摘要
利用多种生物信息学网站与软件,从全基因组水平鉴定桉树焦枯病菌的促分裂原活化蛋白激酶(MAPK),并对其理化性质、亚细胞定位、结构域、二级结构、三级结构等进行分析,并构建系统发育树。结果表明,在桉树焦枯病菌中发现4个MAPK,分别命名为CpKss1、CpSlt2、CpHog1和CpIme2,均属于亲水蛋白,亚细胞定位均在细胞核。拥有高度保守的结构域STKc及位于活化环上的双重磷酸化位点T-X-Y,三级结构显示拥有蛋白特异性底物结合口袋特征。通过系统进化分析,这4个MAPK相应基因分别与酵母Fus3/Kss1、Slt2、Hog1和Ime2类基因同源,均和禾谷镰刀菌中相应的MAPK亲缘关系最近。该研究结果为进一步了解桉树焦枯病菌中MAPK的生物学功能和构建MAPK级联途径奠定基础。同时对桉树焦枯病菌致病、桉树抗病相关重要基因功能及分子机制等研究具有重要意义,最终为桉树焦枯病防治工作提供技术支持。
Using a variety of bioinformatics websites and software, the mitogen-activated protein kinase(MAPK)of C. Pseudoreteaudii from the wh ole genome level was identified, and its physical and chemical properties,subcellular localization, domain, secondary structure and tertiary structure were analyzed, as well as the phylogenetic tree was constructed. The results showed that four MAPKs were identified in C. Pseudoreteaudii and named CpKss1, CpSlt2, CpHog1 and CpIme2, respectively, which were all hydrophilic protein and their subcellular localization were located at nucleus. The four MAPK had a highly conserved domain STKc and a double phosphorylation site T-X-Y located on the activation ring. The tertiary structure showed the characteristics of protein-specific substrate-binding pockets. Through phylogenetic analysis, the corresponding genes of four MAPKs were homologous to the genes of yeast Fus3/Kss1, Slt2, Hog1 and Ime2, respectively, and they also had the closest relationship with the corresponding MAPK in Fusarium graminearum. The results would lay a foundation for further understanding the biological function of MAPK in C. Pseudoreteaudii and constructing the pathway of MAPK cascade. At the same time, it would be of great significance to study the pathogenicity of C.Pseudoreteaudii, the function and molecular mechanism of important genes related to disease resistance of Eucalyptus. Finally, it would provide technical support for the prevention and control of Eucalyptus Cyliudrocladium leaf blight.
引文
Alfenas R.F.,Pereira O.L.,Freitas R.G.,Freitas C.S.,Dita M.A.D.,and Alfenas A.C.,2013,Mass spore production and inoculation of Calonectria pteridis on Eucalyptus spp.under different environmental conditions,Tropical Plant Pathology,38(5):406-413
Bruno K.S.,Tenjo F.,Li L.,Hamer J.E.,and Xu J.R.,2004,Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea,Eukaryot Cell,3(6):1525-1532
Chen L.,Hu W.,Tan S.,Wang M.,Ma Z.,Zhou S.,Deng X.,Zhang Y.,Huang C.,Yang G.,and He G.,2012,Genomewide identification and analysis of MAPK and MAPKKgene families in Brachypodium distachyon,PLo S One,7(10):e46744
Chen Q.,Guo W.,Feng L.,Ye X.,Xie W.,Huang X.,and Liu J.,2015,Transcriptome and proteome analysis of Eucalyptus infected with Calonectria pseudoreteaudii,Journal of Proteomics,115:117-131
Chen Q.Z.,Guo W.S.,Ye X.Z.,Huang X.P.,and Wu Y.Z.,2013,Identification of Calonectria associated with Eucalyptus leaf blight in Fujian Province,Fujian Linxueyuan Xuebao(Journal of Fujian College of Forestry),33(2):176-182(陈全助,郭文硕,叶小真,黄秀萍,吴云忠,2013,福建省桉树焦枯病菌分类鉴定,福建林学院学报,33(2):176-182)
Chen R.E.,and Thorner J.,2007,Function and regulation in MAPK sign aling pathways:lessons learned from the yeast Saccharomyces cerevisiae,Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,1773(8):1311-1340
Chen S.F.,Lombard L.,Roux J.,Xie Y.J.,Wingfield M.J.,and Zhou X.D.,2011,Novel species of Calonectria associated with Eucalyptus leaf blight in Southeast China,Persoonia-Molecular Phylogeny and Evolution of Fungi,26(1):1-12
Cousin A.,Mehrabi R.,Guilleroux M.,Dufresne M.,Van Der Lee T.,Waalwijk C.,Langin T.,and Kema G.H.,2006,The MAP kinase-encoding gene MgFus3 of the non-appressorium phytopathogen Mycosphaerella graminicola is required for penetration and in vitro pycnidia formation,Molecular Plant Pathology,7(4):269-278
Di Pietro A.,García-Maceira F.I.,Meglecz E.,and Roncero M.I.G.,2001,A MAP kinase of the vascular wilt fungus Fusarium oxysporum is essential for root penetration and pathogenesis,Molecular Microbiology,39(5):1140-1152
Fan Y.S.,Liu Y.C.,Gu S.Q.,Gui X.M.,and Dong J.G.,2004,Mitogen activated protein kinase genes and its functions in phytopathogenic fungus,Weishengwu Xuebao(Acta Microbiologica Sinica),44(4):547-551(范永山,刘颖超,谷守芹,桂秀梅,董金皋,2004,植物病原真菌的MAPK基因及其功能,微生物学报,44(4):547-551)
Fujikawa T.,Kuga Y.,Yano S.,Yoshimi A.,Tachiki T.,Abe K.,and Nishimura M.,2009,Dynamics of cell wall components of Magnaporthe griseaduring infectious structure development,Molecular Microbiology,73(4):553-570
Garrido E.,and Pérez-Martín J.,2003,The crk1 gene encodes an Ime2-related protein that is required for morphogenesis in the plant pathogen Ustilago maydis,Molecular Microbiology,47(3):729-743
Garrido E.,Voss U.,Müller P.,Castillo-Lluva S.,Kahmann R.,and Pérez-Martín J.,2004,The induction of sexual development and virulence in the smut fungus Ustilago maydis depends on Crk1,a novel MAPK protein,Genes and Development,18(24):3117-3130
Gong X.D.,Wang Y.,Zhang P.,Fan Y.S.,Gu S.Q.,Han J.M.,and Dong J.G.,2015,Analysis of the genomic location,protein structure prediction and expression of MAPK gene StIME2 in Setosphaeria turcica,Zhongguo Nongye Kexue(Scientia Agricultural Sinica),48(13):2549-2558(巩校东,王玥,张盼,范永山,谷守芹,韩建民,董金皋,2015,玉米大斑病菌MAPK基因StIME2的基因组定位、2蛋白质结构预测及表达分析,中国农业科学,48(13):2549-2558)
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 MAPKKgene families,Trends in Plant Science,11(4):192-198
Hamel L.P.,Nicole M.C.,Duplessis S.,and Ellis B.E.,2012,Mitogen-activated protein kinase signaling in plant-interacting fungi:distinct messages from conserved messengers,The Plant Cell,24(4):1327-1351
Hou Z.,Xue C.,Peng Y.,Katan T.,Kistler H.C.,and Xu J.R.,2002,A mitogen-activated protein kinase gene(MGV1)in Fusarium graminearum is required for female fertility,heterokaryon formation,and plant infection,Molecular PlantM-icrobe Interactions,15(11):1119-1127
Hutchison E.A.,Bueche J.A.,and Glass N.L.,2012,Diversification of a protein kinase cascade:IME-2 is involved in nonself recognition and programmed cell death in Neurospora crassa,Genetics,192(2):467-482
Irniger S.,2011,The Ime2 protein kinase family in fungi:more duties than just meiosis,Molecular Microbiology,80(1):1-13
Kojima K.,Takano Y.,Yoshimi A.,Tanaka C.,Kikuchi T.,and Okuno T.,2004,Fungicide activity through activation of a fungal signalling pathway,Molecular Microbiology,53(6):1785-1796
Lev S.,Sharon A.,Hadar R.,Ma H.,and Horwitz B.A.,1999,Amitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation,appressorium formation,and pathogenicity:diverse roles for mitogen-activated protein kinase homologs in foliar pathogens,Proceedings of the National Academy of Sciences,96(23):13542-13547
Liang W.,Yang B.,Yu B.J.,Zhou Z.,Li C.,Jia M.,Sun Y.,Zhang Y.,Wu F.,Zhang H.,Wang B.,Deyholos M.K.,and Jiang Y.,2013,Identification and analysis of MKK and MPK gene families in canola(Brassica napus L.),BMC Genomics,14(1):392
Mehrabi R.,Van der Lee T.,Waalwijk C.,and Kema G.H.J.,2006a,MgSlt2,a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola,is dispensable for penetration but essential for invasive growth,Molecular Plant-Microbe Interactions,19(4):389-398
Mehrabi R.,Zwiers L.H.,de Waard M.A.,and Kema G.H.,2006b,Mg Hog1 regulates dimorphism and pathogenicity in the fungal wheat patho gen Mycosphaerella graminicola,Molecular Plant-Microbe Interactions,19(11):1262-1269
Miranda-Saavedra D.,and Barton G.J.,2007,Classification and functional annotation of eukaryotic protein kinases,Proteins:Structure,Function,and Bioinformatics,68(4):893-914
Motoyama T.,Ochiai N.,Morita M.,Iida Y.,Usami R.,and Kudo T.,2008,Involvement of putative response regulator genes of the rice blast fungus Magnaporthe oryzae in osmotic stress response,fungicide action,and pathogenicity,Current Genetics,54(4):185-195
Müller P.,Weinzierl G.,Brachmann A.,Feldbrügge M.,and Kahmann R.,2003,Mating and pathogenic development of the smut fungus Ustilago maydis are regulated by one mitogen-activated protein kinase cascade,Eukaryotic Cell,2(6):1187-1199
Segmüller N.,Ellendorf U.,Tudzynski B.,and Tudzynski P.,2007,BcSAK1,a stress-activated mitogen-activated protein kinase,is involved in vegetative differentiation and pathogenicity in Botrytis cinerea,Eukaryotic Cell,6(2):211-221
Solomon P.S.,Waters O.D.,Simmonds J.,Cooper R.M.,and O-liver R.P.,2005,The Mak2 MAP kinase signal transduction pathway is required for pathogenicity in Stagonospora nodorum,Current Genetics,48(1):60-68
Takano Y.,Kikuchi T.,Kubo Y.,Hamer J.E.,Mise K.,and Furusawa I.,2000,The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis,Molecular Plant-Microbe Interactions,13(4):374-383
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,Physiological Reviews,79(1):143-180
Xu J.R.,2000,MAP kinases in fungal pathogens,Fungal Genetics and Biology,31(3):137-152
Yang S.H.,Sharrocks A.D.,and Whitmarsh A.J.,2013,MAP kinase si gnalling cascades and transcriptional regulation,Gene,513(1):1-13
Ye X.,Liu H.,Jin Y.,Guo M.,Huang A.,Chen Q.,Guo W.,Zhang F.,and Feng L.,2017,Transcriptomic analysis of Calonectria pseudoreteaudii during various stages of Eucalyptus infection,PLoS One,12(1):e0169598
Ye X.Z.,Guo W.S.,Feng L.Z.,Jin Y.J.,Yang X.X.,Liu H.Y.,Lian X.K.,and Li H.M.,2016,Optimization on preparation condittion of protoplast from Calonectria pseudoreteaudii,Fujian Nonglin Daxue Xuebao(Ziran Kexue Ban)(Journal of Fujian Agriculture and Forestry University(Natural Science Edition)),45(2):141-145(叶小真,郭文硕,冯丽贞,金亚杰,杨新新,刘宏毅,连鑫坤,李慧敏,2016,桉树焦枯病菌原生质体制备条件的优化,福建农林大学学报(自然科学版),45(2):141-145)
Zhao X.,Kim Y.,Park G.,and Xu J.R.,2005,A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea,The Plant Cell,17(4):1317-1329
Zhao X.,Mehrabi R.,and Xu J.R.,2007,Mitogen-activated protein kinase pathways and fungal pathogenesis,Eukaryotic Cell,6(10):1701-1714
Zhu J.H.,Guo W.S.,Chen H.M.,Wu J.Q.,Chen Q.Z.,and Meng X.M.,2011,Loss estimation of eucalyptus growth cased by eucalyptus dieback,Zhongguo Senlin Bingchong(Forest Pest and Disease),30(5):6-10(朱建华,郭文硕,陈红梅,吴建勤,陈全助,孟祥民,2011,桉树焦枯病对桉树生长量的损失估计研究,中国森林病虫,30(5):6-10)
