花生MAPK13基因的克隆及表达分析研究
|
摘要
为了挖掘花生抗果腐病相关基因,本研究以花生品种花育20号为试验材料,从花生果腐病转录组文库中筛选出促丝裂原活化蛋白激酶13(MAPK13)基因编码区序列设计引物,通过RACE-PCR克隆得到AhMAPK13基因。结果表明AhMAPK13基因序列全长1818 bp,含有3'非编码区593 bp,5'非编码区122 bp,开放阅读框全长1104 bp,编码1条含有368个氨基酸的蛋白序列。预测其分子量为42.5kDa,含有MAPK家族典型的11个结构域,属于MAPK基因家族B组成员。亚细胞定位显示AhMAPK13定位于细胞质和细胞核中。RT-qPCR分析发现AhMAPK13基因在茎中表达量高于其他组织,说明该基因具有组织表达特异性;AhMAPK13基因受干旱、低温、NaCl、ABA诱导时表达量上升,说明该基因可能广泛参与植物非生物胁迫响应过程;AhMAPK13基因受JA、GA、ET、SA诱导时表达量上调,说明该基因可能参与到这些激素介导的抗逆信号转导途径。本研究结果为花生抗果腐病育种研究提供了新的基因资源。
In this article, our aim is to detect peanut genes associated with peanut pod rot resistance. A novel mitogen-activated protein kinase gene, named AhMAPK13, was isolated from leaf of peanut(Arachis hypogaea L. cultivar Huayu20) using the previous peanut pod rot transcriptomic library coding area sequence as template by PACE-PCR. The full-length cDNA of AhMAPK13 is 1818 bp, including a 1104 bp ORF, a 122 bp 5′UTR and a 593 bp 3′ UTR. The number of deduced amino acid was 368 and its predicted molecular weight 42.5 kDa. Sequence analysis revealed that the sequence contain the 11 MAPK motifs, belongs to the group B MAPKs in plants. Further, subcellular localization analysis showed that AhMAPK13 was located in the nucleus and cytoplasm in plant cells. The tissue specific expression analysis by RT-qPCR showed that AhMAPK13 is a tissue specific expression gene with the most abundant level in stems roots. The expressions of AhMAPK13 were induced by PEG6000,NaCl,4℃ and ABA, suggesting that AhMAPK13 may be involved in ABA mediated abiotic stress processes. The increased expression of the AhMAPK13 gene induced by JA, GA, ET, and SA indicates that the gene may participate in these hormones mediated stress response signal transduction pathways. The results of this study provide a new gene resource for peanut resistance breeding.
In this article, our aim is to detect peanut genes associated with peanut pod rot resistance. A novel mitogen-activated protein kinase gene, named AhMAPK13, was isolated from leaf of peanut(Arachis hypogaea L. cultivar Huayu20) using the previous peanut pod rot transcriptomic library coding area sequence as template by PACE-PCR. The full-length cDNA of AhMAPK13 is 1818 bp, including a 1104 bp ORF, a 122 bp 5′UTR and a 593 bp 3′ UTR. The number of deduced amino acid was 368 and its predicted molecular weight 42.5 kDa. Sequence analysis revealed that the sequence contain the 11 MAPK motifs, belongs to the group B MAPKs in plants. Further, subcellular localization analysis showed that AhMAPK13 was located in the nucleus and cytoplasm in plant cells. The tissue specific expression analysis by RT-qPCR showed that AhMAPK13 is a tissue specific expression gene with the most abundant level in stems roots. The expressions of AhMAPK13 were induced by PEG6000,NaCl,4℃ and ABA, suggesting that AhMAPK13 may be involved in ABA mediated abiotic stress processes. The increased expression of the AhMAPK13 gene induced by JA, GA, ET, and SA indicates that the gene may participate in these hormones mediated stress response signal transduction pathways. The results of this study provide a new gene resource for peanut resistance breeding.
引文
[1] FABRA A,CASTRO S,TAURIANI T,et al.Interaction among Arachis hypogaea L.(peanut) and beneficial soil microorganisms:how much is it known [J].Crit Rev Microbiol,2010,36:179-94.
[2] MICHIELSE C,REP M.Pathogen profile update:Fusarium oxysporum [J].Mol Plant Pathol,2009,10:311-24.
[3] CHISHOLM ST,COAKER Q,DAY B,et al.Host-microbe interactions:shaping the evolution of the plant immune response [J].Cell,2006,124:803-814.
[4] DANGL JL,JONES JD.Plant pathogens and integrated defence responses to infection [J].Nature,2001,411:826-833.
[5] ROBERT-SEILANIANTZ A,GRANT M,JONES JD.Hormone crosstalk in plant disease and defense:more than just jasmonate-salicylate antagonism [J].Ann Rev Pltytopathol,2011,49:317-343.
[6] LI A,ZHOU M,WEI D,et al.Transcriptome profiling reveals the negative regulation of multiple plant hormone signaling pathways elicited by overexpression of C-repeat binding factors [J].Front Plant Sci,2017,8:1647.
[7] KALAPOS B,DOBREV P,NAGY T et al.Transcript and hormone analyses reveal the involvement of ABA-signalling,hormone crosstalk and genotype-specific biological processes in cold-shock response in wheat [J].Plant Sci,2016,253:86-97.
[8] LLORENTE F,MUSKETT P,SANCHEZ-VALLET A,et al.Repression of the auxin response pathway increases Arabidopsis susceptibility to necrotrophic fungi [J].MolPlant,2008,1:496-509.
[9] FONSECA S,CHICO JM,SOLANO R.The jasmonate pathway:the ligand,the receptor and the cole signalling module [J].Curr Opin Plant Biol,2009,12:539-547.
[10] ZHANG S,ZHANG D,FAN S,et al.Effect of exogenous GA3 and its inhibitor paclobutrazol on floral formation,endogenous hormones,and flowering-associated genes in 'Fuji' apple (Malus domestica Borkh) [J].Plant PhysiolBiochem,2016,107:178-186.
[11] SAXENA I,SRIKANTH S,CHEN Z.Cross talk between H2O2 and interacting signal molecules under plant stress response [J].Front Plant Sci,2016,7:570.
[12] PITZSCHKE A,SCHIKORA A,HIRT H.MAPK cascade signalling networks in plant defence [J].Curr Opin Plant Biol,2009,12 (4):421-426.
[13] COLCOMBET J,HIRT H.Arabidopsis MAPKs:a complex signalling network involved in multiple biological processes [J].Biochem J,2008,413 (2):217-226.
[14] GOYAL R K,TULPAN D,CHOMISTEK N,et al.Analysis of MAPK and MAPKK gene families in wheat and related Triticeae species [J].BMC Genomics,2018,19(1):178.
[15] MULLER J,BECK M,METTBACH U,et al.Arabidopsis MPK6 is involved in cell division plane control during early root development,and localizes to the pre-prophase band,phragmoplast,trans-Golgi network and plasma membrane[J].Plant J,2010,61:234-248.
[16] ARZEMYSLAW J,MALGORZATA T,AGATA C,et al.Mitogen-activated protein kinase cascades in plant hormone signaling[J].Frontiers in Plant Science,2018,9:1387.
[17] WANG J,PAN C,WANG Y,et al.Genome-wide identification of MAPK,MAPKK,and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber[J].BMC Genomics,2015,16:386.
[18] MAPK GROUP.Mitogen-activated protein kinase cascades in plants:a new nomenclature[J].Trends Plant Sci,2002,7:301-308.
[19] ZHANG X,XU X,YU Y,et al.Integration analysis of MKK and MAPK family members highlights potential MAPK signaling modules in cotton[J].Sci Rep,2016,6:29781.
[20] CHEN L,HU U,TAN S,et al.Genome-wide identification and analysis of MAPK and MAPKK gene families in Brachypodium distachyon[J].PLoS ONE,2012,7(10):e46744.
[21] KONG F,WANG J,CHENG L,et al.Genome-wide analysis of the mitogen-activated protein kinase gene family in Solanum lycopersicum[J].Gene,2012,499:108-120.
[22] REYNA N S,YANG Y.Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection[J].Mol Plant Microbe Interact,2006,19:530-540.
[23] ZHANG L,XI D,LI S,et al.A cotton group C MAP kinase gene,GhMPK2,positively regulates salt and drought tolerance in tobacco[J].Plant Molecular Biology,2011,77(1-2):17-31.
[24] GIMENEZ-IBANEZ S,SOLANO R.Nuclear jasmonate and salicylate signaling and crosstalk indefense against pathogens [J].Front Plant Sci,2013,4.
[2] MICHIELSE C,REP M.Pathogen profile update:Fusarium oxysporum [J].Mol Plant Pathol,2009,10:311-24.
[3] CHISHOLM ST,COAKER Q,DAY B,et al.Host-microbe interactions:shaping the evolution of the plant immune response [J].Cell,2006,124:803-814.
[4] DANGL JL,JONES JD.Plant pathogens and integrated defence responses to infection [J].Nature,2001,411:826-833.
[5] ROBERT-SEILANIANTZ A,GRANT M,JONES JD.Hormone crosstalk in plant disease and defense:more than just jasmonate-salicylate antagonism [J].Ann Rev Pltytopathol,2011,49:317-343.
[6] LI A,ZHOU M,WEI D,et al.Transcriptome profiling reveals the negative regulation of multiple plant hormone signaling pathways elicited by overexpression of C-repeat binding factors [J].Front Plant Sci,2017,8:1647.
[7] KALAPOS B,DOBREV P,NAGY T et al.Transcript and hormone analyses reveal the involvement of ABA-signalling,hormone crosstalk and genotype-specific biological processes in cold-shock response in wheat [J].Plant Sci,2016,253:86-97.
[8] LLORENTE F,MUSKETT P,SANCHEZ-VALLET A,et al.Repression of the auxin response pathway increases Arabidopsis susceptibility to necrotrophic fungi [J].MolPlant,2008,1:496-509.
[9] FONSECA S,CHICO JM,SOLANO R.The jasmonate pathway:the ligand,the receptor and the cole signalling module [J].Curr Opin Plant Biol,2009,12:539-547.
[10] ZHANG S,ZHANG D,FAN S,et al.Effect of exogenous GA3 and its inhibitor paclobutrazol on floral formation,endogenous hormones,and flowering-associated genes in 'Fuji' apple (Malus domestica Borkh) [J].Plant PhysiolBiochem,2016,107:178-186.
[11] SAXENA I,SRIKANTH S,CHEN Z.Cross talk between H2O2 and interacting signal molecules under plant stress response [J].Front Plant Sci,2016,7:570.
[12] PITZSCHKE A,SCHIKORA A,HIRT H.MAPK cascade signalling networks in plant defence [J].Curr Opin Plant Biol,2009,12 (4):421-426.
[13] COLCOMBET J,HIRT H.Arabidopsis MAPKs:a complex signalling network involved in multiple biological processes [J].Biochem J,2008,413 (2):217-226.
[14] GOYAL R K,TULPAN D,CHOMISTEK N,et al.Analysis of MAPK and MAPKK gene families in wheat and related Triticeae species [J].BMC Genomics,2018,19(1):178.
[15] MULLER J,BECK M,METTBACH U,et al.Arabidopsis MPK6 is involved in cell division plane control during early root development,and localizes to the pre-prophase band,phragmoplast,trans-Golgi network and plasma membrane[J].Plant J,2010,61:234-248.
[16] ARZEMYSLAW J,MALGORZATA T,AGATA C,et al.Mitogen-activated protein kinase cascades in plant hormone signaling[J].Frontiers in Plant Science,2018,9:1387.
[17] WANG J,PAN C,WANG Y,et al.Genome-wide identification of MAPK,MAPKK,and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber[J].BMC Genomics,2015,16:386.
[18] MAPK GROUP.Mitogen-activated protein kinase cascades in plants:a new nomenclature[J].Trends Plant Sci,2002,7:301-308.
[19] ZHANG X,XU X,YU Y,et al.Integration analysis of MKK and MAPK family members highlights potential MAPK signaling modules in cotton[J].Sci Rep,2016,6:29781.
[20] CHEN L,HU U,TAN S,et al.Genome-wide identification and analysis of MAPK and MAPKK gene families in Brachypodium distachyon[J].PLoS ONE,2012,7(10):e46744.
[21] KONG F,WANG J,CHENG L,et al.Genome-wide analysis of the mitogen-activated protein kinase gene family in Solanum lycopersicum[J].Gene,2012,499:108-120.
[22] REYNA N S,YANG Y.Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection[J].Mol Plant Microbe Interact,2006,19:530-540.
[23] ZHANG L,XI D,LI S,et al.A cotton group C MAP kinase gene,GhMPK2,positively regulates salt and drought tolerance in tobacco[J].Plant Molecular Biology,2011,77(1-2):17-31.
[24] GIMENEZ-IBANEZ S,SOLANO R.Nuclear jasmonate and salicylate signaling and crosstalk indefense against pathogens [J].Front Plant Sci,2013,4.