human genome[J]. Journal of Lipid Research,2007,48(12):2736-2750.
[3] SOUPENE E,KUYPERS F A.Mammalian long-chain acyl-CoA synthetases[J]. Experimental Biology and M edicine,2008,233(5):507-521.
[4]于莉莉,谭小力,侯文胜.大豆长链脂酰辅酶A合成酶基因GmLACS在酵母中的表达[J].大豆科学,2011,30(5):719-722.
[5] WU M H,LIU H Y,CHEN W,et al.Hepatic expression of long-chain acyl-CoA synthetase 3 is upregulated in hyperlipidemic hamsters[J]. Lipids,2009,44(11):989-998.
[6] LOPES-MARQUES M,CUNHA I,REIS-HENRIQUES M A,et al. Diversity and history of the longchain acyl-CoA synthetase(Acsl)gene family in vertebrates[J]. BM C Evolutionary Biology,2013,13:271.
[7] KANG M J,FUJINO T,SASANO H,et al. A novel arachidonate-preferring acyl-CoA synthetase is present in steroidogenic cells of the rat adrenal,ovary,and testis[J]. Proceedings of the National Academy of Sciences of the United States of America,1997,94(7):2880-2884.
[8] LEWIN T M,VAN HORN C G,KRISANS S K,et al. Rat liver acyl-CoA synthetase 4 is a peripheralmembrane protein located in tw o distinct subcellular organelles,peroxisomes,and mitochondrial-associated membrane[J].Archives of Biochemistry and Biophysics,2002,404(2):263-270.
[9] KOTRONEN A,YKI-JARVINEN H,AMINOFF A,et al.Genetic variation in the ADIPOR2 gene is associated w ith liver fat content and its surrogate markers in three independent cohorts[J].European Journal of Endocrinology,2009,160(4):593-602.
[10] QUINLIVAN V H,FARBER S A. Lipid uptake,metabolism,and transport in the larval zebrafish[J].Frontiers in Endocrinology,2017,8:319
[11] CHENG H L,CHEN S,XU J H,et al.Molecular cloning and nutrient regulation analysis of long chain acyl-CoA synthetase 1 gene in grass carp,Ctenopharyngodon idella L[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and M olecular Biology,2017,204:61-68.
[12]严媛,程汉良,许建和,等.草鱼乙酰辅酶A羧化酶β基因全长cDNA分子克隆与表达分析[J].动物营养学报,2018,30(5):1827-1836.
[13] WEIMAR J D,DIRUSSO C C,DELIO R,et al.Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids. Amino acid residues w ithin the ATP/AM P signature motif of Escherichia coli FadD are required for enzyme activity and fatty acid transport[J]. The Journal of Biological Chemistry,2002,277(33):29369-29376.
[14]郭丽娟.野猪、家猪ACSL4基因的克隆、表达及多态性研究[D].硕士学位论文.哈尔滨:东北农业大学,2008.
[15]王继文,潘志雄,吕佳,等.鹅ACSL4基因克隆及其与肝脂质沉积的关系研究[J].中国畜牧杂志,2012,48(5):6-10.
[16] GOLEJ D L,ASKARI B,KRAMER F,et al. Longchain acyl-CoA synthetase 4 modulates prostaglandin E2release from human arterial smooth muscle cells[J]. Journal of Lipid Research,2011,52(4):782-793.
[17] WU X Y,LI Y R,WANG J H,et al.Long chain fatty acyl-CoA synthetase 4 is a biomarker for and mediator of hormone resistance in human breast cancer[J].PLoS One,2013,8(10):e77060.
[18] MALOBERTI P,CASTILLA R,CASTILLO F,et al.Silencing the expression of mitochondrial acyl-CoA thioesteraseⅠand acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis[J]. The FEBS Journal,2005,272(7):1804-1814.
[19]田丽霞,刘永坚,冯健,等.不同种类淀粉对草鱼生长、肠系膜脂肪沉积和鱼体组成的影响[J].水产学报,2002,26(3):247-251.
[20]郭文英.α-淀粉水平及不同淀粉类型对西伯利亚鲟幼鱼摄食生长和能量收支的影响[D].硕士学位论文.石家庄:河北师范大学,2010.
[21]叶元土,蔡春芳.鱼类营养与饲料配制[M].北京:化学工业出版社,2013:117-118.
[22]张宝龙.饲料中不同水平蛋白、糖、脂肪对鲤鱼生长及相关代谢调控的影响[D].硕士学位论文.天津:天津农学院,2015.
[23] LIN J H,CUI Y B,HUNG S S O,et al.Effect of feeding strategy and carbohydrate source on carbohydrate utilization by w hite sturgeon(Acipenser transmontanus)and hybrid tilapia(Oreochromis niloticus×O.aureus)[J].Aquaculture,1997,148(2/3):201-211.
[24]冯姣.抗性淀粉与OKGM降解产物对齐口裂腹鱼营养成分及脂质代谢相关基因表达的影响[D].硕士学位论文.雅安:四川农业大学,2015.
[25]夏晓杰.多糖对齐口裂腹鱼肌肉品质、脂质代谢及HSP70基因表达的影响[D].硕士学位论文.雅安:四川农业大学,2015.
[26]田娟,涂玮,曾令兵,等.饥饿和再投喂期间尼罗罗非鱼生长、血清生化指标和肝胰脏生长激素、类胰岛素生长因子-Ⅰ和胰岛素mRNA表达丰度的变化[J].水产学报,2012,36(6):900-907.
[27]覃川杰,邵婷,杨洁萍,等.饥饿胁迫对瓦氏黄颡鱼脂肪代谢的影响[J].水生生物学报,2015,39(1):58-65.