日本沼虾几丁质酶1C(MnCht1C)基因的克隆及表达分析
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摘要
本研究从日本沼虾精巢均一化c DNA文库中筛选到一条Mn Cht1C表达序列片段(expressed sequence tags,EST),该序列具有完整的基因编码区,编码区长度为1 473 bp,编码490个氨基酸。Mn Cht1C编码的蛋白质具有完整的几丁质酶结构,N端的前23个氨基酸为信号肽,第24~406位氨基酸序列为几丁质酶催化结构域(catalytic domain),第407~433位氨基酸序列为Ser/Thr富含区(S/T-rich linker),其作用在于连接催化结构域与几丁质结合结构域。q PCR分析Mn Cht1C在日本沼虾各组织和各发育阶段表达情况,研究发现其主要在肝胰腺、肠、眼柄、表皮、精巢5种组织中表达;幼体发育时期不表达,而直至变态时期才开始表达。Mn Cht1C在日本沼虾整个蜕壳周期的表达呈波动性,蜕壳中期表达水平最高,随着蜕壳这一生理过程的完成,其表达水平也相应地下降直至蜕壳间期无表达水平检出,随着新一轮蜕壳的进行,其表达水平又开始逐渐升高。本研究结果为研究几丁质酶基因在日本沼虾和其他甲壳动物蜕壳及生殖发育中的作用提供参考。
In this study,one EST sequence coding MnCht1C was cloned and identified from the Macrobrachium nipponense(M.nipponense)testis c DNA library.The MnCht1C EST had a complete open reading frame(ORF)of 1473 bp,encoding 490 amino acids.The encoded protein by MnCht1C demonstrated complete chitinase structure,with a cleavable signal peptide of 23 amino acid residues in the N-terminal.The encoded protein also comprised catalytic domain and S/T rich linker region to connect the catalytic domain with chitin binding domain,which were on the 24~th~406~th and 407~th~433~th of amino acid sequences,respectively.Expression patterns and tissue distribution of M.nipponense MnCht1C were analyzed by q PCR.The results suggested that Mn Cht1C was expressed in the tissues of hepatopancreas,gut,eyestalk,cuticle and testis.There were no expression of Mn Cht1C in the larval stage and Mn Cht1C has been not expressed until metamorphosis.Mn Cht1C level fluctuated during the molt period,showing the highest level in ecdysis,then it decreased gradually until non-expression in ecdysis.As a new molt cycle started,the expression level of Mn Cht1C was up-regulated slowly.This study might provide important information for further investigations on the functions of chitinase in molting and reproductive development of M.nipponense and other crustaceans.
In this study,one EST sequence coding MnCht1C was cloned and identified from the Macrobrachium nipponense(M.nipponense)testis c DNA library.The MnCht1C EST had a complete open reading frame(ORF)of 1473 bp,encoding 490 amino acids.The encoded protein by MnCht1C demonstrated complete chitinase structure,with a cleavable signal peptide of 23 amino acid residues in the N-terminal.The encoded protein also comprised catalytic domain and S/T rich linker region to connect the catalytic domain with chitin binding domain,which were on the 24~th~406~th and 407~th~433~th of amino acid sequences,respectively.Expression patterns and tissue distribution of M.nipponense MnCht1C were analyzed by q PCR.The results suggested that Mn Cht1C was expressed in the tissues of hepatopancreas,gut,eyestalk,cuticle and testis.There were no expression of Mn Cht1C in the larval stage and Mn Cht1C has been not expressed until metamorphosis.Mn Cht1C level fluctuated during the molt period,showing the highest level in ecdysis,then it decreased gradually until non-expression in ecdysis.As a new molt cycle started,the expression level of Mn Cht1C was up-regulated slowly.This study might provide important information for further investigations on the functions of chitinase in molting and reproductive development of M.nipponense and other crustaceans.
引文
Arakane Y.,and Muthukrishnan S.,2010,Insect chitinase and chitinase-like proteins,Cell Mol.Life Sci.,67(2):201-216
Arakane Y.,Zhu Q.,Matsumiya M.,Muthukrishnan S.,and Kramer K.J.,2003,Properties of catalytic,linker and chitin-binding domains of insect chitinase,Insect Biochem.Mol.Biol.,33(6):631-648
Daimon T.,Hamada K.,Mita K.,Okano K.,Suzuki M.G.,Kobayashi M.,and Shimada T.,2003,A Bombyx mori gene,Bm Chi-h,encodes a protein homologous to bacterial and baculovirus ch itinases,Insect Biochem.Mol.Biol.,33(8):749-759
Gilkes N.R.,Henrissat B.,Kilburn D.G.,Miller R.C.,and Warren R.A.,1991,Domains in microbial beta-1,4-glycanases:sequence conservation,function,and enzyme families,Microbiol.Rev.,55(2):303-315
Hart P.J,Pfluger H.D.,Monzingo A.F.,Hollis T.,and Robertus J.D.,1995,The refined crystal structure of an endochitinase from Hordeum vulgare L.seeds at 1.8 A resolution,J.Mol.Biol.,248(2):402-413
Houston D.R.,Recklies A.D.,Krupa J.C.,and van Aalten D.M.,2003,Structure and ligand-induced conformational change of the 39 k Da glycoprotein from human articular chondrocytes,J.Biol.Chem.,278(32):30206-30212
Huang Q.S.,Yan J.H.,Tang J.Y.,Tao Y.M.,Xie X.L.,Wang Y.,Wei X.Q.,Yan Q.H.,and Chen Q.X.,2010,Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei,Fish Shellfish Immunol.,29(1):75-81
Li D.C.,2006,Review of fungal chitinases,Mycopathologia,161(6):345-360
Li X.G.,Xu Z.Q.,Zhou G.,Lin H.,Zhou J.,Zeng Q.,Mao Z.,and Gu X.H.,2015,Molecular characterization and expression analysis of five chitinases associated with molting in the Chinese mitten crab,Eriocheir sinensis,Comp.Biochem.Physiol.B.Biochem.Mol.Biol.,187:110-120
Lu Y.,Zen K.C.,Muthukrishnan S.,and Kramer K.J.,2002,Site-directed mutagenesis and functional analysis of active site acidic amino acid residues D142,D144 and E146in Manduca sexta(tobacco hornworm)chitinase,Insect Biochem.Mol.Biol.,32(11):1369-1382
Merzendorfer H.,and Zimoch L.,2003,Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases,J.Exp.Biol.,206(24):4393-4412
Pan D.,He N.,Yang Z.,Liu H.,and Xu X.,2005,Differential gene expression profile in hepatopancreas of WSSV-resistant shrimp(Penaeus japonicus)by suppression subtractive hybridization,Dev.Comp.Immunol.,29(2):103-112
Priya T.A.,Li F.,Zhang J.,Wang B.,Zhao C.,and Xiang J.,2009,Molecular characterization and effect of RNA interference of retinoid X receptor(RXR)on E75 and chitinase gene expression in chinese shrimp Fenneropenaeus chinensis,Comp.Biochem.Physiol.B.Biochem.Mol.Biol.,153(1):121-129
Proespraiwong P.,Tassanakajon A.,and Rimphanitchayakit V.,2010,Chitinases from the black tiger shrimp Penaeus monodon:phylogenetics,expression and activities,Comp.Biochem.Physiol.B.Biochem.Mol.Biol.,156(2):86-96
Qiao H.,Fu H.T.,Jin S.,Wu Y.,Jiang S.F.,Gong Y.W.,and Xiong Y.S.,2012,Constructing and random sequencing analysis of normali zed c DNA library of testis tissue from orien tal river prawn(Macrobrachium nipponense),Comp.Biochem.Physiol.Part D Genomics Proteomics,7(3):268-276
Renkema G.H.,Boot R.G.,Muijsers A.O.,Donker-Koopman W.E.,and Aerts J.M.,1995,Purification and characterization of human chitotriosidase,a novel member of the chitinase family of proteins,J.Biol.Chem.,270(5):2198-2202
Salma U.,Uddowla M.H.,Kim M.,Kim J.M.,Kim B.K.,Baek H.J.,Park H.,Mykles D.L.,and Kim H.W.,2012,Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp(Pandalopsis japonica):cloning and effects of eyestalk ablation on gene expression,Comp.Biochem.Physiol.B.Biochem.Mol.Biol.,161(3):197-207
Shao L.,Devenport M.,and Jacobs-Lorena M.,2001,The peritrophic matrix of hematophagous insects,Arch.Insect Biochem.Physiol.,47(2):119-125
Skinner D.M.,1962,The structure and metabolism of a crustacean integumentary tissue during a molt cycle,Biol.Bull.,123(3):635-647
Taira T.,Fujiwara M.,Dennhart N.,Hayashi H.,Onaga S.,Ohnuma T.,Letzel T.,Sakuda S.,and Fukamizo T.,2010,Transglycosylation reaction catalyzed by a class V chitinase from cycad,cycas revoluta:a study involving site-directed mutagenesis,HPLC,and real-time ESI-MS,Biochim.Biophys.Acta.,1804(4):668-675
Tan S.H.,Degnan B.M.,and Lehnert S.A.,2000,The Penaeus monodon chitinase 1 gene is differentially expressed in the hepatopancreas during the molt cycle,Mar.Biotechnol.,2(2):126-315
Tjoelker L.W.,Gosting L.,Frey S.,Hunter C.L.,Trong H.L.,Steiner B.,Brammer H.,and Gray P.W.,2000,Structural and functional definition of the human chitinase chitin-binding domain,J.Biol.Chem.,275(1):514-520
Tronsmo A.,and Harman G.E.,1993,Detection and quantification of N-acetyl-beta-D-glucosaminidase,chitobiosidase,and endochitinase in solutions and on gels,Anal.Biochem.,208(1):74-79
Watanabe T.,and Kono M.,1997,Isolation of a c DNA encoding a chitinase family protein from cuticular tissues of the kuruma prawn Penaeus japonicus,Zoolog.Sci.,14(1):65-68
Watanabe T.,Kobori K.,Miyashita K.,Fujii T.,Sakai H.,Uchida M.,and Tanaka H.,1993,Identification of glutamic acid204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity,J.Biol.Chem.,268(25):18567-18572
Watanabe T.,Kono M.,Aida K.,and Nagasawa H.,1998,Purification and molecular cloning of a chitinase expressed in the hepatopancreas of the penaeid prawn Penaeus japonicus,Biochim.Biophys.Acta.,1382(2):181-185
Wong E.,Vaaje-Kolstad G.,Ghosh A.,Hurtado-Guerrero R.,Konarev P.V.,Ibrahim A.F.,Svergun D.I.,Eijsink V.G.,Chatterjee N.S.,and van Aalten D.M.,2012,The Vibrio cholerae colonization factor Gbp A possesses a modular structure that governs binding to different host surfaces,PLo S Pathoge.,8(1):e1002373
Wu P.,Qi D.,Chen L.Q.,Zhang H.,Zhang X.W.,Qin J.G.,and Hu S.N.,2009,Gene discovery from an ovary c DNA library of oriental river prawn Macrobrachium nipponense by ESTs annotation,Comp.Biochem.Physiol.Part D Genomics Proteomics,4(2):111-120
Yuan H.,Zhou Q.,Zhang S.Y.,Fu H.T.,Sun S.M.,Qiao H.,andZhang W.Y.,2015,Full-length c DNA cloning and spatial-temporal expression analysis of ubiquitin-specific protease 9 gene in oriental river prawn,Macrobrachium nipponense,Zhongguo Nongye Tongbao(Chinese Agricultural Science Bulletin),31(17):28-37(袁晖,周巧,张世勇,傅洪拓,孙盛明,乔慧,张文宜,2015,青虾泛素特异性蛋白酶9基因Usp9全长c DNA序列的克隆及表达分析,中国农学通报,31(17):28-37)
Zhang J.,Sun Y.,Li F.,Huang B.,and Xiang J.,2010,Molecular characterization and expression analysis of chitinase(Fcchi-3)from chinese shrimp,Fenneropenaeus chinensis,Mol.Biol.Rep.,37(4):1913-1921
Zhang Y.P.,Fu H.T.,Qiao H.,Jin S.B.,Jiang S.F.,Xiong Y.W.,Gong Y.S.,and Zhang X.Z.,2013,Molecular cloning and expression analysis of transformer-2 gene during development in Macrobrachium nipponense(de Haan 1849),Journal of the World Aquaculture Society,44(3):338-349
Zou E.,and Bonvillain R.,2004,Chitinase activity in the epidermis of the fiddler crab,Uca pugilator,as an in vivo screen for molt-interfering xenobiotics,Comp.Biochem.Physiol.C Toxicol.Pharmacol.,139(4):225-230
Arakane Y.,Zhu Q.,Matsumiya M.,Muthukrishnan S.,and Kramer K.J.,2003,Properties of catalytic,linker and chitin-binding domains of insect chitinase,Insect Biochem.Mol.Biol.,33(6):631-648
Daimon T.,Hamada K.,Mita K.,Okano K.,Suzuki M.G.,Kobayashi M.,and Shimada T.,2003,A Bombyx mori gene,Bm Chi-h,encodes a protein homologous to bacterial and baculovirus ch itinases,Insect Biochem.Mol.Biol.,33(8):749-759
Gilkes N.R.,Henrissat B.,Kilburn D.G.,Miller R.C.,and Warren R.A.,1991,Domains in microbial beta-1,4-glycanases:sequence conservation,function,and enzyme families,Microbiol.Rev.,55(2):303-315
Hart P.J,Pfluger H.D.,Monzingo A.F.,Hollis T.,and Robertus J.D.,1995,The refined crystal structure of an endochitinase from Hordeum vulgare L.seeds at 1.8 A resolution,J.Mol.Biol.,248(2):402-413
Houston D.R.,Recklies A.D.,Krupa J.C.,and van Aalten D.M.,2003,Structure and ligand-induced conformational change of the 39 k Da glycoprotein from human articular chondrocytes,J.Biol.Chem.,278(32):30206-30212
Huang Q.S.,Yan J.H.,Tang J.Y.,Tao Y.M.,Xie X.L.,Wang Y.,Wei X.Q.,Yan Q.H.,and Chen Q.X.,2010,Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei,Fish Shellfish Immunol.,29(1):75-81
Li D.C.,2006,Review of fungal chitinases,Mycopathologia,161(6):345-360
Li X.G.,Xu Z.Q.,Zhou G.,Lin H.,Zhou J.,Zeng Q.,Mao Z.,and Gu X.H.,2015,Molecular characterization and expression analysis of five chitinases associated with molting in the Chinese mitten crab,Eriocheir sinensis,Comp.Biochem.Physiol.B.Biochem.Mol.Biol.,187:110-120
Lu Y.,Zen K.C.,Muthukrishnan S.,and Kramer K.J.,2002,Site-directed mutagenesis and functional analysis of active site acidic amino acid residues D142,D144 and E146in Manduca sexta(tobacco hornworm)chitinase,Insect Biochem.Mol.Biol.,32(11):1369-1382
Merzendorfer H.,and Zimoch L.,2003,Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases,J.Exp.Biol.,206(24):4393-4412
Pan D.,He N.,Yang Z.,Liu H.,and Xu X.,2005,Differential gene expression profile in hepatopancreas of WSSV-resistant shrimp(Penaeus japonicus)by suppression subtractive hybridization,Dev.Comp.Immunol.,29(2):103-112
Priya T.A.,Li F.,Zhang J.,Wang B.,Zhao C.,and Xiang J.,2009,Molecular characterization and effect of RNA interference of retinoid X receptor(RXR)on E75 and chitinase gene expression in chinese shrimp Fenneropenaeus chinensis,Comp.Biochem.Physiol.B.Biochem.Mol.Biol.,153(1):121-129
Proespraiwong P.,Tassanakajon A.,and Rimphanitchayakit V.,2010,Chitinases from the black tiger shrimp Penaeus monodon:phylogenetics,expression and activities,Comp.Biochem.Physiol.B.Biochem.Mol.Biol.,156(2):86-96
Qiao H.,Fu H.T.,Jin S.,Wu Y.,Jiang S.F.,Gong Y.W.,and Xiong Y.S.,2012,Constructing and random sequencing analysis of normali zed c DNA library of testis tissue from orien tal river prawn(Macrobrachium nipponense),Comp.Biochem.Physiol.Part D Genomics Proteomics,7(3):268-276
Renkema G.H.,Boot R.G.,Muijsers A.O.,Donker-Koopman W.E.,and Aerts J.M.,1995,Purification and characterization of human chitotriosidase,a novel member of the chitinase family of proteins,J.Biol.Chem.,270(5):2198-2202
Salma U.,Uddowla M.H.,Kim M.,Kim J.M.,Kim B.K.,Baek H.J.,Park H.,Mykles D.L.,and Kim H.W.,2012,Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp(Pandalopsis japonica):cloning and effects of eyestalk ablation on gene expression,Comp.Biochem.Physiol.B.Biochem.Mol.Biol.,161(3):197-207
Shao L.,Devenport M.,and Jacobs-Lorena M.,2001,The peritrophic matrix of hematophagous insects,Arch.Insect Biochem.Physiol.,47(2):119-125
Skinner D.M.,1962,The structure and metabolism of a crustacean integumentary tissue during a molt cycle,Biol.Bull.,123(3):635-647
Taira T.,Fujiwara M.,Dennhart N.,Hayashi H.,Onaga S.,Ohnuma T.,Letzel T.,Sakuda S.,and Fukamizo T.,2010,Transglycosylation reaction catalyzed by a class V chitinase from cycad,cycas revoluta:a study involving site-directed mutagenesis,HPLC,and real-time ESI-MS,Biochim.Biophys.Acta.,1804(4):668-675
Tan S.H.,Degnan B.M.,and Lehnert S.A.,2000,The Penaeus monodon chitinase 1 gene is differentially expressed in the hepatopancreas during the molt cycle,Mar.Biotechnol.,2(2):126-315
Tjoelker L.W.,Gosting L.,Frey S.,Hunter C.L.,Trong H.L.,Steiner B.,Brammer H.,and Gray P.W.,2000,Structural and functional definition of the human chitinase chitin-binding domain,J.Biol.Chem.,275(1):514-520
Tronsmo A.,and Harman G.E.,1993,Detection and quantification of N-acetyl-beta-D-glucosaminidase,chitobiosidase,and endochitinase in solutions and on gels,Anal.Biochem.,208(1):74-79
Watanabe T.,and Kono M.,1997,Isolation of a c DNA encoding a chitinase family protein from cuticular tissues of the kuruma prawn Penaeus japonicus,Zoolog.Sci.,14(1):65-68
Watanabe T.,Kobori K.,Miyashita K.,Fujii T.,Sakai H.,Uchida M.,and Tanaka H.,1993,Identification of glutamic acid204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity,J.Biol.Chem.,268(25):18567-18572
Watanabe T.,Kono M.,Aida K.,and Nagasawa H.,1998,Purification and molecular cloning of a chitinase expressed in the hepatopancreas of the penaeid prawn Penaeus japonicus,Biochim.Biophys.Acta.,1382(2):181-185
Wong E.,Vaaje-Kolstad G.,Ghosh A.,Hurtado-Guerrero R.,Konarev P.V.,Ibrahim A.F.,Svergun D.I.,Eijsink V.G.,Chatterjee N.S.,and van Aalten D.M.,2012,The Vibrio cholerae colonization factor Gbp A possesses a modular structure that governs binding to different host surfaces,PLo S Pathoge.,8(1):e1002373
Wu P.,Qi D.,Chen L.Q.,Zhang H.,Zhang X.W.,Qin J.G.,and Hu S.N.,2009,Gene discovery from an ovary c DNA library of oriental river prawn Macrobrachium nipponense by ESTs annotation,Comp.Biochem.Physiol.Part D Genomics Proteomics,4(2):111-120
Yuan H.,Zhou Q.,Zhang S.Y.,Fu H.T.,Sun S.M.,Qiao H.,andZhang W.Y.,2015,Full-length c DNA cloning and spatial-temporal expression analysis of ubiquitin-specific protease 9 gene in oriental river prawn,Macrobrachium nipponense,Zhongguo Nongye Tongbao(Chinese Agricultural Science Bulletin),31(17):28-37(袁晖,周巧,张世勇,傅洪拓,孙盛明,乔慧,张文宜,2015,青虾泛素特异性蛋白酶9基因Usp9全长c DNA序列的克隆及表达分析,中国农学通报,31(17):28-37)
Zhang J.,Sun Y.,Li F.,Huang B.,and Xiang J.,2010,Molecular characterization and expression analysis of chitinase(Fcchi-3)from chinese shrimp,Fenneropenaeus chinensis,Mol.Biol.Rep.,37(4):1913-1921
Zhang Y.P.,Fu H.T.,Qiao H.,Jin S.B.,Jiang S.F.,Xiong Y.W.,Gong Y.S.,and Zhang X.Z.,2013,Molecular cloning and expression analysis of transformer-2 gene during development in Macrobrachium nipponense(de Haan 1849),Journal of the World Aquaculture Society,44(3):338-349
Zou E.,and Bonvillain R.,2004,Chitinase activity in the epidermis of the fiddler crab,Uca pugilator,as an in vivo screen for molt-interfering xenobiotics,Comp.Biochem.Physiol.C Toxicol.Pharmacol.,139(4):225-230