团头鲂肌腱发育相关基因tnmd/xirp2a的克隆和表达
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摘要
为分析肌腱发育相关基因tnmd/xirp2a与团头鲂肌间骨发育之间的相关性,通过克隆获得团头鲂tnmd/xirp2a的cDNA序列,其中tnmd的cDNA全长为1 420bp,开放阅读框为906bp,共编码301个氨基酸;xirp2a的cDNA全长为11 715bp,包括开放阅读框9 522bp,编码3 174个氨基酸。系统进化研究表明,tnmd/xirp2a基因在不同脊椎动物中保守性很低,且相比哺乳类和家禽类,鱼类tnmd基因存在一段氨基酸的缺失。采用荧光定量方法比较分析tnmd/xirp2a在团头鲂成鱼不同组织及肌间骨发生发育4个关键时期的表达情况,结果显示,tnmd/xirp2a在肌肉和肌间骨中的表达量均显著高于其他组织,且肌肉中tnmd基因表达高于肌间骨(P<0.05),而xirp2a在肌间骨中的表达高于肌肉组织(P<0.05)。在肌间骨发育4个关键时期(Ⅰ:尾部尚未出现肌间骨;Ⅱ:尾部出现少量肌间骨;Ⅲ:尾部肌间骨大量出现且长度增加;Ⅳ:肌间骨从尾部到背部全部出现且形态成熟)中,tnmd在第Ⅲ时期的表达水平显著高于另外3个时期;在第Ⅱ、Ⅲ、Ⅳ时期之间,xirp2a的表达水平没有显著性差异。此结果表明,tnmd基因在肌间骨的发育过程中存在潜在作用,而xirp2a基因与肌间骨的发育之间的相关性还需进一步研究。
In order to investigate the relationship between the tendon development related genes(tnmd/xirp2 a)and the intermuscular bone in blunt snout bream(Megalobrama amblycephala),we firstly obtained the cDNA sequence of tnmd/xirp2 a by cloning.The full-length cDNA of tnmdis1 420 bp,including an open reading frame(ORF)of 906 bp encoding 301 amino acids.The cDNA sequences of xirp2 ais 11 715 bp in length,including an ORF of 9 522 bp encoding 3 174 amino acids.Phylogenetic analysis showed that the tnmd gene was lowly conserved among different vertebrates.The tnmd protein had a sequence deletion compared to mammals and poultry.The tnmd/xirp2 aexpression in the muscle and intermuscular bone was significantly higher than that in other tissues(P<0.05).The tnmdexpression in the muscle was significantly higher than that in the intermuscular bone(P<0.05),while the result of xirp2 a was the exact opposite.During intermuscular bone development,the tnmd mRNA was significantly higher at stageⅢthan that at the other three stages.However,there was no significant differences of the xirp2 aexpression among stagesⅡ,Ⅲ and Ⅳ(P>0.05).Our results indicate that the tnmd gene may play possible regulation roles during the intermuscular bone development,while the relationship between the xirp2 agene and the intermuscular bone development needs to be further studied.The results of this study provide theoretical support for the derivation of intermuscular bone from the tendon.
In order to investigate the relationship between the tendon development related genes(tnmd/xirp2 a)and the intermuscular bone in blunt snout bream(Megalobrama amblycephala),we firstly obtained the cDNA sequence of tnmd/xirp2 a by cloning.The full-length cDNA of tnmdis1 420 bp,including an open reading frame(ORF)of 906 bp encoding 301 amino acids.The cDNA sequences of xirp2 ais 11 715 bp in length,including an ORF of 9 522 bp encoding 3 174 amino acids.Phylogenetic analysis showed that the tnmd gene was lowly conserved among different vertebrates.The tnmd protein had a sequence deletion compared to mammals and poultry.The tnmd/xirp2 aexpression in the muscle and intermuscular bone was significantly higher than that in other tissues(P<0.05).The tnmdexpression in the muscle was significantly higher than that in the intermuscular bone(P<0.05),while the result of xirp2 a was the exact opposite.During intermuscular bone development,the tnmd mRNA was significantly higher at stageⅢthan that at the other three stages.However,there was no significant differences of the xirp2 aexpression among stagesⅡ,Ⅲ and Ⅳ(P>0.05).Our results indicate that the tnmd gene may play possible regulation roles during the intermuscular bone development,while the relationship between the xirp2 agene and the intermuscular bone development needs to be further studied.The results of this study provide theoretical support for the derivation of intermuscular bone from the tendon.
引文
[1]DANOS N,WARD A B.The homology and origins of intermuscular bones in fishes:phylogenetic or biomechanical determinants[J].Biological journal of the linnean society,2012,106(3):607-622.
[2]孟庆闻,苏锦祥,李婉端.鱼类比较解剖学[M].北京:科学出版社,1987:102-103.
[3]PATTERSON C,JOHNSON G D.The intermusoular bones and ligaments of teleostean fishes[J].Smlthsonian contribution to zoology,1995(559):1-83.
[4]JOHNSON G D,PATTERSON C.The intermuscular system of acanthomorph fishes:a commentary[J].American museum novitates,2001(3312):1-25.
[5]吕耀平,鲍宝龙,蒋燕,等.低等真骨鱼类肌间骨的比较分析[J].水产学报,2007,31(5):661-668.
[6]秉志.幼鲤大侧肌隔骨针的观察[J].动物学报,1962(2):32-36.
[7]BIRD N C,MABEE P M.Developmental morphology of the axial skeleton of the zebrafish,Danio rerio(Ostariophysi:Cyprinidae)[J].Developmental dynamics,2003,228(3):337-357.
[8]柯中和,张炜,蒋燕,等.鲢肌间小骨发育的形态学观察[J].动物学杂志,2008,43(6):88-96.
[9]万世明,易少奎,仲嘉,等.团头鲂肌间骨发育的形态学观察[J].水生生物学报,2014,38(6):1143-1151.
[10]MOAV R,FINKEL A,WOHLFARTH G.Variability of intermuscular bones,vertebrae,ribs,dorsal fin rays and skeletal disorders in the common carp[J].Theoretical and applied genetics,1975,46(1):33-43.
[11]田雪,王良炎,陈琳,等.OST基因在淇河鲫肌间骨骨化过程中的表达研究[J].水产学报,2016,40(5):673-680.
[12]王良炎,田雪,庞小磊,等.硬化蛋白基因在淇河鲫成鱼不同肌间骨相邻肌组织的表达差异分析[J].中国生物化学与分子生物学报,2016,32(12):1354-1359.
[13]JAVED A,BAE J S,AFZAL F,et al.Structural coupling of smad and runx2for execution of the BMP2osteogenic signal[J].Journal of biological chemistry,2008,283(13):8412-8422.
[14]ZHANG W Z,LAN T,NIE C H,et al.Characterization and spatiotemporal expression analysis of nine bone morphogenetic protein family genes during intermuscular bone development in blunt snout bream[J].Gene,2018,642:116-124.
[15]WAN S M,YI S K,ZHONG J,et al.Dynamic mRNA and miR-NA expression analysis in response to intermuscular bone development of blunt snout bream(Megalobrama amblycephala)[J].Scientific reports,2016,6.DOI:10.1038/srep31050.
[16]LIU H,CHEN C H,GAO Z X,et al.The draft genome of blunt snout bream(Megalobrama amblycephala)reveals the development of intermuscular bone and adaptation to herbivorous diet[J].Giga science,2017,6(7):1-13.
[17]KARSENTY G,WAGNER E F.Reaching agenetic and molecular understanding of skeletal development[J].Developmental cell,2002,2(4):389-406.
[18]BRANDAU O,MEINDL A,FSSLER R,et al.A novel gene,tendin,is strongly expressed in tendons and ligaments and shows high homology with chondromodulin-I[J].Developmental dynamics,2010,221(1):72-80.
[19]SHUKUNAMI C,TAKIMOTO A,NISHIZAKI Y,et al.Scleraxis is a transcriptional activator that regulates the expression of Tenomodulin,a marker of mature tenocytes and ligamentocytes[J].Scientific reports,2018,8:3155.DOI:10.1038/S41598-018-21194-3.
[20]TOLPPANEN A M,PULKKINEN L,KOLEHMAINEN M,et al.Tenomodulin is associated with obesity and diabetes risk:the Finnish diabetes prevention study[J].Obesity,2012,15(5):1082-1088.
[21]TOLPPANEN A M,LAPPALAINEN T J,KOLEHMAINENM,et al.Tenomodulin mRNA levels are correlated with serum and mRNA levels of inflammatory markers:the genobin study[J].Diabetes and vascular disease research,2007,4(suppl 1):19-20.
[22]TOLPPANEN A M,PULKKINEN L,HERDER C,et al.The genetic variation of the tenomodulin gene(TNMD)is associated with serum levels of systemic immune mediators-the Finnish diabetes prevention study[J].Genetics in medicine,2008,10(7):536-544.
[23]米康夫,董耀龄.鱼会患糖尿病吗?---用高碳水化合物饵料,鱼生长缓慢,鰤鱼等越是肉食性强的鱼种耐糖能力越低[J].国外水产,1984,22(8):489-491.
[24]KARSENTY G,WAGNER E F.Reaching agenetic and molecular understanding of skeletal development[J].Developmental cell,2002,2(4):389-406.
[25]MCCLURE J.The effect of diphosphonates on heterotopic ossification in regenerating achilles tendon of the mouse[J].Journal of pathology,2010,139(4):419-430.
[26]ASAI S,OTSURU S,CANDELA M E,et al.Tendon progenitor cells in injured tendons have strong chondrogenic potential:The CD105-negative subpopulation induces chondrogenic degeneration[J].Stem cells,2015,32(12):3266-3277.
[27]CHEN J W,GALLOWAY J L.The development of zebrafish tendon and ligament progenitors[J].Development,2014,141(10):2035-2045.
[28]BRADLEY E W,MCGEE-LAWRENCE M E,WESTENDORFJ J.Hdac-mediated control of endochondral and intramembranous ossification[J].Critical reviews in eukaryotic gene expression,2011,21(2):101-113.
[29]ZHANG J,WANG J H.Mechanobiological response of tendon stem cells:implications of tendon homeostasis and pathogenesis of tendinopathy[J].Journal of orthopaedic research,2010,28(5):639-643.
[30]GEMBALL A S,BRITZ R.Homology of intermuscular bones in acanthomorph fishes[J].American museum novitates,1998(3241):1-25.
[31]ZHANG J,WANG H C.Characterization of differential properties of rabbit tendon stem cells and tenocytes[J].BMC musculoskeletal disorders,2010,11(1):10.
[32]BI Y,EHIRCHIOU D,KILTS T M,et al.Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche[J].Nature medicine,2007,13(10):1219-1227.
[33]RUI Y F,LUI P P,CHAN L S,et al.Does erroneous differentiation of tendon-derived stem cells contribute to the pathogenesis of calcifying tendinopathy?[J].Chinese medical journal,2011,124(4):606-610.
[34]DUCY P,ZHANG R,GEOFFROY V,et al.Osf2/Cbfa1:a transcriptional activator of osteoblast differentiation[J].Cell,1997,89(5):747-754.
[35]NI M,LUI P P Y,RUI Y F,et al.Tendon-derived stem cells(TDSCs)promote tendon repair in a rat patellar tendon window defect model[J].Journal of orthopaedic research,2012,30(4):613-619.
[36]TAN Q,LUI P P Y,RUI Y F,et al.Comparison of potentials of stem cells isolated from tendon and bone marrow for musculoskeletal tissue engineering[J].Tissue engineering Part A,2012,18(7/8):840-851.
[37]KISHIMOTO Y,OHKAWARA B,SAKAI T,et al.Wnt/β-catenin signaling suppresses expressions of Scx,Mkx,and Tnmd in tendon-derived cells[J].PLoS One,2017,12(7):e0182051.
[38]KOMORI T,YAGI H,NOMURA S,et al.Targeted disruption of cbfa1results in a complete lack of bone formation owing to maturational arrest of osteoblasts[J].Cell,1997,89(5):755-764.
[39]OTTEN C,VEN P F V D,LEWRENZ I,et al.Xirp proteins markinjured skeletal muscle in zebrafish[J].PLoS One,2012,7(2):e31041.
[2]孟庆闻,苏锦祥,李婉端.鱼类比较解剖学[M].北京:科学出版社,1987:102-103.
[3]PATTERSON C,JOHNSON G D.The intermusoular bones and ligaments of teleostean fishes[J].Smlthsonian contribution to zoology,1995(559):1-83.
[4]JOHNSON G D,PATTERSON C.The intermuscular system of acanthomorph fishes:a commentary[J].American museum novitates,2001(3312):1-25.
[5]吕耀平,鲍宝龙,蒋燕,等.低等真骨鱼类肌间骨的比较分析[J].水产学报,2007,31(5):661-668.
[6]秉志.幼鲤大侧肌隔骨针的观察[J].动物学报,1962(2):32-36.
[7]BIRD N C,MABEE P M.Developmental morphology of the axial skeleton of the zebrafish,Danio rerio(Ostariophysi:Cyprinidae)[J].Developmental dynamics,2003,228(3):337-357.
[8]柯中和,张炜,蒋燕,等.鲢肌间小骨发育的形态学观察[J].动物学杂志,2008,43(6):88-96.
[9]万世明,易少奎,仲嘉,等.团头鲂肌间骨发育的形态学观察[J].水生生物学报,2014,38(6):1143-1151.
[10]MOAV R,FINKEL A,WOHLFARTH G.Variability of intermuscular bones,vertebrae,ribs,dorsal fin rays and skeletal disorders in the common carp[J].Theoretical and applied genetics,1975,46(1):33-43.
[11]田雪,王良炎,陈琳,等.OST基因在淇河鲫肌间骨骨化过程中的表达研究[J].水产学报,2016,40(5):673-680.
[12]王良炎,田雪,庞小磊,等.硬化蛋白基因在淇河鲫成鱼不同肌间骨相邻肌组织的表达差异分析[J].中国生物化学与分子生物学报,2016,32(12):1354-1359.
[13]JAVED A,BAE J S,AFZAL F,et al.Structural coupling of smad and runx2for execution of the BMP2osteogenic signal[J].Journal of biological chemistry,2008,283(13):8412-8422.
[14]ZHANG W Z,LAN T,NIE C H,et al.Characterization and spatiotemporal expression analysis of nine bone morphogenetic protein family genes during intermuscular bone development in blunt snout bream[J].Gene,2018,642:116-124.
[15]WAN S M,YI S K,ZHONG J,et al.Dynamic mRNA and miR-NA expression analysis in response to intermuscular bone development of blunt snout bream(Megalobrama amblycephala)[J].Scientific reports,2016,6.DOI:10.1038/srep31050.
[16]LIU H,CHEN C H,GAO Z X,et al.The draft genome of blunt snout bream(Megalobrama amblycephala)reveals the development of intermuscular bone and adaptation to herbivorous diet[J].Giga science,2017,6(7):1-13.
[17]KARSENTY G,WAGNER E F.Reaching agenetic and molecular understanding of skeletal development[J].Developmental cell,2002,2(4):389-406.
[18]BRANDAU O,MEINDL A,FSSLER R,et al.A novel gene,tendin,is strongly expressed in tendons and ligaments and shows high homology with chondromodulin-I[J].Developmental dynamics,2010,221(1):72-80.
[19]SHUKUNAMI C,TAKIMOTO A,NISHIZAKI Y,et al.Scleraxis is a transcriptional activator that regulates the expression of Tenomodulin,a marker of mature tenocytes and ligamentocytes[J].Scientific reports,2018,8:3155.DOI:10.1038/S41598-018-21194-3.
[20]TOLPPANEN A M,PULKKINEN L,KOLEHMAINEN M,et al.Tenomodulin is associated with obesity and diabetes risk:the Finnish diabetes prevention study[J].Obesity,2012,15(5):1082-1088.
[21]TOLPPANEN A M,LAPPALAINEN T J,KOLEHMAINENM,et al.Tenomodulin mRNA levels are correlated with serum and mRNA levels of inflammatory markers:the genobin study[J].Diabetes and vascular disease research,2007,4(suppl 1):19-20.
[22]TOLPPANEN A M,PULKKINEN L,HERDER C,et al.The genetic variation of the tenomodulin gene(TNMD)is associated with serum levels of systemic immune mediators-the Finnish diabetes prevention study[J].Genetics in medicine,2008,10(7):536-544.
[23]米康夫,董耀龄.鱼会患糖尿病吗?---用高碳水化合物饵料,鱼生长缓慢,鰤鱼等越是肉食性强的鱼种耐糖能力越低[J].国外水产,1984,22(8):489-491.
[24]KARSENTY G,WAGNER E F.Reaching agenetic and molecular understanding of skeletal development[J].Developmental cell,2002,2(4):389-406.
[25]MCCLURE J.The effect of diphosphonates on heterotopic ossification in regenerating achilles tendon of the mouse[J].Journal of pathology,2010,139(4):419-430.
[26]ASAI S,OTSURU S,CANDELA M E,et al.Tendon progenitor cells in injured tendons have strong chondrogenic potential:The CD105-negative subpopulation induces chondrogenic degeneration[J].Stem cells,2015,32(12):3266-3277.
[27]CHEN J W,GALLOWAY J L.The development of zebrafish tendon and ligament progenitors[J].Development,2014,141(10):2035-2045.
[28]BRADLEY E W,MCGEE-LAWRENCE M E,WESTENDORFJ J.Hdac-mediated control of endochondral and intramembranous ossification[J].Critical reviews in eukaryotic gene expression,2011,21(2):101-113.
[29]ZHANG J,WANG J H.Mechanobiological response of tendon stem cells:implications of tendon homeostasis and pathogenesis of tendinopathy[J].Journal of orthopaedic research,2010,28(5):639-643.
[30]GEMBALL A S,BRITZ R.Homology of intermuscular bones in acanthomorph fishes[J].American museum novitates,1998(3241):1-25.
[31]ZHANG J,WANG H C.Characterization of differential properties of rabbit tendon stem cells and tenocytes[J].BMC musculoskeletal disorders,2010,11(1):10.
[32]BI Y,EHIRCHIOU D,KILTS T M,et al.Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche[J].Nature medicine,2007,13(10):1219-1227.
[33]RUI Y F,LUI P P,CHAN L S,et al.Does erroneous differentiation of tendon-derived stem cells contribute to the pathogenesis of calcifying tendinopathy?[J].Chinese medical journal,2011,124(4):606-610.
[34]DUCY P,ZHANG R,GEOFFROY V,et al.Osf2/Cbfa1:a transcriptional activator of osteoblast differentiation[J].Cell,1997,89(5):747-754.
[35]NI M,LUI P P Y,RUI Y F,et al.Tendon-derived stem cells(TDSCs)promote tendon repair in a rat patellar tendon window defect model[J].Journal of orthopaedic research,2012,30(4):613-619.
[36]TAN Q,LUI P P Y,RUI Y F,et al.Comparison of potentials of stem cells isolated from tendon and bone marrow for musculoskeletal tissue engineering[J].Tissue engineering Part A,2012,18(7/8):840-851.
[37]KISHIMOTO Y,OHKAWARA B,SAKAI T,et al.Wnt/β-catenin signaling suppresses expressions of Scx,Mkx,and Tnmd in tendon-derived cells[J].PLoS One,2017,12(7):e0182051.
[38]KOMORI T,YAGI H,NOMURA S,et al.Targeted disruption of cbfa1results in a complete lack of bone formation owing to maturational arrest of osteoblasts[J].Cell,1997,89(5):755-764.
[39]OTTEN C,VEN P F V D,LEWRENZ I,et al.Xirp proteins markinjured skeletal muscle in zebrafish[J].PLoS One,2012,7(2):e31041.
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