棉花GhMGL11基因的克隆和表达分析
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摘要
为研究陆地棉甲硫氨酸裂解酶(Methionineγ-lyase)基因的结构与功能,本研究以陆地棉中9807叶片为试验材料克隆得到棉花甲硫氨酸裂解酶(Methionineγ-lyase)基因c DNA全长,命名为GhMGL11(Cottongen:Gh_A12G2168)。蛋白序列分析该基因的编码区序列全长1 359 bp,编码452氨基酸;GhMGL11蛋白属于亲水性蛋白,预测含有41个磷酸化位点;亚细胞定位显示位于细胞质,无信号肽。序列比对分析,该基因含有Cys_Met_Meta_PP家族特有的保守结构域Cys_Met_Meta_PP,属于PLP(Pyridoxal phosphate)依赖性氨基转移酶中的一种。系统进化分析,陆地棉GhMGL11蛋白与可可(Theobroma cacao)亲缘关系较近。在陆地棉中发现23个与GhMGL11基因同源的含有Cys_Met_Meta_PP结构域的基因。组织特异性分析,GhMGL11基因在棉花根、茎、叶中均有表达,其中在根中的表达量最高;碱胁迫处理后不同时间内,该基因的表达量先升高后降低,说明该基因表达受碱胁迫影响。
In this study, full-length c DNA of methionine γ-lyase gene was cloned from 9807 leaf in upland cotton, which was named GhMGL11(Cottongen: Gh_A12 G2168) in order to study the structure and function of methionine γ-lyase gene. Sequence analysis of protein showed that the gene had the full length of 1 359 bp in coding region, encoding 452 amino acids. GhMGL11 protein was hydrophilic and predicted to contain 41 phosphorylation sites, of which the subcellular located in the cytoplasm with no signal peptide. Sequence alignment analysis showed that this gene contained the Cys_Met_Meta_PP domain, a specific conserved domain of Cys_Met_Meta_PP family, which was one of pyridoxal-dependent aminotransferases(PLPs). Phylogenetic analysis showed that there was closer evolutionary relationship between GhMGL11 protein in Gossypium hirsutum and Theobroma cacao. Twenty-three genes containing Cys_Met_Meta_PP domain were found in Gossypium hirsutum,which were homologous with GhMGL11 gene. Tissue-specific analysis showed that GhMGL11 gene was expressed in roots, stems and leaves of cotton, and had highest expression level in roots. In different time under alkaline stress, the expression of GhMGL11 gene increased at first and then decreased, indicating that the gene was affected by alkaline stress.
In this study, full-length c DNA of methionine γ-lyase gene was cloned from 9807 leaf in upland cotton, which was named GhMGL11(Cottongen: Gh_A12 G2168) in order to study the structure and function of methionine γ-lyase gene. Sequence analysis of protein showed that the gene had the full length of 1 359 bp in coding region, encoding 452 amino acids. GhMGL11 protein was hydrophilic and predicted to contain 41 phosphorylation sites, of which the subcellular located in the cytoplasm with no signal peptide. Sequence alignment analysis showed that this gene contained the Cys_Met_Meta_PP domain, a specific conserved domain of Cys_Met_Meta_PP family, which was one of pyridoxal-dependent aminotransferases(PLPs). Phylogenetic analysis showed that there was closer evolutionary relationship between GhMGL11 protein in Gossypium hirsutum and Theobroma cacao. Twenty-three genes containing Cys_Met_Meta_PP domain were found in Gossypium hirsutum,which were homologous with GhMGL11 gene. Tissue-specific analysis showed that GhMGL11 gene was expressed in roots, stems and leaves of cotton, and had highest expression level in roots. In different time under alkaline stress, the expression of GhMGL11 gene increased at first and then decreased, indicating that the gene was affected by alkaline stress.
引文
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Chen X.G.,Lu X.K.,Shu N.,Wang S.,Wang J.J.,Wang D.L.,and Guo L.X.,2017,Targeted mutagenesis in cotton(Gossypium hirsutum L.)using the CRISPR/Cas9 system,Sci.Rep.,7:44304
Chen Y.J.,Yu P.,Luo J.C.,and Jiang Y.,2003,Secreted protein prediction system combining CJ-SPHMM,TMHMM and PSORT,Mamm.Genome,14(12):859-865
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Dhas D.B.B.,Ashmi A.H.,Bhat B.V.,Parija S.C.,and Banupriya N.,2015,Modified low cost SNP genotyping technique using cycle threshold(Ct)&melting temperature(Tm)values in allele specific real-time PCR,Indian J.Med.Res.,142(5):555-562
Dou L.L.,Zhang X.H.,Pang C.Y.,Song M.Z.,Wei H.L.,Fan S.L.,and Yu S.X.,2014,Genome-wide analysis of the WRKY gene family in cotton,Mol.Genet.Genomics,289(6):1103-1121
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Horton P.,Park K.J.,Obayashi T.,Fujita N.,Harada H.,AdamsCollier C.J.,and Nakai K.,2007,Wo LF PSORT:protein localization predictor,Nucleic Acids Res.,35(S2):585-587
Jones P.,Binns D.,Chang H.Y.,Fraser M.,Li W.,Mc Anulla C.,Mc William H.,Maslen J.,Mitchell A.,Nuka G.,Pesseat S.,Quinn A.F.,Sangrador-Vegas A.,Scheremetjew M.,Yong S.Y.,Lopez R.,and Hunter S.,2014,Inter Pro Scan 5:genomescale protein function classification,Bioinformatics,30(9):1236-1240
Joshi V.,and Jander G.,2009,Arabidopsis methionine gammalyase is regulated according to isoleucine biosynthesis needs but plays a subordinate role to threonine deaminase,Plant Physiol.,151(1):367-378
Kokkinakis D.M.,2006,Methionine-stress:a pleiotropic approach in enhancing the efficacy of chemotherapy,Cancer Lett.,233(2):195-207
Li F.G.,Fan G.Y.,Lu C.R.,Xiao G.H.,Zou C.S.,Kohel R.J.,Ma Z.Y.,Shang H.H.,Ma X.F.,Wu J.Y.,Liang X.M.,Huang G.,Percy R.G.,Liu K.,Yang W.H.,Chen W.B.,Du X.M.,Shi C.C.,Yuan Y.L.,Ye W.W.,Liu X.,Zhang X.Y.,Liu W.Q.,Wei H.L.,Wei S.J.,Huang G.D.,Zhang X.L.,Zhu S.J.,Zhang H.,Sun F.M.,Wang X.F.,Liang J.,Wang J.H.,He Q.,Huang L.H.,Wang J.,Cui J.J.,Song G.L.,Wang K.B.,Xu X.,Yu J.Z.,Zhu Y.X.,and Yu S.X.,2015,Genome sequence of cultivated upland cotton(Gossypium hirsutum TM-1)provides insights into genome evolution,Nat.Biotechnol.,33(5):524-530
Liang C.,Meng Z.,Meng Z.,Malik W.,Yan R.,Lwin K.M.,Lin F.,Wang Y.,Sun G.,Zhou T.,Zhu T.,Li J.,Jin S.,Guo S.,and Zhang R.,2016,Gh ABF2,a b ZIP transcription factor,confers drought and salinity tolerance in cotton(Gossypium hirsutum L.),Sci.Rep.,6:35040
Liu Z.,Ge X.,Yang Z.,Zhang C.,Zhao G.,Chen E.,Liu J.,Zhang X.,and Li F.,2017,Genome-wide identification and characterization of Sn RK2 gene family in cotton(Gossypium hirsutum L.),BMC Genet.,18(1):54
Lv M.,Ni Z.,Wang J.,Li B.,Luo S.P.,and Fan L.,2010,Tissue specificity express analysis of methyltransferase gene COMTand CCo AOMT in cotton,Henongxue Bao(Journal of Nuclear Agricultural Sciences),24(4):713-719(吕萌,倪志勇,王娟,李波,罗淑萍,范玲,2010,棉花甲基化酶基因COMT和CCoAOMT的组织特异性表达分析,核农学报,24(4):713-719)
Ma B.K.,Tang X.Y.,Huang H.C.,and Shi Y.H.,2006,Cloning of L-methionine-γ-lyase gene,Yixue Yanjiusheng Xuebao(Journal of Medical Postgraduates),19(12):1063-1066(马百坤,唐晓莹,黄惠臣,时永辉,2006,蛋氨酸裂解酶的基因克隆及序列分析,医学研究生学报,19(12):1063-1066)
Milano T.,Paiardini A.,Grgurina I.,and Pascarella S.,2013,TypeⅠpyridoxal 5'-phosphate dependent enzymatic domains embedded within multimodular nonribosomal peptide synthetase and polyketide synthase assembly lines,BMC Struct.Biol.,13(1):26
Mistry J.,and Finn R.,2007,Pfam:a domain-centric method for analyzing proteins and proteomes,Methods Mol.Biol.,396(2):43-58
Mu M.,Lu X.K.,Wang J.J.,Wang D.L.,Yin Z.J.,Wang S.,Fan W.L.,and Ye W.W.,2016,Genome-wide identification and analysis of the stress-resistance function of the TPS(trehalose-6-phosphate synthase)gene family in cotton,BMCGenet.,17(54):1-11
Petersen T.N.,Brunak S.,von Heijne G.,and Nielsen H.,2011,Signal P 4.0:discriminating signal peptides from transmembrane regions,Nat.Methods.,8(10):785-786
Prakash A.,Jeffryes M.,Bateman A.,and Finn R.D.,2017,The HMMER web server for protein sequence similarity search,Curr.Protoc.Bioinformatics,60:1-23
Rebeille F.,Jabrin S.,Bligny R.,Loizeau K.,Gambonnet B.,Van Wilder V.,Douce R.,and Ravanel S.,2006,Methionine catabolism in Arabidopsis cells is initiated by a gamma-cleavage process and leads to S-methylcysteine and isoleucine syntheses,Proc.Natl.Acad.Sci.USA,103(42):15687-15692
Saitou N.,and Nei M.,1987,The neighbor-joining method:a new method for reconstructing phylogenetic trees,Mol.Biol.Evol.,4(4):406-425
Tamura K.,Stecher G.,Peterson D.,Filipski A.,and Kumar S.,2013,MEGA6:molecular evolutionary genetics analysis version6.0,Mol.Biol.Evol.,30(12):2725-2729
Toney M.D.,2005,Reaction specificity in pyridoxal phosphate enzymes,Arch.Biochem.Biophys.,433(1):279-287
Zhang T.B.,Yin Z.J.,Lu X.K.,Chen X.G.,Wang S.,Wang D.L.,Wang J.J.,Fan W.L.,Zhao X.J.,Mu M.,Wang X.G.,and Ye W.W.,2015,Salinity resisted gene ccm C cloning and its expression analysis on Gossypium hirsutum L.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(7):1502-1508(张天豹,阴祖军,陆许可,陈修贵,王帅,王德龙,王俊娟,樊伟莉,赵小洁,穆敏,王晓歌,叶武威,2015,陆地棉线粒体耐盐基因ccm C的克隆与表达分析,分子植物育种,13(7):1502-1508)
Zhao G.,Song Y.,Wang C.X.,Butt H.I.,Wang Q.H.,Zhang C.J.,Yang Z.R.,Liu Z.,Chen E.Y.,Zhang X.Y.,and Li F.G.,2016,Genome-wide identification and functional analysis of the TIFY gene family in response to drought in cotton,Mol.Genet.Genomics.,291(6):2173-2187
Zhao W.S.,Zhai Q.L.,An Z.W.,Fang J.L.,and Huang H.S.,2015,Cloning and characteristics of Hb WRKY9 transcription factorin the rubber tree,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):599-606(赵武帅,翟琪麟,安泽伟,方家林,黄华孙,2015,橡胶树转录因子Hb WRKY9的克隆与特性分析,基因组学与应用生物学,34(3):599-606)
Cedroni M.L.,Cronn R.C.,Adams K.L.,Wilkins T.A.,and Wendel J.F.,2003,Evolution and expression of MYB genes in diploid and polyploid cotton,Plant Mol.Biol.,51(3):313-325
Chen X.G.,Lu X.K.,Shu N.,Wang S.,Wang J.J.,Wang D.L.,and Guo L.X.,2017,Targeted mutagenesis in cotton(Gossypium hirsutum L.)using the CRISPR/Cas9 system,Sci.Rep.,7:44304
Chen Y.J.,Yu P.,Luo J.C.,and Jiang Y.,2003,Secreted protein prediction system combining CJ-SPHMM,TMHMM and PSORT,Mamm.Genome,14(12):859-865
Deinlein U.,Stephan A.B.,Horie T.,Luo W.,Xu G.,and Schroeder J.,2014,Plant salt-tolerance mechanisms,Trends Plant Sci.,19(6):371-379
Dhas D.B.B.,Ashmi A.H.,Bhat B.V.,Parija S.C.,and Banupriya N.,2015,Modified low cost SNP genotyping technique using cycle threshold(Ct)&melting temperature(Tm)values in allele specific real-time PCR,Indian J.Med.Res.,142(5):555-562
Dou L.L.,Zhang X.H.,Pang C.Y.,Song M.Z.,Wei H.L.,Fan S.L.,and Yu S.X.,2014,Genome-wide analysis of the WRKY gene family in cotton,Mol.Genet.Genomics,289(6):1103-1121
Ferla M.P.,and Patrick W.M.,2014,Bacterial methionine biosynthesis,Microbiology,160(8):1571-1584
Goyer A.,Collakova E.,Shachar-Hill Y.,and Hanson A.D.,2007,Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway,Plant Cell Physiol.,48(2):232-242
Horton P.,Park K.J.,Obayashi T.,Fujita N.,Harada H.,AdamsCollier C.J.,and Nakai K.,2007,Wo LF PSORT:protein localization predictor,Nucleic Acids Res.,35(S2):585-587
Jones P.,Binns D.,Chang H.Y.,Fraser M.,Li W.,Mc Anulla C.,Mc William H.,Maslen J.,Mitchell A.,Nuka G.,Pesseat S.,Quinn A.F.,Sangrador-Vegas A.,Scheremetjew M.,Yong S.Y.,Lopez R.,and Hunter S.,2014,Inter Pro Scan 5:genomescale protein function classification,Bioinformatics,30(9):1236-1240
Joshi V.,and Jander G.,2009,Arabidopsis methionine gammalyase is regulated according to isoleucine biosynthesis needs but plays a subordinate role to threonine deaminase,Plant Physiol.,151(1):367-378
Kokkinakis D.M.,2006,Methionine-stress:a pleiotropic approach in enhancing the efficacy of chemotherapy,Cancer Lett.,233(2):195-207
Li F.G.,Fan G.Y.,Lu C.R.,Xiao G.H.,Zou C.S.,Kohel R.J.,Ma Z.Y.,Shang H.H.,Ma X.F.,Wu J.Y.,Liang X.M.,Huang G.,Percy R.G.,Liu K.,Yang W.H.,Chen W.B.,Du X.M.,Shi C.C.,Yuan Y.L.,Ye W.W.,Liu X.,Zhang X.Y.,Liu W.Q.,Wei H.L.,Wei S.J.,Huang G.D.,Zhang X.L.,Zhu S.J.,Zhang H.,Sun F.M.,Wang X.F.,Liang J.,Wang J.H.,He Q.,Huang L.H.,Wang J.,Cui J.J.,Song G.L.,Wang K.B.,Xu X.,Yu J.Z.,Zhu Y.X.,and Yu S.X.,2015,Genome sequence of cultivated upland cotton(Gossypium hirsutum TM-1)provides insights into genome evolution,Nat.Biotechnol.,33(5):524-530
Liang C.,Meng Z.,Meng Z.,Malik W.,Yan R.,Lwin K.M.,Lin F.,Wang Y.,Sun G.,Zhou T.,Zhu T.,Li J.,Jin S.,Guo S.,and Zhang R.,2016,Gh ABF2,a b ZIP transcription factor,confers drought and salinity tolerance in cotton(Gossypium hirsutum L.),Sci.Rep.,6:35040
Liu Z.,Ge X.,Yang Z.,Zhang C.,Zhao G.,Chen E.,Liu J.,Zhang X.,and Li F.,2017,Genome-wide identification and characterization of Sn RK2 gene family in cotton(Gossypium hirsutum L.),BMC Genet.,18(1):54
Lv M.,Ni Z.,Wang J.,Li B.,Luo S.P.,and Fan L.,2010,Tissue specificity express analysis of methyltransferase gene COMTand CCo AOMT in cotton,Henongxue Bao(Journal of Nuclear Agricultural Sciences),24(4):713-719(吕萌,倪志勇,王娟,李波,罗淑萍,范玲,2010,棉花甲基化酶基因COMT和CCoAOMT的组织特异性表达分析,核农学报,24(4):713-719)
Ma B.K.,Tang X.Y.,Huang H.C.,and Shi Y.H.,2006,Cloning of L-methionine-γ-lyase gene,Yixue Yanjiusheng Xuebao(Journal of Medical Postgraduates),19(12):1063-1066(马百坤,唐晓莹,黄惠臣,时永辉,2006,蛋氨酸裂解酶的基因克隆及序列分析,医学研究生学报,19(12):1063-1066)
Milano T.,Paiardini A.,Grgurina I.,and Pascarella S.,2013,TypeⅠpyridoxal 5'-phosphate dependent enzymatic domains embedded within multimodular nonribosomal peptide synthetase and polyketide synthase assembly lines,BMC Struct.Biol.,13(1):26
Mistry J.,and Finn R.,2007,Pfam:a domain-centric method for analyzing proteins and proteomes,Methods Mol.Biol.,396(2):43-58
Mu M.,Lu X.K.,Wang J.J.,Wang D.L.,Yin Z.J.,Wang S.,Fan W.L.,and Ye W.W.,2016,Genome-wide identification and analysis of the stress-resistance function of the TPS(trehalose-6-phosphate synthase)gene family in cotton,BMCGenet.,17(54):1-11
Petersen T.N.,Brunak S.,von Heijne G.,and Nielsen H.,2011,Signal P 4.0:discriminating signal peptides from transmembrane regions,Nat.Methods.,8(10):785-786
Prakash A.,Jeffryes M.,Bateman A.,and Finn R.D.,2017,The HMMER web server for protein sequence similarity search,Curr.Protoc.Bioinformatics,60:1-23
Rebeille F.,Jabrin S.,Bligny R.,Loizeau K.,Gambonnet B.,Van Wilder V.,Douce R.,and Ravanel S.,2006,Methionine catabolism in Arabidopsis cells is initiated by a gamma-cleavage process and leads to S-methylcysteine and isoleucine syntheses,Proc.Natl.Acad.Sci.USA,103(42):15687-15692
Saitou N.,and Nei M.,1987,The neighbor-joining method:a new method for reconstructing phylogenetic trees,Mol.Biol.Evol.,4(4):406-425
Tamura K.,Stecher G.,Peterson D.,Filipski A.,and Kumar S.,2013,MEGA6:molecular evolutionary genetics analysis version6.0,Mol.Biol.Evol.,30(12):2725-2729
Toney M.D.,2005,Reaction specificity in pyridoxal phosphate enzymes,Arch.Biochem.Biophys.,433(1):279-287
Zhang T.B.,Yin Z.J.,Lu X.K.,Chen X.G.,Wang S.,Wang D.L.,Wang J.J.,Fan W.L.,Zhao X.J.,Mu M.,Wang X.G.,and Ye W.W.,2015,Salinity resisted gene ccm C cloning and its expression analysis on Gossypium hirsutum L.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(7):1502-1508(张天豹,阴祖军,陆许可,陈修贵,王帅,王德龙,王俊娟,樊伟莉,赵小洁,穆敏,王晓歌,叶武威,2015,陆地棉线粒体耐盐基因ccm C的克隆与表达分析,分子植物育种,13(7):1502-1508)
Zhao G.,Song Y.,Wang C.X.,Butt H.I.,Wang Q.H.,Zhang C.J.,Yang Z.R.,Liu Z.,Chen E.Y.,Zhang X.Y.,and Li F.G.,2016,Genome-wide identification and functional analysis of the TIFY gene family in response to drought in cotton,Mol.Genet.Genomics.,291(6):2173-2187
Zhao W.S.,Zhai Q.L.,An Z.W.,Fang J.L.,and Huang H.S.,2015,Cloning and characteristics of Hb WRKY9 transcription factorin the rubber tree,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):599-606(赵武帅,翟琪麟,安泽伟,方家林,黄华孙,2015,橡胶树转录因子Hb WRKY9的克隆与特性分析,基因组学与应用生物学,34(3):599-606)