木薯促分裂原激活蛋白激酶MeMAPK2基因的克隆和功能分析
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摘要
木薯(Manihot esculenta Crantz)是热带、亚热带地区最为重要的粮食作物之一,为研究木薯MAPK(Mitogen-activated protein kinases)亚家族基因在木薯抗病抗逆过程中的作用机理,本研究以木薯品种‘SC8’为材料,从叶片中提取总RNA,并利用RT-PCR技术克隆了木薯促分裂原激活蛋白激酶亚家族成员之一的开放阅读框(open reading frame, ORF)基因片段,命名为MeMAPK2 (Phytozome数据库编号:Manes.01G058000)。生物信息学分析表明:MeMAPK2基因全长1 140 bp,编码379个氨基酸,pI值为6.97,推测其相对分子质量约43.35 kD。氨基酸多序列比对和系统进化分析表明,木薯MeMAPK2与多种植物MAPK基因均具有较高的同源性,其中与巴西橡胶树MAPK2基因同源性高达91%,表明了该基因在进化上相对保守。实时荧光定量PCR结果显示,木薯MeMAPK2基因在木薯根,茎,叶中均有表达,但在叶片中的表达明显高于根和茎中的表达量,表明木薯MeMAPK2基因的表达具有明显的组织特异性;另外,利用实时荧光定量PCR分析了MeMAPK2基因在干旱、重金属及激素、病原菌处理下的表达情况,结果表明该基因的表达受到明显的诱导,以上结果初步证明了MeMAPK2基因参与了木薯抗病抗逆反应。本研究为植物应对外界刺激如生物胁迫和非生物胁迫以及基因工程技术改良植物抗病抗逆性提供参考依据。
Manihot esculenta Crantz is one of the most important food crops in the tropical and subtropical areas with high content of starch. In order to figure out the functions of MAPK(Mitogen-activated protein kinases) genes during the cassava resistance to disease and adversity, a cDNA sequence putatively encoding a novel mitogen-activated protein kinase(MAPK), designated MeMAPK2(Phytozome: Manes.01 G058000), was isolated and characterized from the leaves of cassava by RT-PCR. Sequence analysis indicated that MeMAPK2 gene had an open reading frame(ORF) of 1 140 base pairs(bp) encoding 379 amino acids(aa) of 43.35 kD with an isoelectric point(pI) of6.97. Multiple amino acids sequence alignment and phylogenetic evolution revealed that MeMAPK2 shared high identity with many MAPKs in other plants, especially the Hevea brasiliensis MAPK2 with 91% homology. These results indicated that the MAPK was highly conserved evolutionarily. The real-time fluorescent quantitative PCR results showed that the transcription of MeMAPK2 has tissue-specificity and it expressed higher in leaves than other tissues. Moreover, the MeMAPK2 mRNA exhibited an extremely high expression level during the biotic and abiotic stress, like drought, heavy metal, exogenous plant hormone and pathogens, which indicated that the MeMAPK2 may play a key role in response to the environmental biotic and abiotic stresses. The above results preliminarily proved that MeMAPK2 gene were involved in the resistance to disease and adversity of cassava. This study will bring a good foundation to comprehend and further explore the plant response to biotic and abiotic stresses and improve the plant resistance to disease and adversity by bioengineering.
Manihot esculenta Crantz is one of the most important food crops in the tropical and subtropical areas with high content of starch. In order to figure out the functions of MAPK(Mitogen-activated protein kinases) genes during the cassava resistance to disease and adversity, a cDNA sequence putatively encoding a novel mitogen-activated protein kinase(MAPK), designated MeMAPK2(Phytozome: Manes.01 G058000), was isolated and characterized from the leaves of cassava by RT-PCR. Sequence analysis indicated that MeMAPK2 gene had an open reading frame(ORF) of 1 140 base pairs(bp) encoding 379 amino acids(aa) of 43.35 kD with an isoelectric point(pI) of6.97. Multiple amino acids sequence alignment and phylogenetic evolution revealed that MeMAPK2 shared high identity with many MAPKs in other plants, especially the Hevea brasiliensis MAPK2 with 91% homology. These results indicated that the MAPK was highly conserved evolutionarily. The real-time fluorescent quantitative PCR results showed that the transcription of MeMAPK2 has tissue-specificity and it expressed higher in leaves than other tissues. Moreover, the MeMAPK2 mRNA exhibited an extremely high expression level during the biotic and abiotic stress, like drought, heavy metal, exogenous plant hormone and pathogens, which indicated that the MeMAPK2 may play a key role in response to the environmental biotic and abiotic stresses. The above results preliminarily proved that MeMAPK2 gene were involved in the resistance to disease and adversity of cassava. This study will bring a good foundation to comprehend and further explore the plant response to biotic and abiotic stresses and improve the plant resistance to disease and adversity by bioengineering.
引文
Agrawal G.K.,Agrawal S.K.,Shibato J.,Iwahashi H.,and Rakwal R.,2003,Novel rice MAP kinases OsMSRMK3 and OsWJUMK1 involved in encountering diverse environmental stresses an d developmental regulation,Biochem.Biophys.Res.Commun.,300(3):775-783
Agrawal G.K.,Rakwal R.,and Iwahashi H.,2002,Isolation of novel rice(Oryza sativa L.)multiple stress responsive MAP kinase gene,OsMSRMK2,whose m RNA accumulates rapidly in response to environmental cues,Biochem.Biophys.Res.Commun.,294(5):1009-1016
Beckers G.J.,Jaskiewicz M.,Liu Y.,Underwood W.R.,He S.Y.,Zhang S.,and Conrath U.,2009,Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana,Plant Cell,21(3):944-953
Droillard M.,Boudsocq M.,Barbier-Brygoo H.,and Laurière C.,2002,Different protein kinase families are activated by osmotic stresses in Arabidopsis thaliana cell suspensions,FEBS Lett.,527(1-3):43-50
Droillard M.J.,Boudsocq M.,Barbierbrygoo H.,and Laurière C.,2004,Involvement of MPK4 in osmotic stress response pathways in cell suspensions and plantlets of Arabidopsis thaliana:activation by hypoosmolarity and negative role in hyperosmolarity tolerance,FEBS Lett.,574(1):42-48
Duerr B.,Gawienowski M.,Ropp T.,and Jacobs T.,1993,MsERK1:a mitogen-activated protein kinase from a flowering plant,Plant Cell,5(1):87-96
Group M.,Ichimura K.,Shinozaki K.,Tena G.,Sheen J.,Henry Y.,Champion A.,Kreis M.,Zhang S.,and Hirt H.,2002,Mitogen-activated protein kinase cascades in plants:a new nomenclature,Trends Plant Sci.,7(7):301-308
Guan Z.H.,Chen X.,and Wang H.Y.,2015,Cloning of soluble starch synthase promoter in cassava(MeSSΙΙb)and its gradient deletion analysis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(8):1753-1760(关志辉,陈新,王海燕,2015,木薯可溶性淀粉合成酶(MeSSΙΙb)启动子克隆及梯度缺失分析,基因组学与应用生物学,34(8):1753-1760)
Hamel L.P.,Nicole M.C.,Sritubtim S.,Morency M.J.,Ellis M.,Ehlting J.,Beaudoin N.,Barbazuk B.,Klessig D.,Lee J.,Martin G.,Mundy J.,Ohashi Y.,Scheel D.,Sheen J.,Xing T.,Zhang S.,Seguin A.,and Ellis B.E.,2006,Ancient signals:comparative genomics of plant MAPK and MAPKK gene families,Trends Plant Sci.,11(4):192-198
Ichimura K.,Mizoguchi T.,Yoshida R.,Yuasa T.,and Shinozaki K.,2000,Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6,Plant J.,24(5):655-665
Jonak C.,Ligterink W.,and Hirt H.,1999,MAP kinases in plant signal transduction,Cell Mol.Life Sci.,55(2):204-213
Jonak C.,Nakagami H.,and Hirt H.,2004,Heavy metal stress.Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium,Plant Physiol.,136(2):3276-3283
Jonak C.,Okrész L.,Bogre L.,and Hirt H.,2002,Complexity,cross talk and integration of plant MAP kinase signalling,Curr.Opin.Plant Biol.,5(5):415-424
Lopez C.,Soto M.,Restrepo S.,Piégu B.,Cooke R.,Delseny M.,Tohme J.,and Verdier V.,2005,Gene expression profile in response to Xanthomonas axonopodis pv.manihotis in cassava using a cDNA microarray,Plant Mol.Biol.,57(3):393-410
Lu C.,Guevara-Garcia A.,and Fedoroff N.V.,2002,Mitogen-activated protein kinase signaling in postgermination arrest of development by abscisic acid,Proc.Natl.Acad.Sci.USA,99(24):15812
Mizoguchi T.,Ichimura K.,and Shinozaki K.,1997,Environmental stress response in plants:the role of mitogen-activated protein kinases,Trends Biotechnol.,15(1):15-19
Rao K.P.,Richa T.,Kumar K.,Raghuram B.,and Sinha A.K.,2010,In silico analysis reveals 75 members of mitogen-activated protein kinase kinase kinase gene family in rice,DNARes.,17(3):139-153
Reyna N.S.,and Yang Y.N.,2006,Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection,Mol.Plant Microbe Interact.,19(5):530-540
Ulm R.,Ichimura K.,Mizoguchi T.,Peck S.C.,Zhu T.,Wang X.,Shinozaki K.,and Paszkowski J.,2002,Distinct regulation of salinity and genotoxic stress responses by Arabidopsis MAP kinase phosphatase 1,Embo J.,21(23):6483-6493
Verdier V.,Mosquera G.,and AssigbétséK.,1998,Detection of the cassava bacterial blight pathogen,Xanthomonas axonopodis pv.manihotis,by polymerase chain reaction,Plant Dis.,82(1):79-83
Widmann C.,Gibson S.,Jarpe M.B.,and Johnson G.L.,1999,Mitogen-activated protein kinase:conservation of a three-kinase module from yeast to human,Physiol.Rev.,79(1):143-180
Yang Z.Y.,Ma H.G.,Hong H.M.,Yao W.,Xie W.B.,Xiao J.H.,Li X.H.,and Wang S.P.,2015,Transcriptome-based analysis of mitogen-activated protein kinase cascades in the rice response to Xanthomonas oryzae infection,Rice,8(1):4
Yoo S.D.,Cho Y.H.,and Sheen J.,2007,Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis,Nat.Protoc.,2(7):1565-1572
Zhang D.,Jiang S.,Pan J.,Kong X.,Zhou Y.,Liu Y.,and Li D.,2014,The overexpression of a maize mitogen-activated protein kinase gene(ZmMPK5)confers salt stress tolerance and induces defence responses in tobacco,Plant Biol.,16(3):558
Agrawal G.K.,Rakwal R.,and Iwahashi H.,2002,Isolation of novel rice(Oryza sativa L.)multiple stress responsive MAP kinase gene,OsMSRMK2,whose m RNA accumulates rapidly in response to environmental cues,Biochem.Biophys.Res.Commun.,294(5):1009-1016
Beckers G.J.,Jaskiewicz M.,Liu Y.,Underwood W.R.,He S.Y.,Zhang S.,and Conrath U.,2009,Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana,Plant Cell,21(3):944-953
Droillard M.,Boudsocq M.,Barbier-Brygoo H.,and Laurière C.,2002,Different protein kinase families are activated by osmotic stresses in Arabidopsis thaliana cell suspensions,FEBS Lett.,527(1-3):43-50
Droillard M.J.,Boudsocq M.,Barbierbrygoo H.,and Laurière C.,2004,Involvement of MPK4 in osmotic stress response pathways in cell suspensions and plantlets of Arabidopsis thaliana:activation by hypoosmolarity and negative role in hyperosmolarity tolerance,FEBS Lett.,574(1):42-48
Duerr B.,Gawienowski M.,Ropp T.,and Jacobs T.,1993,MsERK1:a mitogen-activated protein kinase from a flowering plant,Plant Cell,5(1):87-96
Group M.,Ichimura K.,Shinozaki K.,Tena G.,Sheen J.,Henry Y.,Champion A.,Kreis M.,Zhang S.,and Hirt H.,2002,Mitogen-activated protein kinase cascades in plants:a new nomenclature,Trends Plant Sci.,7(7):301-308
Guan Z.H.,Chen X.,and Wang H.Y.,2015,Cloning of soluble starch synthase promoter in cassava(MeSSΙΙb)and its gradient deletion analysis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(8):1753-1760(关志辉,陈新,王海燕,2015,木薯可溶性淀粉合成酶(MeSSΙΙb)启动子克隆及梯度缺失分析,基因组学与应用生物学,34(8):1753-1760)
Hamel L.P.,Nicole M.C.,Sritubtim S.,Morency M.J.,Ellis M.,Ehlting J.,Beaudoin N.,Barbazuk B.,Klessig D.,Lee J.,Martin G.,Mundy J.,Ohashi Y.,Scheel D.,Sheen J.,Xing T.,Zhang S.,Seguin A.,and Ellis B.E.,2006,Ancient signals:comparative genomics of plant MAPK and MAPKK gene families,Trends Plant Sci.,11(4):192-198
Ichimura K.,Mizoguchi T.,Yoshida R.,Yuasa T.,and Shinozaki K.,2000,Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6,Plant J.,24(5):655-665
Jonak C.,Ligterink W.,and Hirt H.,1999,MAP kinases in plant signal transduction,Cell Mol.Life Sci.,55(2):204-213
Jonak C.,Nakagami H.,and Hirt H.,2004,Heavy metal stress.Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium,Plant Physiol.,136(2):3276-3283
Jonak C.,Okrész L.,Bogre L.,and Hirt H.,2002,Complexity,cross talk and integration of plant MAP kinase signalling,Curr.Opin.Plant Biol.,5(5):415-424
Lopez C.,Soto M.,Restrepo S.,Piégu B.,Cooke R.,Delseny M.,Tohme J.,and Verdier V.,2005,Gene expression profile in response to Xanthomonas axonopodis pv.manihotis in cassava using a cDNA microarray,Plant Mol.Biol.,57(3):393-410
Lu C.,Guevara-Garcia A.,and Fedoroff N.V.,2002,Mitogen-activated protein kinase signaling in postgermination arrest of development by abscisic acid,Proc.Natl.Acad.Sci.USA,99(24):15812
Mizoguchi T.,Ichimura K.,and Shinozaki K.,1997,Environmental stress response in plants:the role of mitogen-activated protein kinases,Trends Biotechnol.,15(1):15-19
Rao K.P.,Richa T.,Kumar K.,Raghuram B.,and Sinha A.K.,2010,In silico analysis reveals 75 members of mitogen-activated protein kinase kinase kinase gene family in rice,DNARes.,17(3):139-153
Reyna N.S.,and Yang Y.N.,2006,Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection,Mol.Plant Microbe Interact.,19(5):530-540
Ulm R.,Ichimura K.,Mizoguchi T.,Peck S.C.,Zhu T.,Wang X.,Shinozaki K.,and Paszkowski J.,2002,Distinct regulation of salinity and genotoxic stress responses by Arabidopsis MAP kinase phosphatase 1,Embo J.,21(23):6483-6493
Verdier V.,Mosquera G.,and AssigbétséK.,1998,Detection of the cassava bacterial blight pathogen,Xanthomonas axonopodis pv.manihotis,by polymerase chain reaction,Plant Dis.,82(1):79-83
Widmann C.,Gibson S.,Jarpe M.B.,and Johnson G.L.,1999,Mitogen-activated protein kinase:conservation of a three-kinase module from yeast to human,Physiol.Rev.,79(1):143-180
Yang Z.Y.,Ma H.G.,Hong H.M.,Yao W.,Xie W.B.,Xiao J.H.,Li X.H.,and Wang S.P.,2015,Transcriptome-based analysis of mitogen-activated protein kinase cascades in the rice response to Xanthomonas oryzae infection,Rice,8(1):4
Yoo S.D.,Cho Y.H.,and Sheen J.,2007,Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis,Nat.Protoc.,2(7):1565-1572
Zhang D.,Jiang S.,Pan J.,Kong X.,Zhou Y.,Liu Y.,and Li D.,2014,The overexpression of a maize mitogen-activated protein kinase gene(ZmMPK5)confers salt stress tolerance and induces defence responses in tobacco,Plant Biol.,16(3):558