脊尾白虾肌球蛋白重链和肌球蛋白轻链基因的克隆与表达分析
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摘要
肌球蛋白重链和肌球蛋白轻链是粗肌丝的重要组成单位,其表达量的高低影响肌纤维的组成和肌肉生长。为了解析肌球蛋白重链(Myosin heavy chain,MHC)和肌球蛋白轻链(Myosin light chain,MLC)基因在脊尾白虾(Exopalaemon carinicauda)生长发育中的作用,采用RACE技术,克隆了脊尾白虾EcMHC2、EcMLC1全长cDNA序列,采用实时荧光定量PCR方法分析了EcMHC2、EcMLC1在脊尾白虾不同组织、不同幼体发育时期及大小差异个体在不同生长时期的表达特征。研究显示,脊尾白虾EcMHC2基因(GenBank:MG545144)全长5 929bp,开放阅读框5 727bp,编码1 909个氨基酸;脊尾白虾EcMLC1基因(GenBank:MG545145)全长1 506bp,开放阅读框459bp,编码153个氨基酸。脊尾白虾肌球蛋白重链和肌球蛋白轻链在系统进化上与其它十足目甲壳动物的关系较近。脊尾白虾EcMHC2、EcMLC1在各组织中均有表达,但主要在肌肉组织内表达。在脊尾白虾幼体发育的不同时期,EcMHC2、EcMLC1在溞状幼体Ⅱ期的表达量显著高于溞状幼体的其它时期;仔虾第一天,EcMHC2、EcMLC1的表达量显著高于其它时期。在脊尾白虾生长的四个阶段中,大个体组EcMHC2的表达量均显著的高于小个体组;60、80和120日龄时期,大个体组的EcMLC1表达量显著的高于小个体组;在大个体和小个体组中,EcMHC2、EcMLC1在80日龄的表达量最高。脊尾白虾EcMHC2、EcMLC1在大个体组与小个体组的差异表达提示它们在脊尾白虾生长发育过程中发挥重要作用。
Myosin heavy chain(MHC)and myosin light chain(MLC)are the major components of the muscle thick filament.The expression levels of MHCand MLChave significant influences on the formation of thick filaments and muscle development.To study the regulatory roles of MHCand MLCon growth during the development of E.carinicauda,the full-length MHCand MLCcDNA of E.carinicauda(EcMHC2 and EcMLC1)were isolated with the methods of rapid amplification of the cDNA ends(RACE).Quantitative real-time PCR was used to quantify the relative expression level of EcMHC2 and EcMLC1 in different tissues,different larval development stages and heavier group(H)and lighter group at different growth stages.The results showed that the full-length of EcMHC2(MG545144)was5 929 bp in length,containing an open reading frame(ORF)of 5 727 bp encoding a 1 909 amino acid polypeptide,and the full-length of EcMLC1(MG545145)was 1 506 bp in length,containing an ORF of459 bp encoding a 153 amino acid polypeptide.EcMHC2 and EcMLC1 were expressed in various tissues and the highest in the muscle tissue.The EcMHC2 and EcMLC1 mRNA abundance reached the maximum at the first day of postlarval stage,and the EcMHC2 and EcMLC1 expression levels were significantly higher in zoeaⅡthan in other zoea stage during the larval development.EcMHC2 and EcMLC1 strongly expressed in heavier group while weakly expressed in lighter group at different growth stages excepted for EcMLC1 at 100 days of age.The EcMHC2 and EcMLC1 expression levels were higher at 80 days of age than at other days of age.The different expression levels of these two genes in heavier and lighter groups at different growth stages suggested that they played important roles in the growth development of E.carinicauda.
Myosin heavy chain(MHC)and myosin light chain(MLC)are the major components of the muscle thick filament.The expression levels of MHCand MLChave significant influences on the formation of thick filaments and muscle development.To study the regulatory roles of MHCand MLCon growth during the development of E.carinicauda,the full-length MHCand MLCcDNA of E.carinicauda(EcMHC2 and EcMLC1)were isolated with the methods of rapid amplification of the cDNA ends(RACE).Quantitative real-time PCR was used to quantify the relative expression level of EcMHC2 and EcMLC1 in different tissues,different larval development stages and heavier group(H)and lighter group at different growth stages.The results showed that the full-length of EcMHC2(MG545144)was5 929 bp in length,containing an open reading frame(ORF)of 5 727 bp encoding a 1 909 amino acid polypeptide,and the full-length of EcMLC1(MG545145)was 1 506 bp in length,containing an ORF of459 bp encoding a 153 amino acid polypeptide.EcMHC2 and EcMLC1 were expressed in various tissues and the highest in the muscle tissue.The EcMHC2 and EcMLC1 mRNA abundance reached the maximum at the first day of postlarval stage,and the EcMHC2 and EcMLC1 expression levels were significantly higher in zoeaⅡthan in other zoea stage during the larval development.EcMHC2 and EcMLC1 strongly expressed in heavier group while weakly expressed in lighter group at different growth stages excepted for EcMLC1 at 100 days of age.The EcMHC2 and EcMLC1 expression levels were higher at 80 days of age than at other days of age.The different expression levels of these two genes in heavier and lighter groups at different growth stages suggested that they played important roles in the growth development of E.carinicauda.
引文
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[21] Kawasaki H,Kretsinger R H.Structural and functional diversity of EF-hand proteins:Evolutionary perspectives[J].Protein Science a Publication of the Protein Society,2017,26(10):1898-1920.
[22]陈剑清,张耀洲.EF手图像超家族成员——肌钙蛋白C的研究进展[J].生物工程学报,2007,23(3):375-380.Chen J Q,Zhang Y Z.The Research Progress of One Member of the EF-handSuperfamily—Troponin C[J].Chinese Journal of Biotechnology,2007,23(3):375-380.
[23] Kalyanasundaram A,Santiago T C.Identification and characterization of new allergen troponin C(Pen m 6.0101)from Indian black tiger shrimp Penaeus monodon[J].European Food Research&Technology,2015,240(3):509-515.
[24] Tanaka H,Takahashi H,Ojima T.Ca2+-binding properties and regulatory roles of lobster troponin C sites II and IV[J].Febs Letters,2013,587(16):2612.
[25] Pette D,Staron R S.Myosin isoforms,muscle fiber types,and transitions[J].Microscopy Research&Technique,2015,50(6):500-509.
[26] Lowey S,Trybus K M.Role of skeletal and smooth muscle myosin light chains[J].Biophysical Journal,1995,68(4):120S.
[27]农小献,赵发兰,陈敦学,等.翘嘴鳜肌球蛋白轻链3b基因(MLC3b)的分子克隆和特征分析[J].基因组学与应用生物学,2010,29(6):1033-1038.Nong X X,Zhao F L,Chen D X,et al.Molecular cloning and characteristic analysis of myosin light chain 3b(MLC3b)gene fromSiniperca chuasti[J].Genomics and Applied Biology,2010,29(6):1033-1038.
[28]申建梅,胡黎明,宾淑英,等.桔小实蝇肌球蛋白轻链2基因的克隆及表达分析[J].昆虫学报,2011,54(5):508-514.Shen J M,Hu L M,Bin S Y,et al.Cloning and expression profiling of myosin light chain 2gene in Bactrocera dorsalis[J].Acta Entomologica Sinica,2011,54(5):508-514.
[29] Chen X,Luo Y,Huang Z,et al.Akirin2promotes slow myosin heavy chain expression by CaN/NFATc1signaling in porcine skeletal muscle satellite cells[J].Oncotarget,2017,8(15):25158-25166.
[30] Gossett L A,Kelvin D J,Sternberg E A,et al.A new myocytespecific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes[J].Molecular&Cellular Biology,1989,9(11):5022-5033.
[31] Myer A,Olson E N,Klein W H.MyoD cannot compensate for the absence of myogenin during skeletal muscle differentiation in murine embryonic stem cells[J].Developmental Biology,2001,229(2):340-350.
[32] Rooij E V,Quiat D,Johnson B A,et al.A family of micro RNAs encoded by myosin genes governs myosin expression and muscle performance[J].Developmental Cell,2009,17(5):662-673.
[33]王绪峨.脊尾白虾的幼体发育[J].海洋科学,1991,15(1):12-14.Wang X E.The Larval Development of Exopalaemon carinicauda[J].Marine Science,1991,15(1):12-14.
[34]李艳艳,邵芳,吴小娟,等.鹅肌球蛋白重链1基因MyHC1全长cDNA克隆、序列及胚胎期表达特征分析[J].农业生物技术学报,2015,23(3):359-368.Li Y Y,Shao F,Wu X J,et al.Molecular cloning,sequence and expression characterization of myosin heavy chain 1genein goose(Anseranser)at embryonic period[J].Journal of Agricultural Biotechnology,2015,23(3):359-368.
[35]周瑞雪,黄斌,蒙涛,等.鳜碱性肌球蛋白轻链基因cDNA的克隆及其发育表达分析[J].水生生物学报,2009,34(5):927-934.Zhou R X,Shao F,Wu X J,et al.Cloning and ontogenetic expression analysis of the alkali myosin light chain gene in Siniperca chuasti[J].Acta Hydrobiologica Sinica,2009,34(3):927-934.
[36] Hevroy E M,Jordal A E O,Hordvik I,et al.Myosin heavy chain mRNA expression correlates higher with muscle protein accretion than growth in Atlantic salmon,Salmosalar[J].Aquaculture,2006,252(2-4):453-461.
[37]陈之航,董浚键,孙成飞,等.基于转录组测序对翘嘴鳜(Sinipercachuatsi)2种肌球蛋白重链基因的克隆与分析[J].渔业科学进展,2017,38(3):51-61.Chen Z H,Dong J J,Sun F C,et al.cDNA cloning and analyses of Two myosin heavy chain isoforms of mandarin fish(Sinipercachuatsi)based on transcriptome sequencing[J].Progress in Fishery Sciences,2017,38(3):51-61.
[38] Ren X,Yu X,Gao B,et al.iTRAQ-based identification of differentially expressed proteins related to growth in the swimming crab,Portunus trituberculatus[J].Aquaculture Research,2017,48(6):1-11.
[2] Jung H,Lyons R E,Hurwood D A,et al.Genes and growth performance in crustacean species:A review of relevant genomic studies in crustaceans and other taxa[J].Reviews in Aquaculture,2013,5(2):77-110.
[3]阎斌伦,薛蓓,赵莲,等.甲壳类生长相关基因研究进展[J].海洋湖沼通报,2016(4):97-104.Yan Bin Lun,XueBei,Zhao Lian,et al.Progress on the research of the growth-related genes in crustaceans[J].Transactions of Oceanology and Limnology,2016(4):97-104.
[4] Ge Q Q,Li J,Duan Y F,et al.Isolation of prawn(Exopalaemon carinicauda)lipopolysaccharide andβ-1,3-Glucan binding protein gene and its expressionin responding to bacterial and viral infections[J].Journal of Ocean University of China,2016,15(2):288-296.
[5] Knight A E,Molloy J E.Muscle,myosin and single molecules[J].Essays in Biochemistry,2000,35(35):43.
[6] Heissler S M,Sellers J R.Various themes of myosin regulation[J].Journal of Molecular Biology,2016,428(9):1927-1946.
[7] Santos M,Moura R S,Gonzaga S,et al.Embryonic essential myosin light chain regulates fetal lung development in rats[J].American Journal of Respiratory Cell&Molecular Biology,2007,37(3):330-338.
[8] Andruchov O,Andruchova O,Wang Y,et al.Dependence of cross-bridge kinetics on myosin light chain isoforms in rabbit and rat skeletal muscle fibres[J].The Journal of Physiology,2006,571(1):231-242.
[9] Heissler S M,Sellers J R.Kinetic adaptations of myosins for their diverse cellular functions[J].Traffic,2016,17(8):839-859.
[10] Berg J S,Powell B C,Cheney R E.A millennial myosin census[J].Molecular Biology of the Cell,2001,12(4):780.
[11] Kim G D,Yang H S,Jeong J Y.Comparison of myosin heavy chain content determined by label-free quantification between porcine longissimus thoracis,psoas major and semimembranosus muscles[J].Food Research International,2017,10:504-513.
[12] Kang J H,Bang W Y,Eunjung K,et al.Expression of porcine myosin heavy chain 1gene in Berkshire loins with a high pH24value[J].African Journal of Biotechnology,2016,9(44):7568-7572.
[13] Asaduzzaman M,Akolkar D B,Kinoshita S,et al.The expression of multiple myosin heavy chain genes during skeletal muscle development of torafuguTakifugurubripes embryos and larvae[J].Gene,2013,515(1):144-154.
[14]李文兰,盛小伟,高永华,等.凡纳滨对虾肌球蛋白轻链基因MLC的克隆及表达分析[J].西南农业学报,2011,24(4):1558-1563.Li W L,Sheng X W,Gao Y H,et al.Clone and expression analysis of myosin light chain in Litopenaeus vannamei[J].Southwest China Journal of Agricultural Sciences,2011,24(4):1558-1563.
[15] Koyama H,Akolkar D B,Piyapattanakorn S,et al.Cloning,expression,and localization of two types of fast skeletal myosin heavy chain genes from black tiger and Pacific white shrimps[J].Journal of Experimental Zoology Part A Ecological Genetics&Physiology,2012,317(10):608-621.
[16]蒲红双,高祥刚,侯红漫,等.甲壳动物蜕皮相关基因的研究进展[J].水产科学,2015,34(6):391-398.Pu S H,Gao X G,Hou H M,et al.Advances in Genes Related to Molting in Crustacean[J].Fisheries Science,2015,34(6):391-398.
[17]梁象秋,李亚娟,周昭曼.脊尾白虾的幼体发育[J].水产学报,1988,12(2):157-168.Liang X Q,Li Y J,Zhou Z M,The larval development of Exopalaemon carinicauda[J].Journal of Fisheries of China,1988,12(2):157-168.
[18] Duan Y,Liu P,Li J,et al.cDNA cloning,characterization and expression analysis of peroxiredoxin 5gene in the ridgetail white prawn Exopalaemon carinicauda[J].Molecular Biology Reports,2013,40:6569-6577.
[19] Gordon A M,Homsher E,Regnier M.Regulation of contraction in striated muscle[J].Physiological Reviews,2000,80(2):853-924.
[20] Rayment I,Holden H M,Whittaker M,et al.Structure of the actin-myosin complex and its implications for muscle contraction[J].Science,1993,261(5117):58-65.
[21] Kawasaki H,Kretsinger R H.Structural and functional diversity of EF-hand proteins:Evolutionary perspectives[J].Protein Science a Publication of the Protein Society,2017,26(10):1898-1920.
[22]陈剑清,张耀洲.EF手图像超家族成员——肌钙蛋白C的研究进展[J].生物工程学报,2007,23(3):375-380.Chen J Q,Zhang Y Z.The Research Progress of One Member of the EF-handSuperfamily—Troponin C[J].Chinese Journal of Biotechnology,2007,23(3):375-380.
[23] Kalyanasundaram A,Santiago T C.Identification and characterization of new allergen troponin C(Pen m 6.0101)from Indian black tiger shrimp Penaeus monodon[J].European Food Research&Technology,2015,240(3):509-515.
[24] Tanaka H,Takahashi H,Ojima T.Ca2+-binding properties and regulatory roles of lobster troponin C sites II and IV[J].Febs Letters,2013,587(16):2612.
[25] Pette D,Staron R S.Myosin isoforms,muscle fiber types,and transitions[J].Microscopy Research&Technique,2015,50(6):500-509.
[26] Lowey S,Trybus K M.Role of skeletal and smooth muscle myosin light chains[J].Biophysical Journal,1995,68(4):120S.
[27]农小献,赵发兰,陈敦学,等.翘嘴鳜肌球蛋白轻链3b基因(MLC3b)的分子克隆和特征分析[J].基因组学与应用生物学,2010,29(6):1033-1038.Nong X X,Zhao F L,Chen D X,et al.Molecular cloning and characteristic analysis of myosin light chain 3b(MLC3b)gene fromSiniperca chuasti[J].Genomics and Applied Biology,2010,29(6):1033-1038.
[28]申建梅,胡黎明,宾淑英,等.桔小实蝇肌球蛋白轻链2基因的克隆及表达分析[J].昆虫学报,2011,54(5):508-514.Shen J M,Hu L M,Bin S Y,et al.Cloning and expression profiling of myosin light chain 2gene in Bactrocera dorsalis[J].Acta Entomologica Sinica,2011,54(5):508-514.
[29] Chen X,Luo Y,Huang Z,et al.Akirin2promotes slow myosin heavy chain expression by CaN/NFATc1signaling in porcine skeletal muscle satellite cells[J].Oncotarget,2017,8(15):25158-25166.
[30] Gossett L A,Kelvin D J,Sternberg E A,et al.A new myocytespecific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes[J].Molecular&Cellular Biology,1989,9(11):5022-5033.
[31] Myer A,Olson E N,Klein W H.MyoD cannot compensate for the absence of myogenin during skeletal muscle differentiation in murine embryonic stem cells[J].Developmental Biology,2001,229(2):340-350.
[32] Rooij E V,Quiat D,Johnson B A,et al.A family of micro RNAs encoded by myosin genes governs myosin expression and muscle performance[J].Developmental Cell,2009,17(5):662-673.
[33]王绪峨.脊尾白虾的幼体发育[J].海洋科学,1991,15(1):12-14.Wang X E.The Larval Development of Exopalaemon carinicauda[J].Marine Science,1991,15(1):12-14.
[34]李艳艳,邵芳,吴小娟,等.鹅肌球蛋白重链1基因MyHC1全长cDNA克隆、序列及胚胎期表达特征分析[J].农业生物技术学报,2015,23(3):359-368.Li Y Y,Shao F,Wu X J,et al.Molecular cloning,sequence and expression characterization of myosin heavy chain 1genein goose(Anseranser)at embryonic period[J].Journal of Agricultural Biotechnology,2015,23(3):359-368.
[35]周瑞雪,黄斌,蒙涛,等.鳜碱性肌球蛋白轻链基因cDNA的克隆及其发育表达分析[J].水生生物学报,2009,34(5):927-934.Zhou R X,Shao F,Wu X J,et al.Cloning and ontogenetic expression analysis of the alkali myosin light chain gene in Siniperca chuasti[J].Acta Hydrobiologica Sinica,2009,34(3):927-934.
[36] Hevroy E M,Jordal A E O,Hordvik I,et al.Myosin heavy chain mRNA expression correlates higher with muscle protein accretion than growth in Atlantic salmon,Salmosalar[J].Aquaculture,2006,252(2-4):453-461.
[37]陈之航,董浚键,孙成飞,等.基于转录组测序对翘嘴鳜(Sinipercachuatsi)2种肌球蛋白重链基因的克隆与分析[J].渔业科学进展,2017,38(3):51-61.Chen Z H,Dong J J,Sun F C,et al.cDNA cloning and analyses of Two myosin heavy chain isoforms of mandarin fish(Sinipercachuatsi)based on transcriptome sequencing[J].Progress in Fishery Sciences,2017,38(3):51-61.
[38] Ren X,Yu X,Gao B,et al.iTRAQ-based identification of differentially expressed proteins related to growth in the swimming crab,Portunus trituberculatus[J].Aquaculture Research,2017,48(6):1-11.