百脉根钙调素蛋白(LjCaM3)基因的克隆及表达分析
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摘要
钙调素(calmodulin,CaM)是一种广泛存在于真核生物中的钙感受器,参与各种植物生理活动的信号转导。在高等植物中存在多个钙调素基因编码多个不同蛋白亚型的现象,并且不同的蛋白亚型在各器官的不同发育阶段可能有着不同的表达模式或转录调控作用。本研究通过3'-RACE技术克隆获得LjCaM3-1和LjCaM3-2分别为637 bp和746 bp,并且编码同一个蛋白LjCaM3。LjCaM3-2的3'-UTR中具有AU富集元件可能对其mRNA的降解起着调节作用。通过荧光定量PCR实验,发现LjCaM3蛋白在百脉根的根及根瘤中的相对表达量显著高于茎、叶、花和豆荚。LjCaM3很有可能参与了百脉根根的生长发育,为进一步探讨该基因的生物学功能提供了一定的理论基础。
Calmodulin(CaM) is a calcium receptor widely found in eukaryotes,which participates in signal transduction of various plant physiologic activities.In higher plants,the multiple calmodulin genes encode multiple different isoforms which have different expression patterns or transcriptional regulations at different stages of plant development.In present study,LjCaM3-1 and LjCaM3-2 were cloned by 3'-RACE,which were 637 bp and 746 bp,respectively,and encoded the same protein named LjCaM3.The 3'-UTR of LjCaM3-2 had AU-enriched element which might regulate its mRNA degradation.The qRT-PCR found that the relative expression of mRNA in LjCaM3 in roots and nodules of Lotus japonicus was significantly higher than those in stem,leaf,flower and pods.LjCaM3 was likely to be involved in the growth and development of the roots of L.japonicus,which could provide certain theoretical basis for further study of the biological function of the gene.
Calmodulin(CaM) is a calcium receptor widely found in eukaryotes,which participates in signal transduction of various plant physiologic activities.In higher plants,the multiple calmodulin genes encode multiple different isoforms which have different expression patterns or transcriptional regulations at different stages of plant development.In present study,LjCaM3-1 and LjCaM3-2 were cloned by 3'-RACE,which were 637 bp and 746 bp,respectively,and encoded the same protein named LjCaM3.The 3'-UTR of LjCaM3-2 had AU-enriched element which might regulate its mRNA degradation.The qRT-PCR found that the relative expression of mRNA in LjCaM3 in roots and nodules of Lotus japonicus was significantly higher than those in stem,leaf,flower and pods.LjCaM3 was likely to be involved in the growth and development of the roots of L.japonicus,which could provide certain theoretical basis for further study of the biological function of the gene.
引文
Babu Y.S.,Bu gg C.E.,an d Cook W.J.,1988,Structure of calmodulin refined at 2.2 A resolution,Journal of Molecular Biology,204(1):191-204
Charbonneau H.,and Cormier M.J.,1979,Purification of plan calmodulin by fluphenazine-sepharose affinity chromatography,Biochemical and Biophysical Research Communications,90(3):1039-1047
Cheng Y.D.,Ge W.,Yan H.B.,Yang K.,and Guan J.F.,2016,Clonin g and expression analysis of calmodulin gene from'Yali'pears,Hebei Nongye Daxue Xuebao(Journal of Agricultural University of Hebei),39(2):58-63(程玉豆,葛文雅,闫洪波,杨坤,关军峰,2016,‘鸭梨’钙调素基因的克隆与表达分析,河北农业大学学报,39(2):58-63)
Chul C.,and Dae-Jin.,2009,Calcium and calmodulin-mediated regulation of gene expression in plants,Molecular Plant,2(1):13-21
Di Noia J.M.,D'Orso I.,S觃nchez D.O.,and Frasch A.C.C.,2000,A U-rich elements in the 3'-untranslated region of a new mucin-type gene family of trypanosoma cruzi confers m RNA instability and modulates translation efficiency,Journal of Biological Chemistry,275(14):10218-10227
Eibl C.,Zou Z.R.,Beck A.,Kim M.,Mullet J.,and Koop H.U.,1999,In vivo analysis of plastid psb A,rbc L and rpl32 UTRelements by chloroplast transformation:tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency,Plant Journal,19(3):333-345
Frohman M.A.,Dush M.K.,and Martin G.R.,1988,Rapid production of full-length c DNAs from rare transcripts:amplification using a single gene-specific oligonucleotide primer,Proc.Natl.Acad.Sci.USA,85(23):8998-9002
Geurts R.,and Bisseling T.,2002,Rhizobium nod factor perception and signalling,The Plant Cell,14:239-249
Huang Q.Q.,Ren X.Y.,Tan Y.M.,Wei H.M.,Yi Y.,and Liu Y.X.,2015,Cloning and sequence analysis of the full length Ku70 DNA in Shiraia bambusicola P.Hennings,Jiyin Zuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(12):2593-2600(黄青青,任锡毅,谭玉梅,魏洪美,乙引,刘永翔,2015,竹黄菌Ku70基因全长克隆及序列分析,基因组学与应用生物学,34(12):2593-2600)
Huang Y.T.,Qian W.J.,Wang Y.C.,Cao H.L.,Wang L.,Hao X.Y.,Wang X.C.,and Yang Y.J.,2016,Molecular cloning and expression of Ca M family gene Cs Ca Ms under cold stress in tea plant(Camellia sinensis(L.)O.Kuntze),Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),17(5):906-912(黄玉婷,钱文俊,王玉春,曹红利,王璐,郝心愿,王新超,杨亚军,2016,茶树钙调素基因Cs Ca Ms的克隆及其低温胁迫下的表达分析,植物遗传资源学报,17(5):906-912)
Johnson J.M.,and Reichelt M.,and Vadassery J.,2014,An Arabidopsis mutant impaired in intracellular calcium elevation is sensitive to biotic and abiotic stress,BMC Plant Biology,14(1):1-19
Kim M.C.,Chung,W.S.,Yun D.J.,and Cho M.J.,2009,Calcium and calmodulin-mediated regulation of gene expression in plants,Molecular Plant,2(1):13-21
Kislauskis E.H.,Zhu X.,and Singer R.H.,1994,Sequences responsible for intracellular localization ofβ-Actin messenger RNA also affect cell phenotype,Journal of Cel Biology,127(2):441-451
Kumar S.,Stecher G.,and Tamura K.,2016,MEGA7:Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets,Molecular Biology&Evolution,33(7):1870-1874
Lee S.H.,Johnson J.D.,Walsh M.P.,Lierop J.E.,Cindy S.,Xus A.,Snedden W.A.,Kosk-kosicka D.,Fromm H.,Narayanan N.,and Cho M.J.,2000,Differential regulation of Ca2+calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+concentration,Biochemical Journal350:299-306
Lee S.H.,Kim J.C.,Lee M.S.,Heo W.D.,Seo H.Y.,Yoon H.W.Hong J.,Lee S.Y.,Bahk J.D.,Hwang I.,and Cho J.M.1995,Identification of a novel divergent calmodulin isoform from soybean which has differential ability to activate Calmodulin-dependent enzymes,Journal of Biologica Chemistry,270(37):21806-21812
Liao J.Q.,Singh S.,Hossain M.S.,Andersen S.U.,Ross L.Bonetta D.,Zhou Y.H.,Sato S.,Tabata S.,Stougaard J.Szczyglowski K.,and Parniske M.,2012,Negative regulation of CCa MK is essential for symbiotic infection,Plan Journal,72(4):572-584
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTMethod,Methods,25(4):402-408
Ma L.,and Sun D.,1997,The effects of extracellular calmodulin on initiation of Hippeastrum rutilum pollen germination and tube growth,Planta,202(3):336-340
Mao G.H.,Hou L.X.,Ding C.B.,Cui S.J.,and Sun D.Y.,2005Characterization of a c DNA coding for an extracellular calmodulin-binding protein from suspension-cultured cells of Angelica dahurica,Planta,222(3):428-437
Mccormack E.,Tsai Y.C.,and Braam J.,2005,Handling calcium signaling:Arabidopsis,Ca Ms and CMLs,Trends in Plan Science,10(8):383-389
Mccormak E.,and Braam J.,2003,Calmodulins and related poten tial calcium sensors of Arabidopsis,New Phytologist,159(3):585-598
Means A.R.,and Dedman J.R.,1980,Calmodulin--an intracellular calcium receptor,Nature,285(5760):73-77
Miller J.B.,Pratap A.,Miyahara A.,Zhou L.,Bornemann S.and Morris R.J.,2013,Calcium/Calmodulin-dependen protein kinase is negatively and positively regulated by calcium,providing a mechanism for decoding calcium responses during symbiosis signaling,The Plant Cell,25(12):5053-5066
Oldroyd G.E.D.,and Downie J.A.,2004,Calcium,kinases and nodulation signalling in legumes,Nature Reviews Molecular Cell Biology,5(7):566-576
Rott R.,Liveanu V.,Drager R.G.,Stem D.B.,and Schuster G.,1998,The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of m RNA accumulation and stability,Plant Molecular Biology,36(2):307-314
Shaw G.,and Kamen R.,1986,A conserved AU sequence from the 3'-untranslated region of GM-CSF m RNA mediates selective m RNA degradation,Cell,46(5):659-667
Shimoda Y.,Han L.,Yamazaki T.,Suzuki R.,Hayashi M.,and Imaizumi-Anraku H.,2012,Rhizobial and fungal symbioses show different requirements for calmodulin binding to calcium calmodulin-dependent protein kinase in Lotus japonicus,The Plant cell,24(1):304-321
Sun D.Y.,Li H.B.,and Cheng G.,1994,Extracellular calmodulin accelerates the proliferation of suspension-cultured cells of Angelica dahurica,Plant Science,99(1):1-8
Suzuki Y.,Mae T.,and Makino A.,2008,RNA extraction from various recalcitrant plant tissues with a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment,Bioscience Biotechnology&Biochemistry,72(7):1951-1953
Szczyglowski K.,and Stougaard J.,2008,Lotus genome:pod of gold for legume research,Trends in Plant Science,13(10):515-517
Takezawad D.,Liu Z.H.,and An G.,1995,Calmodulin gene family in potato:developmental and touch-induced expression of the m RNA encoding a novel isoform,Plant Molecular Biology,27(4):693-703
Tang J.Y.,Zhao H.,He A.H.,Zhou J.C.,Jia G.,Liu G.M.,Chen X.L.,and Cai J.Y.,2016,Cloning,identification and tissue m RNA relative expression level analysis of porcine selenoprotein P gene,Dongwu Yingyang Xuebao(Chinese Journal of Animal Nutrition)28(3):858-863(汤加勇,赵华,何爱华,周继昌,贾刚,刘光芒,陈小玲,蔡景义,2016,猪硒蛋白P基因克隆,鉴定及组织m RNA相对表达量分析,动物营养学报,28(3):858-863)
Watillon B.,Kettman R.,Boxus P.,and Burny A.,1992,Cloning and characterization of an apple(Malus domestica Borkh)calmodulin gene,Plant Science,82(2):201-212
Yang T.,Levyadun S.,Feldman M.,and Fromm H.,1998,Developmentally regulated organ-,tissue-,and cell-specific expression of calmodulin genes in common wheat,Plant Molecular Biology,37(1):109-120
Yang T.,Segal G.,Abbo S.,Feidman M.,and Fromm H.,1996,Characterization of the calmodulin gene family in wheat:structure,chromosomal location,and evolutionary aspects,Molecular Genetics and Genomics,252(6):684-694
Zeng H.Q.,Xu L.Q.,Singh A.,Wang H.Z.,Du L.Q.,and Poovaiah B.W.,2015,Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses,Frontiers in Plant Science,6(660):600
Zhang W.,and Sun D.Y.,2009,Molecular and genetic evidence for the key role of At Ca M3 in heat-shock signal transduction in Arabidopsis,Plant Physiol.,149(4):1773-1784
Zimmer W.E.,Schloss J.A.,Silflow C.D.,Youngblom J.,and Watterson C.D.,1988,Structural organization,DNAsequence,and expression of the calmodulin gene,Journal of Biological Chemistry,263(36):19370-19383
Charbonneau H.,and Cormier M.J.,1979,Purification of plan calmodulin by fluphenazine-sepharose affinity chromatography,Biochemical and Biophysical Research Communications,90(3):1039-1047
Cheng Y.D.,Ge W.,Yan H.B.,Yang K.,and Guan J.F.,2016,Clonin g and expression analysis of calmodulin gene from'Yali'pears,Hebei Nongye Daxue Xuebao(Journal of Agricultural University of Hebei),39(2):58-63(程玉豆,葛文雅,闫洪波,杨坤,关军峰,2016,‘鸭梨’钙调素基因的克隆与表达分析,河北农业大学学报,39(2):58-63)
Chul C.,and Dae-Jin.,2009,Calcium and calmodulin-mediated regulation of gene expression in plants,Molecular Plant,2(1):13-21
Di Noia J.M.,D'Orso I.,S觃nchez D.O.,and Frasch A.C.C.,2000,A U-rich elements in the 3'-untranslated region of a new mucin-type gene family of trypanosoma cruzi confers m RNA instability and modulates translation efficiency,Journal of Biological Chemistry,275(14):10218-10227
Eibl C.,Zou Z.R.,Beck A.,Kim M.,Mullet J.,and Koop H.U.,1999,In vivo analysis of plastid psb A,rbc L and rpl32 UTRelements by chloroplast transformation:tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency,Plant Journal,19(3):333-345
Frohman M.A.,Dush M.K.,and Martin G.R.,1988,Rapid production of full-length c DNAs from rare transcripts:amplification using a single gene-specific oligonucleotide primer,Proc.Natl.Acad.Sci.USA,85(23):8998-9002
Geurts R.,and Bisseling T.,2002,Rhizobium nod factor perception and signalling,The Plant Cell,14:239-249
Huang Q.Q.,Ren X.Y.,Tan Y.M.,Wei H.M.,Yi Y.,and Liu Y.X.,2015,Cloning and sequence analysis of the full length Ku70 DNA in Shiraia bambusicola P.Hennings,Jiyin Zuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(12):2593-2600(黄青青,任锡毅,谭玉梅,魏洪美,乙引,刘永翔,2015,竹黄菌Ku70基因全长克隆及序列分析,基因组学与应用生物学,34(12):2593-2600)
Huang Y.T.,Qian W.J.,Wang Y.C.,Cao H.L.,Wang L.,Hao X.Y.,Wang X.C.,and Yang Y.J.,2016,Molecular cloning and expression of Ca M family gene Cs Ca Ms under cold stress in tea plant(Camellia sinensis(L.)O.Kuntze),Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),17(5):906-912(黄玉婷,钱文俊,王玉春,曹红利,王璐,郝心愿,王新超,杨亚军,2016,茶树钙调素基因Cs Ca Ms的克隆及其低温胁迫下的表达分析,植物遗传资源学报,17(5):906-912)
Johnson J.M.,and Reichelt M.,and Vadassery J.,2014,An Arabidopsis mutant impaired in intracellular calcium elevation is sensitive to biotic and abiotic stress,BMC Plant Biology,14(1):1-19
Kim M.C.,Chung,W.S.,Yun D.J.,and Cho M.J.,2009,Calcium and calmodulin-mediated regulation of gene expression in plants,Molecular Plant,2(1):13-21
Kislauskis E.H.,Zhu X.,and Singer R.H.,1994,Sequences responsible for intracellular localization ofβ-Actin messenger RNA also affect cell phenotype,Journal of Cel Biology,127(2):441-451
Kumar S.,Stecher G.,and Tamura K.,2016,MEGA7:Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets,Molecular Biology&Evolution,33(7):1870-1874
Lee S.H.,Johnson J.D.,Walsh M.P.,Lierop J.E.,Cindy S.,Xus A.,Snedden W.A.,Kosk-kosicka D.,Fromm H.,Narayanan N.,and Cho M.J.,2000,Differential regulation of Ca2+calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+concentration,Biochemical Journal350:299-306
Lee S.H.,Kim J.C.,Lee M.S.,Heo W.D.,Seo H.Y.,Yoon H.W.Hong J.,Lee S.Y.,Bahk J.D.,Hwang I.,and Cho J.M.1995,Identification of a novel divergent calmodulin isoform from soybean which has differential ability to activate Calmodulin-dependent enzymes,Journal of Biologica Chemistry,270(37):21806-21812
Liao J.Q.,Singh S.,Hossain M.S.,Andersen S.U.,Ross L.Bonetta D.,Zhou Y.H.,Sato S.,Tabata S.,Stougaard J.Szczyglowski K.,and Parniske M.,2012,Negative regulation of CCa MK is essential for symbiotic infection,Plan Journal,72(4):572-584
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTMethod,Methods,25(4):402-408
Ma L.,and Sun D.,1997,The effects of extracellular calmodulin on initiation of Hippeastrum rutilum pollen germination and tube growth,Planta,202(3):336-340
Mao G.H.,Hou L.X.,Ding C.B.,Cui S.J.,and Sun D.Y.,2005Characterization of a c DNA coding for an extracellular calmodulin-binding protein from suspension-cultured cells of Angelica dahurica,Planta,222(3):428-437
Mccormack E.,Tsai Y.C.,and Braam J.,2005,Handling calcium signaling:Arabidopsis,Ca Ms and CMLs,Trends in Plan Science,10(8):383-389
Mccormak E.,and Braam J.,2003,Calmodulins and related poten tial calcium sensors of Arabidopsis,New Phytologist,159(3):585-598
Means A.R.,and Dedman J.R.,1980,Calmodulin--an intracellular calcium receptor,Nature,285(5760):73-77
Miller J.B.,Pratap A.,Miyahara A.,Zhou L.,Bornemann S.and Morris R.J.,2013,Calcium/Calmodulin-dependen protein kinase is negatively and positively regulated by calcium,providing a mechanism for decoding calcium responses during symbiosis signaling,The Plant Cell,25(12):5053-5066
Oldroyd G.E.D.,and Downie J.A.,2004,Calcium,kinases and nodulation signalling in legumes,Nature Reviews Molecular Cell Biology,5(7):566-576
Rott R.,Liveanu V.,Drager R.G.,Stem D.B.,and Schuster G.,1998,The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of m RNA accumulation and stability,Plant Molecular Biology,36(2):307-314
Shaw G.,and Kamen R.,1986,A conserved AU sequence from the 3'-untranslated region of GM-CSF m RNA mediates selective m RNA degradation,Cell,46(5):659-667
Shimoda Y.,Han L.,Yamazaki T.,Suzuki R.,Hayashi M.,and Imaizumi-Anraku H.,2012,Rhizobial and fungal symbioses show different requirements for calmodulin binding to calcium calmodulin-dependent protein kinase in Lotus japonicus,The Plant cell,24(1):304-321
Sun D.Y.,Li H.B.,and Cheng G.,1994,Extracellular calmodulin accelerates the proliferation of suspension-cultured cells of Angelica dahurica,Plant Science,99(1):1-8
Suzuki Y.,Mae T.,and Makino A.,2008,RNA extraction from various recalcitrant plant tissues with a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment,Bioscience Biotechnology&Biochemistry,72(7):1951-1953
Szczyglowski K.,and Stougaard J.,2008,Lotus genome:pod of gold for legume research,Trends in Plant Science,13(10):515-517
Takezawad D.,Liu Z.H.,and An G.,1995,Calmodulin gene family in potato:developmental and touch-induced expression of the m RNA encoding a novel isoform,Plant Molecular Biology,27(4):693-703
Tang J.Y.,Zhao H.,He A.H.,Zhou J.C.,Jia G.,Liu G.M.,Chen X.L.,and Cai J.Y.,2016,Cloning,identification and tissue m RNA relative expression level analysis of porcine selenoprotein P gene,Dongwu Yingyang Xuebao(Chinese Journal of Animal Nutrition)28(3):858-863(汤加勇,赵华,何爱华,周继昌,贾刚,刘光芒,陈小玲,蔡景义,2016,猪硒蛋白P基因克隆,鉴定及组织m RNA相对表达量分析,动物营养学报,28(3):858-863)
Watillon B.,Kettman R.,Boxus P.,and Burny A.,1992,Cloning and characterization of an apple(Malus domestica Borkh)calmodulin gene,Plant Science,82(2):201-212
Yang T.,Levyadun S.,Feldman M.,and Fromm H.,1998,Developmentally regulated organ-,tissue-,and cell-specific expression of calmodulin genes in common wheat,Plant Molecular Biology,37(1):109-120
Yang T.,Segal G.,Abbo S.,Feidman M.,and Fromm H.,1996,Characterization of the calmodulin gene family in wheat:structure,chromosomal location,and evolutionary aspects,Molecular Genetics and Genomics,252(6):684-694
Zeng H.Q.,Xu L.Q.,Singh A.,Wang H.Z.,Du L.Q.,and Poovaiah B.W.,2015,Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses,Frontiers in Plant Science,6(660):600
Zhang W.,and Sun D.Y.,2009,Molecular and genetic evidence for the key role of At Ca M3 in heat-shock signal transduction in Arabidopsis,Plant Physiol.,149(4):1773-1784
Zimmer W.E.,Schloss J.A.,Silflow C.D.,Youngblom J.,and Watterson C.D.,1988,Structural organization,DNAsequence,and expression of the calmodulin gene,Journal of Biological Chemistry,263(36):19370-19383