豫西脂尾羊CIDEa基因的克隆、序列分析与组织表达
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摘要
试验旨在对豫西脂尾羊诱导细胞凋亡DNA片段化因子45样效应因子A(cell death-inducing DNA fragmentation factor 45-like effectors A,CIDEa)基因进行克隆及生物信息学分析,并检测其在豫西脂尾羊不同组织中的表达。以豫西脂尾羊脂肪组织RNA为模板,采用RT-PCR方法扩增并克隆豫西脂尾羊CIDEa基因完整CDS区序列,对测序结果进行了相关生物信息学分析,并对CIDEa基因在豫西脂尾羊心脏、肝脏、肺脏、脾脏、肾脏、瘤胃、小肠、背最长肌、皮下和内脏脂肪组织中的表达进行了分析。结果显示,豫西脂尾羊CIDEa基因CDS区序列长660 bp,编码219个氨基酸;豫西脂尾羊与绵羊、水牛、黄牛、藏羚羊、猪和人的CIDEa基因同源性分别为99.1%、96.8%、96.2%、98.8%、85.0%和79.4%。蛋白理化性质分析表明,CIDEa蛋白分子质量为24.38 ku,理论等电点(pI)为9.12,属于碱性蛋白。跨膜结构和信号肽预测分析表明,CIDEa蛋白不含跨膜结构和信号肽。亚细胞定位分析表明,豫西脂尾羊CIDEa蛋白分布在细胞质(47.8%)、线粒体(26.1%)、细胞核(17.4%)、液泡(4.3%)和内质网(4.3%)。蛋白质三级结构预测发现,CIDEa蛋白结构具有2个α-螺旋、4个β-折叠及一些无规则卷曲。实时荧光定量PCR结果表明,CIDEa基因在豫西脂尾羊脂肪组织(皮下和内脏脂肪组织)中表达量较高,其他组织中表达量从高到低依次为小肠、肝脏、脾脏、瘤胃、心脏、肾脏、肺脏、背最长肌。这些结果可为进一步研究CIDEa基因在肉羊脂质代谢中的功能及羊肉品质调控提供基础资料。
This study was aimed to clone cell death-inducing DNA fragmentation factor 45-like effector A(CIDEa),analyze the sequences by bioinformatics software,and detect the expression of CIDEa gene in different tissues of Yuxi fat-tailed sheep.Using RNA of the adipose tissue in Yuxi fat-tailed sheep as a template,the complete CDS sequence of CIDEa gene in Yuxi fat-tailed sheep was amplified and cloned,and the sequencing result was analyzed by bioinformatics.The expression of CIDEa gene in heart,liver,lung,spleen,kidney,rumen,small intestine,longissimus dorsal muscle,subcutaneous adipose and visceral adipose tissue of Yuxi fat-tailed sheep were analyzed.The results showed that the length of CDS sequence of CIDEa gene in Yuxi fat-tailed sheep was 660 bp and encoded 219 amino acids.The homology of CIDEa gene in Yuxi fat-tailed sheep were 99.1%,96.8%,96.2%,98.8%,85.0% and 79.4% with Ovis aries,Bubalus bubalis,Bos taurus,Pantholops hodgsonii,Sus scrofa and Homo sapiens,respectively.The physical and chemical properties of CIDEa protein showed that the molecular weight was 24.38 ku and the theoretical isoelectric point(pI) was 9.12,which belonged to alkaline protein.There was no transmembrane structure and signal peptide in CIDEa protein.The subcellular localization results showed that CIDEa protein was located in the cytoplasm(47.8%),mitochondria(26.1%),nuclear(17.4%),vacuolar(4.3%) and endoplasmic reticulum(4.3%).The tertiary structure of CIDEa protein mainly consisted of 2 alpha helices,4 beta foldings and random coils.Real-time quantitative PCR results showed that the expression of CIDEa gene in subcutaneous adipose and visceral adipose were obviously higher than other tissues,which followed by intestine,liver,spleen,rumen,heart,kidney,lung and longissimus dorsi muscle ranged from high to low.This results provided basic data for further study on the function of CIDEa gene in lipid metabolism and regulation of mutton quality in sheep.
This study was aimed to clone cell death-inducing DNA fragmentation factor 45-like effector A(CIDEa),analyze the sequences by bioinformatics software,and detect the expression of CIDEa gene in different tissues of Yuxi fat-tailed sheep.Using RNA of the adipose tissue in Yuxi fat-tailed sheep as a template,the complete CDS sequence of CIDEa gene in Yuxi fat-tailed sheep was amplified and cloned,and the sequencing result was analyzed by bioinformatics.The expression of CIDEa gene in heart,liver,lung,spleen,kidney,rumen,small intestine,longissimus dorsal muscle,subcutaneous adipose and visceral adipose tissue of Yuxi fat-tailed sheep were analyzed.The results showed that the length of CDS sequence of CIDEa gene in Yuxi fat-tailed sheep was 660 bp and encoded 219 amino acids.The homology of CIDEa gene in Yuxi fat-tailed sheep were 99.1%,96.8%,96.2%,98.8%,85.0% and 79.4% with Ovis aries,Bubalus bubalis,Bos taurus,Pantholops hodgsonii,Sus scrofa and Homo sapiens,respectively.The physical and chemical properties of CIDEa protein showed that the molecular weight was 24.38 ku and the theoretical isoelectric point(pI) was 9.12,which belonged to alkaline protein.There was no transmembrane structure and signal peptide in CIDEa protein.The subcellular localization results showed that CIDEa protein was located in the cytoplasm(47.8%),mitochondria(26.1%),nuclear(17.4%),vacuolar(4.3%) and endoplasmic reticulum(4.3%).The tertiary structure of CIDEa protein mainly consisted of 2 alpha helices,4 beta foldings and random coils.Real-time quantitative PCR results showed that the expression of CIDEa gene in subcutaneous adipose and visceral adipose were obviously higher than other tissues,which followed by intestine,liver,spleen,rumen,heart,kidney,lung and longissimus dorsi muscle ranged from high to low.This results provided basic data for further study on the function of CIDEa gene in lipid metabolism and regulation of mutton quality in sheep.
引文
[1] XU L,ZHOU L,LI P.CIDE proteins and lipid metabolism[J].Arteriosclerosis Thrombosis and Vascular Biology,2012,32(5):1094-1098.
[2] XU W Y,WU L Z,YU M,et al.Differential roles of cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins in promoting lipid droplet fusion and growth in subpopulations of hepatocytes[J].Journal of Biological Chemistry,2016,291(9):4282-4293.
[3] ZHOU Z,TOH S Y,CHEN Z M,et al.Cidea-deficient mice have lean phenotype and are resistant to obesity[J].Nature Genetics,2003,35(1):49-56.
[4] QI J,GONG J,ZHAO T,et al.Downregulation of AMP-activated protein kinase by Cidea-mediated ubiquitination and degradation in brown adipose tissue[J].EMBO Journal,2014,27(11):1537-1548.
[5] 龚婧怡.Cidea以及Fsp27在脂肪代谢中的功能以及分子机制[D].北京:清华大学,2010.GONG J Y.Function and molecular mechanism of Cidea and Fsp27 in lipid metabolism[D].Beijing:Tsinghua University,2010.(in Chinese)
[6] 王卫林,韩向敏,权凯.‘豫西脂尾羊’屠宰性能及肉质分析[J].甘肃农业大学学报,2018,53(2):37-42.WANG W L,HAN X M,QUAN K.Slaughter performance and meat quality of Yuxi fat-tailed sheep[J].Journal of Gansu Agricultural University,2018,53(2):37-42.(in Chinese)
[7] 梁莎莎,庞春英,邓廷贤,等.奶水牛固醇携带蛋白2基因克隆、生物信息学分析及其组织表达检测[J].中国畜牧兽医,2018,45(9):2358-2367.LIANG S S,PANG C Y,DENG T X,et al.Cloning,bioinformatics and tissue expression analysis of SCP2 gene in dairy buffalo[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2358-2367.(in Chinese)
[8] 黄韵琪,邓廷贤,唐辉,等.水牛SND1基因克隆及生物信息学分析[J].中国畜牧兽医,2018,45(7):1759-1767.HUANG Y Q,DENG T X,TANG H,et al.Cloning and bioinformatics analysis of SND1 gene in buffalo[J].China Animal Husbandry & Veterinary Medicine,2018,45(7):1759-1767.(in Chinese)
[9] BONNET M,BERNARD L,BES S,et al.Selection of reference genes for quantitative Real-time PCR normalisation in adipose tissue,muscle,liver and mammary gland from ruminants[J].Animal,2013,7(8):1344-1353.
[10] 赵东宇,郁苗,汪文敏,等.Cidea和Cidec促进肝脏中脂类的积累[J].现代生物医学进展,2018,18(5):801-806.ZHAO D Y,YU M,WANG W M,et al.Cidea and Cidec promote the lipid storage in liver[J].Progress in Modern Biomedicine,2018,18(5):801-806.(in Chinese)
[11] HE Q,DIAO Y,ZHAO T,et al.SREBP1c mediates the effect of acetaldehyde on Cidea expression in alcoholic fatty liver mice[J].Scientific Reports,2018,8(1):1200-1210.
[12] ABREU-VIEIRA G,FISCHER AW,MATTSSON C,et al.Cidea improves the metabolic profile through expansion of adipose tissue[J].Nature Communications,2015,6:7433.
[13] CHEN Z,GUO K,TOH S Y,et al.Mitochondria localization and dimerization are required for CIDE-B to induce apoptosis[J].Journal of Biological Chemistry,2000,275(30):22619-22622.
[14] MONTASTIER E,DEJEAN S,GALL C L,et al.Adipose tissue CIDEA is associated,independently of weight variation,to change in insulin resistance during a longitudinal weight control dietary program in obese individuals[J].PLoS One,2014,9(7):e98707.
[15] NISHIMOTO Y,TAMORI Y.CIDE family-mediated unique lipid droplet morphology in white adipose tissue and brown adipose tissue determines the adipocyte energy metabolism[J].Journal of Atherosclerosis & Thrombosis,2017,24(10):989-998.
[16] NIELSEN H,ENGELBRECHT J,BRUNAK S,et al.Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites[J].Protein Engineering,1997,10(1):1-6.
[17] NIELSEN H,KROGH A.Prediction of signal peptides and signal anchors by a hidden Markov model[A].Proceedings/International conference on Intelligent Systems for Molecular Biology[C].1998.
[18] BOSTROM P,ANDERSSON L,RUTBERG M,et al.SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity[J].Nature Cell Biology,2007,9(11):1286-1293.
[19] WANG W,LV N,ZHANG S,et al.Cidea is an essential transcriptional coactivator regulating mammary gland secretion of milk lipids[J].Nature Medicine,2012,18(2):235-243.
[20] CHRISTIANSON J L,BOUTET E,PURI V,et al.Identification of the lipid droplet targeting domain of the Cidea protein[J].Journal of Lipid Research,2010,51(12):3455-3462.
[21] 李艳华.猪CIDE家族基因的克隆及功能研究[D].武汉:华中农业大学,2008.LI Y H.Cloning and functional research of porcine CIDEs family[D].Wuhan:Huazhong Agricultural University,2008.(in Chinese)
[22] 黄柳梅.CIDEa和CIDEc基因在肉鸡中的表达分析[D].南昌:江西农业大学,2016.HUANG L M.Expression analysis of CIDEa and CIDEc genes in broiler chickens[D].Nanchang:Jiangxi Agricultural University,2016.(in Chinese)
[23] LI Y H,LEI T,CHEN X D,et al.Molecular cloning,chromosomal location and expression pattern of porcine CIDEa,and CIDEc[J].Molecular Biology Reports,2009,36(3):575-582.
[2] XU W Y,WU L Z,YU M,et al.Differential roles of cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins in promoting lipid droplet fusion and growth in subpopulations of hepatocytes[J].Journal of Biological Chemistry,2016,291(9):4282-4293.
[3] ZHOU Z,TOH S Y,CHEN Z M,et al.Cidea-deficient mice have lean phenotype and are resistant to obesity[J].Nature Genetics,2003,35(1):49-56.
[4] QI J,GONG J,ZHAO T,et al.Downregulation of AMP-activated protein kinase by Cidea-mediated ubiquitination and degradation in brown adipose tissue[J].EMBO Journal,2014,27(11):1537-1548.
[5] 龚婧怡.Cidea以及Fsp27在脂肪代谢中的功能以及分子机制[D].北京:清华大学,2010.GONG J Y.Function and molecular mechanism of Cidea and Fsp27 in lipid metabolism[D].Beijing:Tsinghua University,2010.(in Chinese)
[6] 王卫林,韩向敏,权凯.‘豫西脂尾羊’屠宰性能及肉质分析[J].甘肃农业大学学报,2018,53(2):37-42.WANG W L,HAN X M,QUAN K.Slaughter performance and meat quality of Yuxi fat-tailed sheep[J].Journal of Gansu Agricultural University,2018,53(2):37-42.(in Chinese)
[7] 梁莎莎,庞春英,邓廷贤,等.奶水牛固醇携带蛋白2基因克隆、生物信息学分析及其组织表达检测[J].中国畜牧兽医,2018,45(9):2358-2367.LIANG S S,PANG C Y,DENG T X,et al.Cloning,bioinformatics and tissue expression analysis of SCP2 gene in dairy buffalo[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2358-2367.(in Chinese)
[8] 黄韵琪,邓廷贤,唐辉,等.水牛SND1基因克隆及生物信息学分析[J].中国畜牧兽医,2018,45(7):1759-1767.HUANG Y Q,DENG T X,TANG H,et al.Cloning and bioinformatics analysis of SND1 gene in buffalo[J].China Animal Husbandry & Veterinary Medicine,2018,45(7):1759-1767.(in Chinese)
[9] BONNET M,BERNARD L,BES S,et al.Selection of reference genes for quantitative Real-time PCR normalisation in adipose tissue,muscle,liver and mammary gland from ruminants[J].Animal,2013,7(8):1344-1353.
[10] 赵东宇,郁苗,汪文敏,等.Cidea和Cidec促进肝脏中脂类的积累[J].现代生物医学进展,2018,18(5):801-806.ZHAO D Y,YU M,WANG W M,et al.Cidea and Cidec promote the lipid storage in liver[J].Progress in Modern Biomedicine,2018,18(5):801-806.(in Chinese)
[11] HE Q,DIAO Y,ZHAO T,et al.SREBP1c mediates the effect of acetaldehyde on Cidea expression in alcoholic fatty liver mice[J].Scientific Reports,2018,8(1):1200-1210.
[12] ABREU-VIEIRA G,FISCHER AW,MATTSSON C,et al.Cidea improves the metabolic profile through expansion of adipose tissue[J].Nature Communications,2015,6:7433.
[13] CHEN Z,GUO K,TOH S Y,et al.Mitochondria localization and dimerization are required for CIDE-B to induce apoptosis[J].Journal of Biological Chemistry,2000,275(30):22619-22622.
[14] MONTASTIER E,DEJEAN S,GALL C L,et al.Adipose tissue CIDEA is associated,independently of weight variation,to change in insulin resistance during a longitudinal weight control dietary program in obese individuals[J].PLoS One,2014,9(7):e98707.
[15] NISHIMOTO Y,TAMORI Y.CIDE family-mediated unique lipid droplet morphology in white adipose tissue and brown adipose tissue determines the adipocyte energy metabolism[J].Journal of Atherosclerosis & Thrombosis,2017,24(10):989-998.
[16] NIELSEN H,ENGELBRECHT J,BRUNAK S,et al.Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites[J].Protein Engineering,1997,10(1):1-6.
[17] NIELSEN H,KROGH A.Prediction of signal peptides and signal anchors by a hidden Markov model[A].Proceedings/International conference on Intelligent Systems for Molecular Biology[C].1998.
[18] BOSTROM P,ANDERSSON L,RUTBERG M,et al.SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity[J].Nature Cell Biology,2007,9(11):1286-1293.
[19] WANG W,LV N,ZHANG S,et al.Cidea is an essential transcriptional coactivator regulating mammary gland secretion of milk lipids[J].Nature Medicine,2012,18(2):235-243.
[20] CHRISTIANSON J L,BOUTET E,PURI V,et al.Identification of the lipid droplet targeting domain of the Cidea protein[J].Journal of Lipid Research,2010,51(12):3455-3462.
[21] 李艳华.猪CIDE家族基因的克隆及功能研究[D].武汉:华中农业大学,2008.LI Y H.Cloning and functional research of porcine CIDEs family[D].Wuhan:Huazhong Agricultural University,2008.(in Chinese)
[22] 黄柳梅.CIDEa和CIDEc基因在肉鸡中的表达分析[D].南昌:江西农业大学,2016.HUANG L M.Expression analysis of CIDEa and CIDEc genes in broiler chickens[D].Nanchang:Jiangxi Agricultural University,2016.(in Chinese)
[23] LI Y H,LEI T,CHEN X D,et al.Molecular cloning,chromosomal location and expression pattern of porcine CIDEa,and CIDEc[J].Molecular Biology Reports,2009,36(3):575-582.
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