翘嘴鲌胰岛素样生长因子-Ⅰ的克隆及序列分析
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摘要
为探讨胰岛素样生长因子-Ⅰ(Insulin like growth factor-Ⅰ,IGF-Ⅰ)对于翘嘴鲌(Culter alburnus)生长性状的影响,对其DNA序列进行了克隆。翘嘴鲌IGF-Ⅰ全长14567 bp,由5个外显子和4个内含子组成。其5个外显子长度分别为298、160、182、36和1360 bp。推测的阅读框为486 bp,编码由161个氨基酸组成的IGF-Ⅰ前体蛋白。前体肽由信号肽、成熟肽、E肽三部分组成,其中信号肽44个氨基酸,成熟肽70个氨基酸,E肽47个氨基酸。成熟肽由B、C、A、D四个区域组成,其中B结构域和A结构域的保守性最高,在这2个区域包含由6个半胱氨酸残基形成的3个二硫键。翘嘴鲌B区域还包含保守的IGF-Ⅰ受体识别序列(Phe B23-Tyr B24-Phe B25)。E肽的长度表明翘嘴鲌IGF-Ⅰ属Ea-2型。同源性分析表明翘嘴鲌与鲤科鱼类的IGF-Ⅰ编码氨基酸同源性较高,为94%—100%,但在聚类分析中翘嘴鲌并不是首先和鲌亚科的鱼类聚集在一起。Real-time qPCR组织特异性表达结果显示IGF-ⅠmRNA在肝脏组织中的表达量最高,脾、心脏、精巢、脑次之,肾、鳃、胃和卵巢中表达量较低。翘嘴鲌IGF-Ⅰ基因4个内含子长度分别为1170、9364、251和1746 bp。相对外显子来说,种间内含子变异较大,其中第三内含子变异最大。翘嘴鲌IGF-Ⅰ基因中包含6个微卫星,(GATG)5AATAT(ATAG)11位于第一内含子中,(CT)8、(TTA)5、(AC)13、(TG)12和(ATT)5位于第二内含子中。其中4个微卫星位点具有多态性,将它们在120尾同塘养殖的翘嘴鲌中进行基因型与生长性状的关联性分析,均未达到显著水平(P>0.05)。结果为进一步研究该基因的表达、功能及其转录调控特征奠定了分子基础。
In order to study the effect of IGF-Ⅰ gene on growth traits of topmouth culter(Culter alburnus Basilewsky),IGF-Ⅰ gene was cloned from the genomic DNA of topmouth culter in this study.The gene spanned 14567 bp and comprised five exons and five introns.The five exons of IGF-Ⅰ were 298,160,182,36,and 1360 bp long,respectively.The hypothetical open reading frame of the IGF-Ⅰ cDNA precursor was 486 bp,encoding a putative protein of161 amino acids.The precursor peptide included a signal peptide(44 amino acids),a mature peptide(70 amino acids),and an E peptide(47 amino acids).The mature peptide comprised four regions:B,C,A,and D.The A and B domains were conserved with six cysteine residues in these two regions to form three disulfide bonds.The B region contained a conserved IGF-Ⅰ receptor recognition sequence(Phe B23-Tyr B24-Phe B25).Analysis of the E peptide showed that IGF-Ⅰof the topmouth culter was an Ea-2 type.The IGF-Ⅰ amino acid sequence of the topmouth culter had high sequence identity with IGF-Ⅰ proteins from Cyprinidae fish,ranging from 94 to 100%.Phylogenetic analysis of IGF-Ⅰ amino acid sequences showed that IGF-Ⅰ of the topmouth culter does not cluster with the fish from subfamily Cultrinae.IGF-Ⅰis expressed in all 10 tested tissues with the highest mRNA level in liver,modest level in spleen,heart,testis,and brain,and low level in kidney,gill,stomach,and ovary.The lengths of the four introns of topmouth culter IGF-Ⅰ were 1170,9364,251,and 1746 bp,respectively.The variation in the sequence of the introns among species was greater than that of the exons with the highest variation in the third intron.Six microsatellite loci were found in the introns of IGF-Ⅰwith(GATG)5 AATAT(ATAG)11 in intron 1,and(CT)8,(TTA)5,(AC)13,(TG)12,and(ATT)5 in intron 2.Four of 6 microsatellite loci were polymorphic,and the genotypes of the four microsatellite loci had no significant correlation with the growth traits of 120 topmouth culter cultured in the same pond(P>0.05).These results provide molecular basis for studying the expression pattern,function and transcriptional regulation of the IGF-Ⅰ gene.
In order to study the effect of IGF-Ⅰ gene on growth traits of topmouth culter(Culter alburnus Basilewsky),IGF-Ⅰ gene was cloned from the genomic DNA of topmouth culter in this study.The gene spanned 14567 bp and comprised five exons and five introns.The five exons of IGF-Ⅰ were 298,160,182,36,and 1360 bp long,respectively.The hypothetical open reading frame of the IGF-Ⅰ cDNA precursor was 486 bp,encoding a putative protein of161 amino acids.The precursor peptide included a signal peptide(44 amino acids),a mature peptide(70 amino acids),and an E peptide(47 amino acids).The mature peptide comprised four regions:B,C,A,and D.The A and B domains were conserved with six cysteine residues in these two regions to form three disulfide bonds.The B region contained a conserved IGF-Ⅰ receptor recognition sequence(Phe B23-Tyr B24-Phe B25).Analysis of the E peptide showed that IGF-Ⅰof the topmouth culter was an Ea-2 type.The IGF-Ⅰ amino acid sequence of the topmouth culter had high sequence identity with IGF-Ⅰ proteins from Cyprinidae fish,ranging from 94 to 100%.Phylogenetic analysis of IGF-Ⅰ amino acid sequences showed that IGF-Ⅰ of the topmouth culter does not cluster with the fish from subfamily Cultrinae.IGF-Ⅰis expressed in all 10 tested tissues with the highest mRNA level in liver,modest level in spleen,heart,testis,and brain,and low level in kidney,gill,stomach,and ovary.The lengths of the four introns of topmouth culter IGF-Ⅰ were 1170,9364,251,and 1746 bp,respectively.The variation in the sequence of the introns among species was greater than that of the exons with the highest variation in the third intron.Six microsatellite loci were found in the introns of IGF-Ⅰwith(GATG)5 AATAT(ATAG)11 in intron 1,and(CT)8,(TTA)5,(AC)13,(TG)12,and(ATT)5 in intron 2.Four of 6 microsatellite loci were polymorphic,and the genotypes of the four microsatellite loci had no significant correlation with the growth traits of 120 topmouth culter cultured in the same pond(P>0.05).These results provide molecular basis for studying the expression pattern,function and transcriptional regulation of the IGF-Ⅰ gene.
引文
[1]Hu X S,Yu M,Li C T,et al.Two IGF-I gene microsatellite loci with high polymorphism and their association with growth-related traits in common carp(Cyprinus carpio L.)[J].Journal of Fishery Sciences of China,2014,21(5):872-882[胡雪松,于淼,李池陶,等.鲤IGF-I基因2个高度多态微卫星位点的鉴定及其与生长相关性状的关联分析.中国水产科学,2014,21(5):872-882]
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[3]Uchida K,Kajimura S,Riley L G,et al.Effects of fasting hormone/insulin-like growth factor-I axis in the intilapia,Oreochromis mosssambicus[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2003,134(2):429-439
[4]Ruan R X,Yu J H,Li H X,et al.Effects of IGF-1 genotype on weight gain and body shape in GIFT strain Oreochromis niloticus[J].Journal of Fishery Sciences of China,2011,18(3):682-688[阮瑞霞,俞菊华,李红霞,等.吉富罗非鱼IGF-1基因的基因型对生长和体型的影响.中国水产科学,2011,18(3):682-688]
[5]Li X H,Bai J J,Ye X,et al.Polymorohisms of intron 1,3and 4 of insulin-like growth factor I gene in largemouth bass[J].Journal of Shanghai Ocean University,2009,18(1):8-13[李小慧,白俊杰,叶星,等.大口黑鲈IGF-Ⅰ基因内含子1、3和4序列多态性研究.上海海洋大学学报,2009,18(1):8-13]
[6]Zhang K H.Function and application of intron[J].China Animal Husbandry and Veterinary Medicine,2012,39(7):80-83[张开慧.内含子的功能及应用.中国畜牧兽医,2012,39(7):80-83]
[7]Tsai H Y,Hamilton A,Guy D R,et al.Single nucleotide polymorphisms in the insulin-like growth factor 1(IGF1) gene are associated with growth-related traits in farmed Atlantic salmon[J].Animal Genetics,2014,45(5):709-715
[8]Yue P Q,Luo Y L.Preliminary studies on phylogeny of subfamily Cultrinae(Cypriniformes:Cyprinidae)[J].Acta Hydrobiologica Sinica,1996,20(2):182-185[乐佩琦,罗云林.鲌亚科鱼类系统发育初探(鲤形目:鲤科).水生生物学报,1996,20(2):182-185]
[9]Liu S L,Gu Z M,Jia Y Y,et al.Isolation and characterization of 32 microsatellite loci for topmouth culter(Culter alburnus Basilewsky)[J].Genetics and Molecular Research,2014,13(3):7480-7483
[10]Liu S L,Gu Z M,Zhao J L,et al.Characterization of microsatellites and repetitive flanking sequences(ReFS)from the topmouth culter(Culter alburnus Basilewsky)[J].Biochemical Systematics and Ecology,2015,63(5):127-135
[11]Liu S L,Jia Y Y,Jiang W P,et al.Cloning and sequence analysis of growth hormone gene and its flanking region in topmouth culter(Culter alburnus Basilewsky)[J].Acta Agriculturae Zhejiangensis,2017,29(8):1281-1289[刘士力,贾永义,蒋文枰,等.翘嘴鲌生长激素(GH)基因与侧翼区的克隆及分析.浙江农业学报,2017,29(8):1281-1289]
[12]Liu S L,Zhang A J,Lian Q P,et al.Cloning and tissue expression analysis of the cytochrome P450c17-Ⅰ(CYP17-Ⅰ)gene from mud loach(Misgurnus anguillicaudatus)and large-scale loach(Paramisgurnus dabryanus)[J].Acta Agriculturae Zhejiangensis,2016,28(11):1853-1861[刘士力,张爱菊,练青平,等.泥鳅和大鳞副泥鳅细胞色素P450c17-Ⅰ(CYP17-Ⅰ)基因的克隆及组织表达分析.浙江农业学报,2016,28(11):1853-1861]
[13]Livak K J,Schmittgen T D.Analysis of relative gene expression data using Real-Time Quantitative PCR and the2-DDCT method[J].Methods,2001,25(4):402-408
[14]Blacket M J,Robin C,Good R T,et al.Universal Primers for fluorescent labelling of PCR fragments-an efficient and cost effective approach to genotyping by fluorescence[J].Molecular Ecology Resources,2012,12(3):456-463
[15]Hashimoto H,Mikawa S,Takayama E,et al.Molecular cloning and growth hormone-regulated gene expression of carp insulin-like growth factor-I[J].Biochemistry and Molecular Biology International,1997,41(5):877-886
[16]Rotwein P.Two insulin-like growth factor I messenger RNAs are expressed in human liver[J].Proceedings of the National Academy of Sciences of the United States of America,1986,83(1):77-81
[17]Shimatsu A,Rotwein P.Mosaic evolution of the insulinlike growth factors.Organization,sequence,and expression of the rat insulin-like growth factor I gene[J].Journal of Biological Chemistry,1987,262(16):7894-7900
[18]Kajimoto Y,Rotwein P.Structure of the chicken insulinlike growth factor I gene reveals conserved promoter elements[J].Journal of Biological Chemistry,1991,266(15):9724-9731
[19]Wallis A E,Devlin R H.Duplicate insulin-like growth factor-I genes in salmon display alternative splicing pathways[J].Molecular Endocrinology,1993,7(3):409-422
[20]Vong Q P,Chan K M,Leung K,et al.Common carp insulin-like growth factor-I gene:complete nucleotide sequence and functional characterization of the 5′-flanking region[J].Gene,2003,322(1):145-156
[21]Clay L A,Wang S Y,Wolters W R,et al.Molecular characterization of the insulin-like growth factor-I(IGF-I)gene in channel catfish(Ictalurus punctatus)[J].Biochimica et Biophysica Acta(BBA)/Gene Structure and Expression,2005,1731(3):139-148
[22]Feng X,Yu X,Tong J.Novel single nucleotide polymorphisms of the insulin-like growth factor-I gene and their associations with growth traits in common carp(Cyprinus carpio L.)[J].International Journal of Molecular Sciences,2014,15(12):22471-22482
[23]Cao Y B,Wu L Q,Shao L X,et al.Expression and purification of recombinant insulin-like growth factor-Ⅱof naked carp(Gymnocypris przewalskii)in Lake Qinghai[J].Acta Hydrobiologica Sinica,2010,34(2):459-462[曹诣斌,吴兰亲,邵邻相,等.青海湖裸鲤类胰岛素生长因子IGF-Ⅱ的原核表达.水生生物学报,2010,34(2):459-462]
[24]Hober S,Forsberg G,Palm G,et al.Disulfide exchange folding of insulin-like growth factor I[J].Biochemistry,1992,31(6):1749-1756
[25]Magee B A,Shooter G K,Wallace J C,et al.Insulin-like growth factor I and its binding proteins:a study of the binding interface using B-domain analogues[J].Biochemistry,1999,38(48):15863-15870
[26]Gao F Y,Lu M X,Huang Z H,et al.Cloning and sequence of two cDNAs encoding insulin-like growth factors I and II genes(IGF-I and IGF-Ⅱ)of Zanzibar tilapia(Oreochromis hornorum)and their tissue distribution[J].Journal of Agricultural Biotechnology,2012,20(2):171-180[高风英,卢迈新,黄樟翰,等.荷那龙罗非鱼两种胰岛素样生长因子基因(IGF-Ⅰ和IGF-Ⅱ)的克隆、序列分析及组织表达特征.农业生物技术学报,2012,20(2):171-180]
[27]Tao M,Zhong H,Liu S J,et al.The cloning of IGF-1cDNA and tissue distribution in Japanese crucian carp(Carassius cuvieri)[J].Life Science Research,2012,16(4):295-300[陶敏,钟欢,刘少军,等.日本白鲫IGF-1基因全长cDNA克隆及组织表达分析.生命科学研究,2012,16(4):295-300]
[28]Wang X Y,Ji F,Xu L M,et al.Cloning,sequence analysis and tissue expression of insulin-like growth factor I in Hucho taimen[J].Freshwater Fisheries,2016,46(4):19-24[王晓玉,纪锋,徐黎明,等.哲罗鲑胰岛素样生长因子-I(IGF-I)cDNA分子克隆、序列分析及组织表达.淡水渔业,2016,46(4):19-24]
[29]Zhu X F,Guo X G,Zhang D Y,et al.Cloning and expression analysis of insulin-like growth factor I gene in Pampus Argenteus[J].Oceanologia et Limnologia Sinica,2016,47(6):1221-1232[朱晓芳,郭晓鸽,张鼎元,等.银鲳(Pampus argenteus)胰岛素样生长因子-I基因的克隆和表达分析.海洋与湖沼,2016,47(6):1221-1232]
[30]Qian K,Wen H S,Chi M L,et al.Isolation of full-length cDNA of insulin-like growth factor-1(IGF-1)gene of Japanese sea bass(Lateolabrax japonicas)and its expression analysis[J].Periodical of Ocean University of China,2014,44(2):27-34[钱焜,温海深,迟美丽,等.花鲈类胰岛素生长因子-I基因的全长cDNA分离与表达分析.中国海洋大学学报,2014,44(2):27-34]
[2]Upton Z,Yandell C A,Degger B G,et al.Evolution of insulin-like growth factor-I(IGF-I)action:in vitro characterization of vertebrate IGF-I proteins[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,1998,121(1):35-41
[3]Uchida K,Kajimura S,Riley L G,et al.Effects of fasting hormone/insulin-like growth factor-I axis in the intilapia,Oreochromis mosssambicus[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2003,134(2):429-439
[4]Ruan R X,Yu J H,Li H X,et al.Effects of IGF-1 genotype on weight gain and body shape in GIFT strain Oreochromis niloticus[J].Journal of Fishery Sciences of China,2011,18(3):682-688[阮瑞霞,俞菊华,李红霞,等.吉富罗非鱼IGF-1基因的基因型对生长和体型的影响.中国水产科学,2011,18(3):682-688]
[5]Li X H,Bai J J,Ye X,et al.Polymorohisms of intron 1,3and 4 of insulin-like growth factor I gene in largemouth bass[J].Journal of Shanghai Ocean University,2009,18(1):8-13[李小慧,白俊杰,叶星,等.大口黑鲈IGF-Ⅰ基因内含子1、3和4序列多态性研究.上海海洋大学学报,2009,18(1):8-13]
[6]Zhang K H.Function and application of intron[J].China Animal Husbandry and Veterinary Medicine,2012,39(7):80-83[张开慧.内含子的功能及应用.中国畜牧兽医,2012,39(7):80-83]
[7]Tsai H Y,Hamilton A,Guy D R,et al.Single nucleotide polymorphisms in the insulin-like growth factor 1(IGF1) gene are associated with growth-related traits in farmed Atlantic salmon[J].Animal Genetics,2014,45(5):709-715
[8]Yue P Q,Luo Y L.Preliminary studies on phylogeny of subfamily Cultrinae(Cypriniformes:Cyprinidae)[J].Acta Hydrobiologica Sinica,1996,20(2):182-185[乐佩琦,罗云林.鲌亚科鱼类系统发育初探(鲤形目:鲤科).水生生物学报,1996,20(2):182-185]
[9]Liu S L,Gu Z M,Jia Y Y,et al.Isolation and characterization of 32 microsatellite loci for topmouth culter(Culter alburnus Basilewsky)[J].Genetics and Molecular Research,2014,13(3):7480-7483
[10]Liu S L,Gu Z M,Zhao J L,et al.Characterization of microsatellites and repetitive flanking sequences(ReFS)from the topmouth culter(Culter alburnus Basilewsky)[J].Biochemical Systematics and Ecology,2015,63(5):127-135
[11]Liu S L,Jia Y Y,Jiang W P,et al.Cloning and sequence analysis of growth hormone gene and its flanking region in topmouth culter(Culter alburnus Basilewsky)[J].Acta Agriculturae Zhejiangensis,2017,29(8):1281-1289[刘士力,贾永义,蒋文枰,等.翘嘴鲌生长激素(GH)基因与侧翼区的克隆及分析.浙江农业学报,2017,29(8):1281-1289]
[12]Liu S L,Zhang A J,Lian Q P,et al.Cloning and tissue expression analysis of the cytochrome P450c17-Ⅰ(CYP17-Ⅰ)gene from mud loach(Misgurnus anguillicaudatus)and large-scale loach(Paramisgurnus dabryanus)[J].Acta Agriculturae Zhejiangensis,2016,28(11):1853-1861[刘士力,张爱菊,练青平,等.泥鳅和大鳞副泥鳅细胞色素P450c17-Ⅰ(CYP17-Ⅰ)基因的克隆及组织表达分析.浙江农业学报,2016,28(11):1853-1861]
[13]Livak K J,Schmittgen T D.Analysis of relative gene expression data using Real-Time Quantitative PCR and the2-DDCT method[J].Methods,2001,25(4):402-408
[14]Blacket M J,Robin C,Good R T,et al.Universal Primers for fluorescent labelling of PCR fragments-an efficient and cost effective approach to genotyping by fluorescence[J].Molecular Ecology Resources,2012,12(3):456-463
[15]Hashimoto H,Mikawa S,Takayama E,et al.Molecular cloning and growth hormone-regulated gene expression of carp insulin-like growth factor-I[J].Biochemistry and Molecular Biology International,1997,41(5):877-886
[16]Rotwein P.Two insulin-like growth factor I messenger RNAs are expressed in human liver[J].Proceedings of the National Academy of Sciences of the United States of America,1986,83(1):77-81
[17]Shimatsu A,Rotwein P.Mosaic evolution of the insulinlike growth factors.Organization,sequence,and expression of the rat insulin-like growth factor I gene[J].Journal of Biological Chemistry,1987,262(16):7894-7900
[18]Kajimoto Y,Rotwein P.Structure of the chicken insulinlike growth factor I gene reveals conserved promoter elements[J].Journal of Biological Chemistry,1991,266(15):9724-9731
[19]Wallis A E,Devlin R H.Duplicate insulin-like growth factor-I genes in salmon display alternative splicing pathways[J].Molecular Endocrinology,1993,7(3):409-422
[20]Vong Q P,Chan K M,Leung K,et al.Common carp insulin-like growth factor-I gene:complete nucleotide sequence and functional characterization of the 5′-flanking region[J].Gene,2003,322(1):145-156
[21]Clay L A,Wang S Y,Wolters W R,et al.Molecular characterization of the insulin-like growth factor-I(IGF-I)gene in channel catfish(Ictalurus punctatus)[J].Biochimica et Biophysica Acta(BBA)/Gene Structure and Expression,2005,1731(3):139-148
[22]Feng X,Yu X,Tong J.Novel single nucleotide polymorphisms of the insulin-like growth factor-I gene and their associations with growth traits in common carp(Cyprinus carpio L.)[J].International Journal of Molecular Sciences,2014,15(12):22471-22482
[23]Cao Y B,Wu L Q,Shao L X,et al.Expression and purification of recombinant insulin-like growth factor-Ⅱof naked carp(Gymnocypris przewalskii)in Lake Qinghai[J].Acta Hydrobiologica Sinica,2010,34(2):459-462[曹诣斌,吴兰亲,邵邻相,等.青海湖裸鲤类胰岛素生长因子IGF-Ⅱ的原核表达.水生生物学报,2010,34(2):459-462]
[24]Hober S,Forsberg G,Palm G,et al.Disulfide exchange folding of insulin-like growth factor I[J].Biochemistry,1992,31(6):1749-1756
[25]Magee B A,Shooter G K,Wallace J C,et al.Insulin-like growth factor I and its binding proteins:a study of the binding interface using B-domain analogues[J].Biochemistry,1999,38(48):15863-15870
[26]Gao F Y,Lu M X,Huang Z H,et al.Cloning and sequence of two cDNAs encoding insulin-like growth factors I and II genes(IGF-I and IGF-Ⅱ)of Zanzibar tilapia(Oreochromis hornorum)and their tissue distribution[J].Journal of Agricultural Biotechnology,2012,20(2):171-180[高风英,卢迈新,黄樟翰,等.荷那龙罗非鱼两种胰岛素样生长因子基因(IGF-Ⅰ和IGF-Ⅱ)的克隆、序列分析及组织表达特征.农业生物技术学报,2012,20(2):171-180]
[27]Tao M,Zhong H,Liu S J,et al.The cloning of IGF-1cDNA and tissue distribution in Japanese crucian carp(Carassius cuvieri)[J].Life Science Research,2012,16(4):295-300[陶敏,钟欢,刘少军,等.日本白鲫IGF-1基因全长cDNA克隆及组织表达分析.生命科学研究,2012,16(4):295-300]
[28]Wang X Y,Ji F,Xu L M,et al.Cloning,sequence analysis and tissue expression of insulin-like growth factor I in Hucho taimen[J].Freshwater Fisheries,2016,46(4):19-24[王晓玉,纪锋,徐黎明,等.哲罗鲑胰岛素样生长因子-I(IGF-I)cDNA分子克隆、序列分析及组织表达.淡水渔业,2016,46(4):19-24]
[29]Zhu X F,Guo X G,Zhang D Y,et al.Cloning and expression analysis of insulin-like growth factor I gene in Pampus Argenteus[J].Oceanologia et Limnologia Sinica,2016,47(6):1221-1232[朱晓芳,郭晓鸽,张鼎元,等.银鲳(Pampus argenteus)胰岛素样生长因子-I基因的克隆和表达分析.海洋与湖沼,2016,47(6):1221-1232]
[30]Qian K,Wen H S,Chi M L,et al.Isolation of full-length cDNA of insulin-like growth factor-1(IGF-1)gene of Japanese sea bass(Lateolabrax japonicas)and its expression analysis[J].Periodical of Ocean University of China,2014,44(2):27-34[钱焜,温海深,迟美丽,等.花鲈类胰岛素生长因子-I基因的全长cDNA分离与表达分析.中国海洋大学学报,2014,44(2):27-34]