山羊FGF9基因克隆及其在成肌细胞分化中的表达模式研究
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摘要
旨在克隆山羊FGF9基因序列并对其进行生物信息学分析,阐明FGF9基因组织表达特性及其在成肌细胞分化过程中的表达差异。试验动物为简州大耳羊,利用RT-PCR技术克隆FGF9基因序列,再用实时荧光定量PCR(qRT-PCR)技术检测FGF9在山羊各组织中的表达特性及其在成肌细胞不同分化阶段的表达情况。结果显示,克隆得到山羊FGF9基因序列818 bp,其中ORF区627 bp,编码208个氨基酸,其CDS区核苷酸序列与牛和绵羊有99%的同源性。FGF9蛋白具有1个跨膜结构域和1个FGF家族同源性结构域,为不稳定亲水蛋白。FGF9基因在山羊各组织中均有表达,在肾脏中表达水平最高,极显著高于其他组织(P<0.01)。FGF9基因在诱导分化第2天表达水平显著高于分化前(P<0.05),且在第4天达到极显著水平(P<0.01)。推测其可作为调控山羊成肌细胞分化的候选基因。为进一步探究FGF9基因在山羊肌肉生长中的作用提供理论依据。
The aim of this study was to clone the goat FGF9 gene sequence and perform bioinformatics analysis,so as to elucidate the tissue expression characteristics of FGF9 and its differential expression during myoblast differentiation. The experimental animal of this study was the Jianzhou Big-eared sheep. The FGF9 gene sequence was cloned by RT-PCR. Real-time quantitative PCR(qRT-PCR) was performed to detect the expression of FGF9 in different tissues of goat and at different stages of myoblast differentiation. The results showed that the sequence of goat FGF9 gene was 818 bp,with an ORF region of 627 bp,encoding 208 amino acids. The nucleotide sequence of the CDS region was 99% homologous to Bostaurus and Ovisaries. The FGF9 protein had a transmembrane domain and a FGF family homology domain,which was an unstable hydrophilic protein. The FGF9 gene could express in all tissues of goats,and the expression level in the kidney was the highest,which was significantly higher than in other tissues(P<0.01). The expression level of FGF9 gene in the 2 nd day of induced differentiation was significantly higher than that before differentiation(P<0.05),and reached a very significant level in the 4 th day(P<0.01). This study provided the important data for further exploring the role of FGF9 gene in goat muscle growth.
The aim of this study was to clone the goat FGF9 gene sequence and perform bioinformatics analysis,so as to elucidate the tissue expression characteristics of FGF9 and its differential expression during myoblast differentiation. The experimental animal of this study was the Jianzhou Big-eared sheep. The FGF9 gene sequence was cloned by RT-PCR. Real-time quantitative PCR(qRT-PCR) was performed to detect the expression of FGF9 in different tissues of goat and at different stages of myoblast differentiation. The results showed that the sequence of goat FGF9 gene was 818 bp,with an ORF region of 627 bp,encoding 208 amino acids. The nucleotide sequence of the CDS region was 99% homologous to Bostaurus and Ovisaries. The FGF9 protein had a transmembrane domain and a FGF family homology domain,which was an unstable hydrophilic protein. The FGF9 gene could express in all tissues of goats,and the expression level in the kidney was the highest,which was significantly higher than in other tissues(P<0.01). The expression level of FGF9 gene in the 2 nd day of induced differentiation was significantly higher than that before differentiation(P<0.05),and reached a very significant level in the 4 th day(P<0.01). This study provided the important data for further exploring the role of FGF9 gene in goat muscle growth.
引文
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[13] Fakhry A,Ratisoontorn C,Vedhachalam C,Salhab I,Koyama E,Leboy P,Pacifici M,Kirschner R E,Nah H D.Effectsof FGF-2/-9 in calvarial bone cell cultures:differentiationstage-dependent-mitogeniceffect,inverse regulation of BMP-2 and noggin,and en-hancement of osteogenic potential[J].Bone,2005,36(2):254-266.doi:10.1016/j.bone.2004.10.003.
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[15] Behr B,Leucht P,Longaker M T,Quarto N.Fgf-9 is required for angiogenesis and osteogenesis in long bone repair[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(26):11853-11858.doi:10.1073/pnas.1003317107.
[16] 易善勇,杨晶,官丽莉,王艳芳,黄建,王立勇,李海燕,李校堃,姜潮.成纤维细胞生长因子9(FGF9)的研究进展[J].中国生物工程杂志,2015,35(7):94-101.doi:10.13523/j.cb.20150713.Yi S Y,Yang J,Gong L L,Wang Y F,Huang J,Wang L Y,Li H Y,Li X K,Jiang C.Research progresses on the fibroblast growth factor 9[J].China Biotechnology,2015,35(7):94-101.
[17] 廖冰,吴宁,韩凤桐,林秀坤.牛性别决定新基因Fgf9的克隆及生物信息学分析[J].中国生物工程杂志,2009,29(8):45-50.doi:10.13523/j.cb.20090808.Liao B,Wu N,Han F T,Lin X K.Cloning and bioinformatics analysis of Fgf9,a novel gene related to sex determination in cow[J].China Biotechnology,2009,29(8):45-50.
[18] Karuppaiah K,Yu K,Lim J,Chen J,Smith C,Long F,Ornitz D M.FGF signaling in the osteoprogenitor lineage non-autonomously regulates postnatal chondrocyte proliferation and skeletal growth[J].Development,2016,143(10):1811-1822.doi:10.1242/dev.131722.
[19] 于丽云,周学瀛.成纤维细胞生长因子9及其受体对骨骼的作用[J].国外医学(内分泌学分册),2005,25(5):324-327.Yu L Y,Zhou X Y.Effect of fibroblast growth factor 9 and its receptor on bone[J].Foreign Medicine(Endocrinology),2005,25(5):324-327.
[20] Rodriguez Z M,Aza C M,Rivera P C I,Belinchón A,Guerrero Z I,Barraza G J,Vallespin E,Lu M,Del Pozo A,Glucksman M J,Santos S F,Heath K E.FGF9 mutation causes craniosynostosis along with multiple synostoses[J].Human Mutation,2017,38(11):1471-1476.doi:10.1002/humu.23292.
[21] 裴育,孟迅吾,周学瀛,邢小平,夏维波.成纤维细胞生长因子9影响成骨细胞核心结合因子α1基因表达的作用[J].中国临床康复,2005,9(10):32-34.Pei Y,Meng X W,Zhou X Y,Xin X P,Xia W B.Effect of fibroblast growth factor 9 on the expression of core binding factor α1 gene in osteoblasts[J].Chinese Journal of Clinical Rehabilitation,2005,9(10):32-34.
[22] Frontini M J,Nong Z,Gros R,Drangova M,O′Neil C,Rahman M N,Akawi O,Yin H,Ellis C G,Pickering J G.Fibroblast growth factor 9 delivery during angiogenesis produces durable,vasoresponsive microvessels wrapped by smooth muscle cells[J].Nature Biotechnology,2011,29(5):421-427.doi:10.1038/nbt.1845.
[23] Xie B,Tassi E,Swift M R,McDonnell K,Bowden E T,Wang S,Ueda Y,Tomita Y,Riegel A T,Wellstein A.Identification of the fibroblast growth factor(FGF)-interacting domain in a secreted FGF-binding protein by phage display[J].Journal of Biological Chemistry,2006,281(2):1137-1144.doi:10.1074/jbc.M510754200.
[24] Revest J M,Demoerlooze L,Dickson C.Fibroblast growth factor 9 secretion is mediated by a non-cleaved amino-terminal signal sequence[J].Journal of Biological Chemistry,2000,275(11):8083-8090.doi:10.1074/jbc.275.11.8083.
[25] Lai M S,Wang C Y,Yang S H,Wu C C,Sun H S,Tsai S J,Chuang J I,Chen Y C,Huang B M.The expression profiles of fibroblast growth factor 9 and its receptors in developing mice testes[J].Organogenesis,2016,12(2):61-77.doi:10.1080/15476278.2016.1171448.
[26] 陈晓怡,吴晓林,顾鸣敏,陆顺元,王铸钢.TFGF9转基因小鼠的功能及组织表达谱的研究[J].诊断学理论与实践,2009,8(5):496-501.doi:10.16150/j.1671-2870.2009.05.006.Chen X Y,Wu X L,Gu M M,Lu S Y,Wang Z G.Investigation on function and tissue expression profile of transgenic mice with mutated FGF9 gene[J].Journal of Diagnostics Concepts & Practice,2009,8(5):496-501.
[27] Zimna A,Janeczek A,Rozwadowska N,Fraczek M,Kucharzewska P,Rucinski M,Mietkiewski T,Kurpisz M.Biological properties of human skeletal myoblasts genetically modified to simultaneously overexpress the pro-angiogenic factors vascular endothelial growth factor-a and fibroblast growth factor-4[J].Journal of Physiology and Pharmacology,2014,65(2):193-207.
[28] Bao W L,Yao R Y,He Q,Guo Z X,Bao C,Wang Y F,Wang Z G.Cloning,molecular characterization,and expression pattern of FGF5 in Cashmere goat (Capra hircus)[J].Genetics and Molecular Research,2015,14(3):11154-11161.doi:10.4238/2015.September.22.9.
[29] Han D,Zhao H,Parada C,Hacia J G,Bringas P Jr,Chai Y.A TGF β-Smad4-Fgf6 signaling cascade controls myogenic differentiation and myoblast fusion during tongue development[J].Development,2012,139(9):1640-1650.doi:10.1242/dev.076653.
[30] Colvin J S,Feldman B,Nadeau J H,Goldfarb M,Ornitz D M.Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene[J].Developmental Dynamics:an Official Publication of the American Association of Anatomists,1999,216(1):72-88.doi:10.1002/(SICI)1097-0177(199909)216:1<72::AID-DVDY9>3.0.CO;2-9.
[2] Deng S,Azevedo M,Baylies M.Acting on identity:Myoblast fusion and the formation of the syncytial muscle fiber[J].Seminars in Cell & Developmental Biology,2017,72:45-55.doi:10.1016/j.semcdb.2017.10.033.
[3] 文逸凡,贺花,宋成创,彭术军,雷初朝,陈宏,黄永震.家养动物肌肉发育相关基因的研究进展[J].基因组学与应用生物学,2017,09(12):1-8.doi:10.13417/j.gab.037.002825.Wen Y F,He H,Song C C,Peng S J,Lei C C,Chen H,Huang Y Z.The research progress of genes related to the domestic animals muscle development[J].Genomics and Applied Biology,2017,09(12):1-8.
[4] Gnatenko D A,Kopantzev E P,Sverdlov E D.Fibroblast growth factors and their effects in pancreas organogenesis[J].Biomeditsinskaia Khimiia,2017,63(3):211-218.doi:10.18097/PBMC20176303211.
[5] Chang M M,Lai M S,Hong S Y,Pan B S,Huang H,Yang S H,Wu C C,Sun H S,Chuang J I,Wang C Y,Huang B M.FGF9/FGFR2 increase cell proliferation by activating ERK1/2,Rb/E2F1,and cell cycle pathways in mouse Leydig tumor cells[J].Cancer Science,2018,109(11):3503-3518.doi:10.1111/cas.13793.
[6] Korsensky L,Ron D.Regulation of FGF signaling:Recent insights from studying positive and negative modulators[J].Seminars in Cell & Developmental Biology,2016,53:101-114.doi:10.1016/j.semcdb.2016.01.023.
[7] Oisen S K,Garbi M,Zampieri N,Eliseenkova A V,Ornitz D M,Goldfarb M,Mohammadi M.Fibroblast growth factor(FGF)homologous factors share structural but not functional homology with FGFs[J].Journal of Biological Chemistry,2003,278(36):34226-34236.doi:10.1074/jbc.M303183200.
[8] Dunstan C R,Zhou H,Seibel M J.Fibroblast growth factor 23:a phosphatonin regulating phosphate homeostasis[J].Endocrinology,2004,145(7):3084-3086.doi:10.1210/en.2004-0354.
[9] Naruo K,Seko C,Kuroshima K,Matsutani E,Sasada R,Kondo T,Kurokawa T.Novel secretory heparin-binding factors from human glioma-cells (glia-activating factors) involved in glial-cell growth-purification and biological properties[J].Journal of Biological Chemistry,1993,268(4):2857-2864.
[10] Itoh N,Ohta H,Nakayama Y,Konishi M.Roles of FGF signals in heart development,health,and disease[J].Frontiers in Cell and Developmental Biology,2016,4:110.doi:10.3389/fcell.2016.00110.
[11] Colvin J S,Green R P,Schmahl J,Capel B,Ornitz D M.Male-to-female sex reversal in mice lacking fibroblast growth factor 9[J].Cell,2001,104:875-889.doi:10.1016/s0092-8674(01)00284-7.
[12] Abdel-Rahman W M,Kalinina J,Shoman S,Eissa S,Ollikainen M,Elomaa O,Eliseenkova A V,Bützow R,Mohammadi M,Peltom?ki P.Somatic FGF9 mutations in colorectal and endometrial carcinomas associated with membranous beta-catenin[J].Human Mutation,2008,29(3):390-397.doi:10.1002/humu.20653.
[13] Fakhry A,Ratisoontorn C,Vedhachalam C,Salhab I,Koyama E,Leboy P,Pacifici M,Kirschner R E,Nah H D.Effectsof FGF-2/-9 in calvarial bone cell cultures:differentiationstage-dependent-mitogeniceffect,inverse regulation of BMP-2 and noggin,and en-hancement of osteogenic potential[J].Bone,2005,36(2):254-266.doi:10.1016/j.bone.2004.10.003.
[14] Hung I H,Schoenwolf G C,Lewandoski M,Ornitz D M.A combined series of Fgf9 and Fgf18 mutant alleles identifies unique and redundant roles in skeletal development[J].Developmental Biology,2016,411(1):72-84.doi:10.1016/j.ydbio.2016.01.008.
[15] Behr B,Leucht P,Longaker M T,Quarto N.Fgf-9 is required for angiogenesis and osteogenesis in long bone repair[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(26):11853-11858.doi:10.1073/pnas.1003317107.
[16] 易善勇,杨晶,官丽莉,王艳芳,黄建,王立勇,李海燕,李校堃,姜潮.成纤维细胞生长因子9(FGF9)的研究进展[J].中国生物工程杂志,2015,35(7):94-101.doi:10.13523/j.cb.20150713.Yi S Y,Yang J,Gong L L,Wang Y F,Huang J,Wang L Y,Li H Y,Li X K,Jiang C.Research progresses on the fibroblast growth factor 9[J].China Biotechnology,2015,35(7):94-101.
[17] 廖冰,吴宁,韩凤桐,林秀坤.牛性别决定新基因Fgf9的克隆及生物信息学分析[J].中国生物工程杂志,2009,29(8):45-50.doi:10.13523/j.cb.20090808.Liao B,Wu N,Han F T,Lin X K.Cloning and bioinformatics analysis of Fgf9,a novel gene related to sex determination in cow[J].China Biotechnology,2009,29(8):45-50.
[18] Karuppaiah K,Yu K,Lim J,Chen J,Smith C,Long F,Ornitz D M.FGF signaling in the osteoprogenitor lineage non-autonomously regulates postnatal chondrocyte proliferation and skeletal growth[J].Development,2016,143(10):1811-1822.doi:10.1242/dev.131722.
[19] 于丽云,周学瀛.成纤维细胞生长因子9及其受体对骨骼的作用[J].国外医学(内分泌学分册),2005,25(5):324-327.Yu L Y,Zhou X Y.Effect of fibroblast growth factor 9 and its receptor on bone[J].Foreign Medicine(Endocrinology),2005,25(5):324-327.
[20] Rodriguez Z M,Aza C M,Rivera P C I,Belinchón A,Guerrero Z I,Barraza G J,Vallespin E,Lu M,Del Pozo A,Glucksman M J,Santos S F,Heath K E.FGF9 mutation causes craniosynostosis along with multiple synostoses[J].Human Mutation,2017,38(11):1471-1476.doi:10.1002/humu.23292.
[21] 裴育,孟迅吾,周学瀛,邢小平,夏维波.成纤维细胞生长因子9影响成骨细胞核心结合因子α1基因表达的作用[J].中国临床康复,2005,9(10):32-34.Pei Y,Meng X W,Zhou X Y,Xin X P,Xia W B.Effect of fibroblast growth factor 9 on the expression of core binding factor α1 gene in osteoblasts[J].Chinese Journal of Clinical Rehabilitation,2005,9(10):32-34.
[22] Frontini M J,Nong Z,Gros R,Drangova M,O′Neil C,Rahman M N,Akawi O,Yin H,Ellis C G,Pickering J G.Fibroblast growth factor 9 delivery during angiogenesis produces durable,vasoresponsive microvessels wrapped by smooth muscle cells[J].Nature Biotechnology,2011,29(5):421-427.doi:10.1038/nbt.1845.
[23] Xie B,Tassi E,Swift M R,McDonnell K,Bowden E T,Wang S,Ueda Y,Tomita Y,Riegel A T,Wellstein A.Identification of the fibroblast growth factor(FGF)-interacting domain in a secreted FGF-binding protein by phage display[J].Journal of Biological Chemistry,2006,281(2):1137-1144.doi:10.1074/jbc.M510754200.
[24] Revest J M,Demoerlooze L,Dickson C.Fibroblast growth factor 9 secretion is mediated by a non-cleaved amino-terminal signal sequence[J].Journal of Biological Chemistry,2000,275(11):8083-8090.doi:10.1074/jbc.275.11.8083.
[25] Lai M S,Wang C Y,Yang S H,Wu C C,Sun H S,Tsai S J,Chuang J I,Chen Y C,Huang B M.The expression profiles of fibroblast growth factor 9 and its receptors in developing mice testes[J].Organogenesis,2016,12(2):61-77.doi:10.1080/15476278.2016.1171448.
[26] 陈晓怡,吴晓林,顾鸣敏,陆顺元,王铸钢.TFGF9转基因小鼠的功能及组织表达谱的研究[J].诊断学理论与实践,2009,8(5):496-501.doi:10.16150/j.1671-2870.2009.05.006.Chen X Y,Wu X L,Gu M M,Lu S Y,Wang Z G.Investigation on function and tissue expression profile of transgenic mice with mutated FGF9 gene[J].Journal of Diagnostics Concepts & Practice,2009,8(5):496-501.
[27] Zimna A,Janeczek A,Rozwadowska N,Fraczek M,Kucharzewska P,Rucinski M,Mietkiewski T,Kurpisz M.Biological properties of human skeletal myoblasts genetically modified to simultaneously overexpress the pro-angiogenic factors vascular endothelial growth factor-a and fibroblast growth factor-4[J].Journal of Physiology and Pharmacology,2014,65(2):193-207.
[28] Bao W L,Yao R Y,He Q,Guo Z X,Bao C,Wang Y F,Wang Z G.Cloning,molecular characterization,and expression pattern of FGF5 in Cashmere goat (Capra hircus)[J].Genetics and Molecular Research,2015,14(3):11154-11161.doi:10.4238/2015.September.22.9.
[29] Han D,Zhao H,Parada C,Hacia J G,Bringas P Jr,Chai Y.A TGF β-Smad4-Fgf6 signaling cascade controls myogenic differentiation and myoblast fusion during tongue development[J].Development,2012,139(9):1640-1650.doi:10.1242/dev.076653.
[30] Colvin J S,Feldman B,Nadeau J H,Goldfarb M,Ornitz D M.Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene[J].Developmental Dynamics:an Official Publication of the American Association of Anatomists,1999,216(1):72-88.doi:10.1002/(SICI)1097-0177(199909)216:1<72::AID-DVDY9>3.0.CO;2-9.