山羊CPT1B基因的克隆、表达及其与肌内脂肪含量的相关性分析
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摘要
肌内脂肪含量是动物育种的关键指标,利用分子生物学手段研究脂代谢调控机制是一项重要工作。本研究旨在克隆山羊(Capra hircus)肉碱脂酰转移酶Ⅰ肌肉亚型基因(carnitine palmityl transferase 1B, CPT1B)序列,并利用qRT-PCR技术检测山羊不同组织和肌内前体脂肪细胞中CPT1B基因的表达模式,为深入探讨CPT1B基因在山羊脂肪酸代谢调控中的功能提供实验数据。首先选取1周岁的健康简州大耳羊(Jianzhou Big-eared goat) 7只,采集心、肝、脾、肺、肾、背最长肌、股二头肌、臂三头肌、皮下脂肪和腹间脂肪并保存于液氮中,以索氏提取法检测背最长肌、股二头肌、臂三头肌的肌内脂肪含量;利用Trizol试剂提取上述保存的组织样品总RNA,利用反转录PCR方法克隆CPT1B序列并进行生物信息学分析;利用qRT-PCR方法检测CPT1B基因在山羊各组织的表达,分析其表达水平与肌内脂肪含量的相关性。结果显示,克隆得到山羊CPT1B基因序列2 619 bp (GenBank No. MH340532),包含完整的CDS区2 313 bp、5'UTR序列52 bp、3'UTR序列254 bp,编码771个氨基酸。各组织qRT-PCR结果显示,CPT1B在简州大耳羊心脏、背最长肌、股二头肌和臂三头肌有较高水平的基因表达,在肝脏、脾脏、肺脏、肾脏、皮下脂肪和腹间脂肪的表达水平较低;肌内前体脂肪细胞的检测结果显示,前体脂肪细胞分化期间的CPT1B表达量均极显著高于分化前和分化后(P<0.01);相关性分析显示,CPT1B基因表达量与各肌肉组织肌内脂肪含量均显著正相关(P<0.05)。综上所述,CPT1B可能在山羊肌内脂肪沉积过程中具有调控作用,上述结果为CPT1B基因在肉用山羊育种中的应用提供了基础资料。
Intramuscular fat content is a key indicator of animal breeding. It is an important task to study the regulation mechanism of lipid metabolism by molecular biology. The present study aims to clone the carnitine palmityl transferase 1 B gene(CPT1 B) and determine its expression in various tissues and intramuscular precursor adipose cells of goats(Capra hircus), which will lay the foundation for further study of CPT1 B gene in regulating goat fatty acid metabolism. Seven healthy Jianzhou Big-eared goats aged 1 year old were selected, and the tissue samples of heart, liver, spleen, lung, kidney, back longissimus dorsi, biceps femoris,triceps, subcutaneous fat and abdominal fat were quickly collected and stored in liquid nitrogen. The intramuscular fat contents of back longissimus dorsi, biceps femoris, triceps were detected by Soxhlet extraction method. The total RNA of the above-mentioned tissue samples was extracted using Trizol reagent and the sequence of CPT1 B was cloned by RT-PCR following by the sequence bioinformatics analysis. The expression of CPT1 B in various muscle tissues were determined by qRT-PCR method, which were then used for correlation analysis with the intramuscular fat content. The results showed that the goat sequence was 2619 bp(GenBank No. MH340532), including 2 313 bp CDS, 52 bp 5'UTR and 254 bp 3' UTR, encoding 771 amino acid residues. Tissue quantification results showed that CPT1 B was expressed higher in heart,longissimus dorsi, biceps femoris and tricep, and expressed lower in liver, spleen, lung, kidney, subcutaneous fat and abdominal fat of Jianzhou Big-eared goat. The qRT-PCR results of intramuscular precursor adipose cells showed that the expression of CPT1 B during differentiation of preadipocytes was significantly higher than that before and after differentiation(P<0.01). The expression of CPT1 B gene was significantly positively correlated with the intramuscular fat content in all the 3 muscle tissues(P<0.05). In summary,CPT1 B might have a regulatory role in goat intramuscular fat deposition, which also lays a foundation for the application of CPT1 B gene in meat goat breeding.
Intramuscular fat content is a key indicator of animal breeding. It is an important task to study the regulation mechanism of lipid metabolism by molecular biology. The present study aims to clone the carnitine palmityl transferase 1 B gene(CPT1 B) and determine its expression in various tissues and intramuscular precursor adipose cells of goats(Capra hircus), which will lay the foundation for further study of CPT1 B gene in regulating goat fatty acid metabolism. Seven healthy Jianzhou Big-eared goats aged 1 year old were selected, and the tissue samples of heart, liver, spleen, lung, kidney, back longissimus dorsi, biceps femoris,triceps, subcutaneous fat and abdominal fat were quickly collected and stored in liquid nitrogen. The intramuscular fat contents of back longissimus dorsi, biceps femoris, triceps were detected by Soxhlet extraction method. The total RNA of the above-mentioned tissue samples was extracted using Trizol reagent and the sequence of CPT1 B was cloned by RT-PCR following by the sequence bioinformatics analysis. The expression of CPT1 B in various muscle tissues were determined by qRT-PCR method, which were then used for correlation analysis with the intramuscular fat content. The results showed that the goat sequence was 2619 bp(GenBank No. MH340532), including 2 313 bp CDS, 52 bp 5'UTR and 254 bp 3' UTR, encoding 771 amino acid residues. Tissue quantification results showed that CPT1 B was expressed higher in heart,longissimus dorsi, biceps femoris and tricep, and expressed lower in liver, spleen, lung, kidney, subcutaneous fat and abdominal fat of Jianzhou Big-eared goat. The qRT-PCR results of intramuscular precursor adipose cells showed that the expression of CPT1 B during differentiation of preadipocytes was significantly higher than that before and after differentiation(P<0.01). The expression of CPT1 B gene was significantly positively correlated with the intramuscular fat content in all the 3 muscle tissues(P<0.05). In summary,CPT1 B might have a regulatory role in goat intramuscular fat deposition, which also lays a foundation for the application of CPT1 B gene in meat goat breeding.
引文
李倩,林亚秋,朱江江,等.2017.山羊FGF21基因克隆及其在肌内脂肪细胞中的表达模式研究[J].畜牧兽医学报,48(1):31-38.(Li Q,Lin Y Q,Zhu J J,et al.2017.Cloning of goat FGF21 gene and its expression pattern in intramuscular adipocyte[J].Acta Veterinaria et Zootechnica Sinica,48(1):31-38.)
陶璇,顾以韧,梁艳,等.2017.藏猪和杜洛克猪SCD、CPT1B基因表达差异及其与IMF含量相关性分析[J].黑龙江畜牧兽医,(13):82-85.(Tao X,Gu Y R,Liang Y,et al.2017.Differential expression of SCD and CPT1B genes and their correlation analysis with IMF contents in Tibet and Duroc pigs[J].Heilongjiang Animal Science and Veterinary Medicine,(13):82-85.)
王珍.2016.不同强度跑台运动对大鼠心肌细胞糖脂代谢关键因子的影响[D].硕士学位论文,河北医科大学,导师:曹雪滨,pp.25-27.(Wang Z.2016.The effect of different intensity of treadmill exercise on mitochondrial glucose and fatty acid oxidation key factors in rats'myocytes[D].Thesis for M.S.,Hebei Medical University,Supervisor:Cao X B,pp.25-27.)
许晴,林森,朱江江,等.2018.山羊肌内前体脂肪细胞诱导分化过程中内参基因的表达稳定性分析[J].畜牧兽医学报,49(05):907-918.(Xu Q,Lin S,Zhu J J,et al.2018.The expression stability analysis of reference genes in the process of goat intramuscular preadipocytes differentiation in goat[J].Acta Veterinaria et Zootechnica Sinica,49(05):907-918.)
杨家大,陈祥,龙威海等.2015.放牧山羊CPT1B基因mRNA的组织分布研究[J].上海农业学报,31(03):53-57.(Yang J D,Chen X,Long H W,et al.2015.Study on the tissue distribution of CPT1B mRNA in grazing goats[J].Acta Agriculturae Shanghai,31(03):53-57.)
于宝莉.2012.CPT基因在绵羊和山羊不同肌肉组织的表达分析[D].硕士学位论文,内蒙古农业大学,导师:李金泉,pp.25-28.(Yu B L.2012.CPT gene expression and analysis in muscle tissue of goat and sheep[D].Thesis for M.S.,Inner Mongolia Agricultural University,Supervisor:Li J Q,pp.25-28.)
张艳芳.2010.CPT1基因对猪脂肪沉积的影响及其调控机制的研究[D].博士学位论文,浙江大学,导师:汪以真,pp.55-98.(Zhang Y F.2010.Effect of CPT1 gene on porcine fat deposition and its molecular mechanism[D].Thesis for M.S.,Zhejiang University,Supervisor:Wang Y Z,pp.55-98.)
张永峰.2016.成纤维细胞因子21(FGF21)对山羊成熟脂肪细胞代谢的调控作用[D].硕士学位论文,四川农业大学,导师:李利,pp.13.(Zhang Y F.2016.The regulation mechanism of fibroblast growth factor 21 on lipid metabolism in fully differentiated adipocyte in goat[D].Thesis for M.S.,Sichuan Agricultural University,Supervisor:Li L,pp:13.)
朱武政,林亚秋,江明锋,等.2016.肉用山羊脂代谢相关基因与肌内脂肪含量的相关性分析[J].畜牧兽医学报,47(07):1333-1341.(Zhu W Z,Lin Y Q,Jiang M F,et al.2016.Association analysis of intramuscular fat content and the expression of genes related to lipid metabolism in meat goat[J].Acta Veterinaria et Zootechnica Sinica,47(07):1333-1341.)
Bartelds B,Takens J,Smid G B,et al.2004.Myocardial carnitine palmitoyl transferaseⅠexpression and long-chain fatty acid oxidation in fetal and newborn lambs[J].American Journal of Physiology-Heart and Circulatory Physiology,286(6):2243-2248.
Bonnet M,Bernard L,Bes S,et al.2013.Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue,muscle,liver and mammary gland from ruminants[J].Animal,7(8):1344-1353.
Britton C H,Mackey D W,Esser V,et al.1997.Fine chromosome mapping of the genes for human liver and muscle carnitine palmitoyltransferaseⅠ(CPT1A and CPT1B)[J].Genomics,40(1):209-211.
Bruce C R,Hoy A J,Turner N,et al.2009.Overexpression of carnitine palmitoyltransferase-1 in skeletal muscle is sufficient to enhance fatty acid oxidation and improve high-fat diet-induced insulin resistance[J].Diabetes,58(3):550-558.
Eaton S,Fukumoto K,Paladio D N,et al.2001.Carnitine palmitoyl transferaseⅠand the control of myocardial betaoxidation flux[J].Biochemical Society Transactions,29(2):245-249.
Gregoire F M,Smas C M,Sul H S.1998.Understanding adipocyte differentiation[J].Physiological Reviews,78(3):783-809.
Jensen C H,Teisner B,H?jrup P,et al.1993.Studies on the isolation,structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal-specific cDNA,pG2[J].Human Reproduction,8(4):635-641.
Lyvers P P,Lin X,Jacobi S K,et al.2007.Ontogeny of carnitine palmitoyl transferaseⅠactivity,carnitine-Km,and mRNA abundance in pigs throughout growth and development[J].Journal of Nutrition,137(4):898-903.
MascaróC,Acosta E,Ortiz J A,et al.1999.Characterization of a response element for peroxisomal proliferator activated receptor(PPRE)in human muscle-type carnitine palmitoyltransferaseⅠ[J].Advances in Experimental Medicine and Biology,466:79-85.
McGarry J D,Brown N F.2000.Reconstitution of purified,active and malonyl-CoA-sensitive rat liver carnitine palmitoyltransferaseⅠ:Relationship between membrane environment and malonyl-CoA sensitivity[J].Biochemical Journal,349(Pt 1):179-187.
Mcmanus B L,Korpela R,Speakman J R,et al.2015.Bovine serum albumin as the dominant form of dietary protein reduces subcutaneous fat mass,plasma leptin and plasma corticosterone in high fat-fed C57/BL6J mice[J].British Journal of Nutrition,114(4):654-662.
Miljkovic I,Yerges Lm,Li H,et al.2009.Association of the CPT1B gene with skeletal muscle fat infiltration in AfroCaribbean men[J].Obesity,17(7):1396-1401.
Morash A J,Mcclelland G B.2011.Regulation of carnitine palmitoyltransferase(CPT)I during fasting in rainbow trout(Oncorhynchus mykiss)promotes increased mitochondrial fatty acid oxidation[J].Physiological&Biochemical Zoology,84(6):625-633.
SebastiáN D,Guitart M,GarcíA-MartíNez C,et al.2009.Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells[J].Journal of Lipid Research,50(9):1789-1799.
Sepa-Kishi D M,Wu M V,Uthayakumar A,et al.2016.Antilipolytic and anti-lipogenic effects of the CPT-1b inhibitor oxfenicine in the white adipose tissue of rats[J].American Journal of Physiology-Regulatory Integrative and Comparative Physiology,311(4):779-787.
van der Leij F R,Cox K B,Jackson V N,et al.2002.Structural and functional genomics of the CPT1B gene for muscle-type carnitine palmitoyltransferaseⅠin mammals[J].Journal of Biological Chemistry,277(30):26994-27005.
van der Leij F R,Kram A M,Bartelds B,et al.1999.Cytological evidence that the C-terminus of carnitine palmitoyltransferaseⅠis on the cytosolic face of the mitochondrial outer membrane[J].Biochemical Journal,341(3):777-784.
Warfel J D,Vandanmagsar B,Dubuisson O S,et al.2017.Examination of carnitine palmitoyl transferase 1 abundance in white adipose tissue:Implications in obesity research[J].American Journal of Physiology-Regulatory Integrative and Comparative Physiology,312(5):816-820.
Yamazaki N,Shinohara Y,Shima A,et al.1996.Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferaseⅠ[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,1307(2):157-161.
Zhao S M,Ren L J,Chen L,et al.2009.Differential expression of lipid metabolism related genes in porcine muscle tissue leading to different intramuscular fat deposition[J].Lipids,44(11):1029-1037.
陶璇,顾以韧,梁艳,等.2017.藏猪和杜洛克猪SCD、CPT1B基因表达差异及其与IMF含量相关性分析[J].黑龙江畜牧兽医,(13):82-85.(Tao X,Gu Y R,Liang Y,et al.2017.Differential expression of SCD and CPT1B genes and their correlation analysis with IMF contents in Tibet and Duroc pigs[J].Heilongjiang Animal Science and Veterinary Medicine,(13):82-85.)
王珍.2016.不同强度跑台运动对大鼠心肌细胞糖脂代谢关键因子的影响[D].硕士学位论文,河北医科大学,导师:曹雪滨,pp.25-27.(Wang Z.2016.The effect of different intensity of treadmill exercise on mitochondrial glucose and fatty acid oxidation key factors in rats'myocytes[D].Thesis for M.S.,Hebei Medical University,Supervisor:Cao X B,pp.25-27.)
许晴,林森,朱江江,等.2018.山羊肌内前体脂肪细胞诱导分化过程中内参基因的表达稳定性分析[J].畜牧兽医学报,49(05):907-918.(Xu Q,Lin S,Zhu J J,et al.2018.The expression stability analysis of reference genes in the process of goat intramuscular preadipocytes differentiation in goat[J].Acta Veterinaria et Zootechnica Sinica,49(05):907-918.)
杨家大,陈祥,龙威海等.2015.放牧山羊CPT1B基因mRNA的组织分布研究[J].上海农业学报,31(03):53-57.(Yang J D,Chen X,Long H W,et al.2015.Study on the tissue distribution of CPT1B mRNA in grazing goats[J].Acta Agriculturae Shanghai,31(03):53-57.)
于宝莉.2012.CPT基因在绵羊和山羊不同肌肉组织的表达分析[D].硕士学位论文,内蒙古农业大学,导师:李金泉,pp.25-28.(Yu B L.2012.CPT gene expression and analysis in muscle tissue of goat and sheep[D].Thesis for M.S.,Inner Mongolia Agricultural University,Supervisor:Li J Q,pp.25-28.)
张艳芳.2010.CPT1基因对猪脂肪沉积的影响及其调控机制的研究[D].博士学位论文,浙江大学,导师:汪以真,pp.55-98.(Zhang Y F.2010.Effect of CPT1 gene on porcine fat deposition and its molecular mechanism[D].Thesis for M.S.,Zhejiang University,Supervisor:Wang Y Z,pp.55-98.)
张永峰.2016.成纤维细胞因子21(FGF21)对山羊成熟脂肪细胞代谢的调控作用[D].硕士学位论文,四川农业大学,导师:李利,pp.13.(Zhang Y F.2016.The regulation mechanism of fibroblast growth factor 21 on lipid metabolism in fully differentiated adipocyte in goat[D].Thesis for M.S.,Sichuan Agricultural University,Supervisor:Li L,pp:13.)
朱武政,林亚秋,江明锋,等.2016.肉用山羊脂代谢相关基因与肌内脂肪含量的相关性分析[J].畜牧兽医学报,47(07):1333-1341.(Zhu W Z,Lin Y Q,Jiang M F,et al.2016.Association analysis of intramuscular fat content and the expression of genes related to lipid metabolism in meat goat[J].Acta Veterinaria et Zootechnica Sinica,47(07):1333-1341.)
Bartelds B,Takens J,Smid G B,et al.2004.Myocardial carnitine palmitoyl transferaseⅠexpression and long-chain fatty acid oxidation in fetal and newborn lambs[J].American Journal of Physiology-Heart and Circulatory Physiology,286(6):2243-2248.
Bonnet M,Bernard L,Bes S,et al.2013.Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue,muscle,liver and mammary gland from ruminants[J].Animal,7(8):1344-1353.
Britton C H,Mackey D W,Esser V,et al.1997.Fine chromosome mapping of the genes for human liver and muscle carnitine palmitoyltransferaseⅠ(CPT1A and CPT1B)[J].Genomics,40(1):209-211.
Bruce C R,Hoy A J,Turner N,et al.2009.Overexpression of carnitine palmitoyltransferase-1 in skeletal muscle is sufficient to enhance fatty acid oxidation and improve high-fat diet-induced insulin resistance[J].Diabetes,58(3):550-558.
Eaton S,Fukumoto K,Paladio D N,et al.2001.Carnitine palmitoyl transferaseⅠand the control of myocardial betaoxidation flux[J].Biochemical Society Transactions,29(2):245-249.
Gregoire F M,Smas C M,Sul H S.1998.Understanding adipocyte differentiation[J].Physiological Reviews,78(3):783-809.
Jensen C H,Teisner B,H?jrup P,et al.1993.Studies on the isolation,structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal-specific cDNA,pG2[J].Human Reproduction,8(4):635-641.
Lyvers P P,Lin X,Jacobi S K,et al.2007.Ontogeny of carnitine palmitoyl transferaseⅠactivity,carnitine-Km,and mRNA abundance in pigs throughout growth and development[J].Journal of Nutrition,137(4):898-903.
MascaróC,Acosta E,Ortiz J A,et al.1999.Characterization of a response element for peroxisomal proliferator activated receptor(PPRE)in human muscle-type carnitine palmitoyltransferaseⅠ[J].Advances in Experimental Medicine and Biology,466:79-85.
McGarry J D,Brown N F.2000.Reconstitution of purified,active and malonyl-CoA-sensitive rat liver carnitine palmitoyltransferaseⅠ:Relationship between membrane environment and malonyl-CoA sensitivity[J].Biochemical Journal,349(Pt 1):179-187.
Mcmanus B L,Korpela R,Speakman J R,et al.2015.Bovine serum albumin as the dominant form of dietary protein reduces subcutaneous fat mass,plasma leptin and plasma corticosterone in high fat-fed C57/BL6J mice[J].British Journal of Nutrition,114(4):654-662.
Miljkovic I,Yerges Lm,Li H,et al.2009.Association of the CPT1B gene with skeletal muscle fat infiltration in AfroCaribbean men[J].Obesity,17(7):1396-1401.
Morash A J,Mcclelland G B.2011.Regulation of carnitine palmitoyltransferase(CPT)I during fasting in rainbow trout(Oncorhynchus mykiss)promotes increased mitochondrial fatty acid oxidation[J].Physiological&Biochemical Zoology,84(6):625-633.
SebastiáN D,Guitart M,GarcíA-MartíNez C,et al.2009.Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells[J].Journal of Lipid Research,50(9):1789-1799.
Sepa-Kishi D M,Wu M V,Uthayakumar A,et al.2016.Antilipolytic and anti-lipogenic effects of the CPT-1b inhibitor oxfenicine in the white adipose tissue of rats[J].American Journal of Physiology-Regulatory Integrative and Comparative Physiology,311(4):779-787.
van der Leij F R,Cox K B,Jackson V N,et al.2002.Structural and functional genomics of the CPT1B gene for muscle-type carnitine palmitoyltransferaseⅠin mammals[J].Journal of Biological Chemistry,277(30):26994-27005.
van der Leij F R,Kram A M,Bartelds B,et al.1999.Cytological evidence that the C-terminus of carnitine palmitoyltransferaseⅠis on the cytosolic face of the mitochondrial outer membrane[J].Biochemical Journal,341(3):777-784.
Warfel J D,Vandanmagsar B,Dubuisson O S,et al.2017.Examination of carnitine palmitoyl transferase 1 abundance in white adipose tissue:Implications in obesity research[J].American Journal of Physiology-Regulatory Integrative and Comparative Physiology,312(5):816-820.
Yamazaki N,Shinohara Y,Shima A,et al.1996.Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferaseⅠ[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,1307(2):157-161.
Zhao S M,Ren L J,Chen L,et al.2009.Differential expression of lipid metabolism related genes in porcine muscle tissue leading to different intramuscular fat deposition[J].Lipids,44(11):1029-1037.