am真菌和胞囊线虫对大豆的防御反应

am真菌和胞囊线虫对大豆的防御反应

安迪蒙斯基亚（am）、自由配置的碳灰岩共享系统，拥有大多数二级森林、乔伊尔和硬件互联系统的root系统。跑动着模拟的非马蹄莲，以进入所有世界。am和未动未指的是，它是一个缓慢的目标，缓慢的目标是建立在一个没有陆地的地方。am和未动未指的是，它是一个缓慢的目标，缓慢的目标是建立在一个缓慢的地方。一般，这是雅歌劳动洲的特征。这是秋分墨冶异常发展的结果。进度、秋分墨冶鱼、东科等。2003年（美国科学家、乔恩等人，1986；博塔利亚等人，1998；李等人，2002；亚历斯科学士第七个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八个阶段，第八。Plantchitinase(EC4),alyticenzymethatcatalyzesthehydrolysisofchitin,isconsideredasoneoftheimportantcomponentsinvolvedinthedefenseresponsesagainstpathogens(Busametal.,1997).Theirtransientactivationaswellastheinductionofnewacidicisoformsanddifferentialexpressionwasshowedindifferentmycorrhizalplants(Pozoetal.,2002;Liuetal.,2003;Salzeretal.,2004).However,theexactroleandfunctionofmycorrhiza-inducedchitinaseisoformsarestillunclear.Overexpressionofchitinasegenesintransgenictobaccosdidnotaffecttheestablishmentandfunctioningofmycorrhizas,whilesuchplantsshowedanincreaseresistancetopathogens(Gianinazzi-Pearsonetal.,1994;Vierheiligetal.,1995).Thus,itisreasonabletopostulatethatsomechitinasesinducedbyAMfungimayplayaroleinprotectionagainstsoil-bornepathogens.Theactivationofphenylpropanoidmetabolismisoneoftheinitialdiseaseresistancereactionsinmanyplants(Dixon&Paiva,1995).PALcatalysesphenylalaninetotrans-cinnamicacidwhichisthefirststepinthebiosynthesisofphenylpropanoidsleadingtoadiversegroupofplantsecondarymetabolitesincludinglignins,phytoalexins,andflavonoids(Hahlbrock&Sccheel,1989).SoybeanrootscolonizedbyGlomusmosseaeorG.fasciculatumaccumulatedmoreoftheisoflavonoidphytoalexinglyceollinIthanthatofthenonmycorrhizalones(Morandietal.,1984).Inaddition,flavonoidaccumulationandactivitiesofPALincreasewasshowedinMedicagosativarootscolonizedbytheAMfungusG.intraradix(Volpinetal.,1994).However,theroleofPALassociatedwiththedefenseresponsesinducedbyAMfungiremainsunclear.Soybeancystnematode(SCN,Heteroderaglycines),themostfrequentlyfoundnematodespeciesinsoybeanfields,seriouslysuppressestheyieldofsoybeanbyasmuchasabout60%forthesusceptiblecultivars,andabout30%fortheresistantones.WepreviouslyshowedthattheAMfungusG.fasciculatumconferreduponthehostsoybeancultivar‘Ludou4’againsttheSCNattack(Lietal.,2002).Inthiswork,weextendedourstudytotestourhypothesisthatchitinaseandPALgenesChib1andPAL5mightbeinducedforadefensivereasonagainsttheSCNbyinoculationwiththeAMfungusG.fasciculatumintherootsofsoybean.1杏仁杏仁1.1新型价值规制Seedsofthesoybean[Glycinemax(L.)Merr.]cv‘Ludou4’whichissusceptibletoH.glycinesrace4weresurface-sterilizedwith2%NaOClfor2minandgerminatedonsterilizedwetfilterpaperat27℃for12h.Seedlingswereasepticallyplantedinpots(500mL),eachofwhichwasfilledwiththemixtureofsteam-sterilized(121℃,2h)sandand8000inoculumpotentialunits(IPU,oneIPUincludingthequantitativesporesandhyphaoftheAMfunguswhichwasasepticallyco-culturedwithtobaccosinsterilizedsand)ofGlomusversiformeBerch(Liu&Luo1994).Incontrolpots,thesamerootsegmentswithsandfromtobaccoseedlingsgrowninsterilizedsandwithoutAMfungusinoculationwereaddedinordertokeepthesamemicroorganismfloraexcludingAMfungiintherhizosphere.Eachofthegrapevineseedlingswasraisedinasinglepotplacedinagreenhouseatday/nighttemperatureof25/15℃witha16-hlight,andatarelativehumidityof60%.PotswerewateredeverydayandfertilizedwithHoagland’snutrientseverythreedays.At25daftergermination,halvesfromeithercontrolorAMplantswereinoculatedwith3000ofthesecond-stagejuveniles(J1.2非织造材料revilusingThecolonizationpercentageoftheAMfunguswasmeasuredaccordingtotheproceduredescribedbyBiermannandLinderman(1981):theAMrootswereobservedafterKOH(5%)digestionat90℃for45minandtrypanbluestaining.MeasurementofcolonizationstatuswasconductedundertheBX50OlympusmicroscopewithPM-30AutomaticPhotomicrographicSystem(Japan).TheinfectionrateofSCNwascountedaspreviouslydescribed(Liu,1995):thewholerootsystemwasstainedwithacidfuchsininlactophenolfor3min,thenumberofnematodesintherootsystemsofeachplantwerecountedafterexcisingtherootswithsharpdissectionneedle.1.3sphenylahene.关于MethoddescribedbyBolleretal.(1983)wasappliedtoassaychitinase:onegramfreshrootwasgroundwithapestleinamotarcontainingliquidnitrogen,suspendedin5mL0.1Mcitratesodiumbuffer,pH5.0,andcentrifuged(15000gfor15min)at4℃.Thereactionmixturecontained0.2mLappropriatelydilutedcrudeenzymepreparation,0.2mL(0.8mg)colloidalchitinand0.4mL0.1Maceticacidbuffer(pH5.2).Themixturewasincubatedinawaterbathat37℃for1h.Thereactionwasstoppedbycentrifugation.Oftheresultingsupernatant,0.4mLwasincubatedwith0.05mLsnailgutjuice(sigma)and0.05mLNa-Phosphatebuffertohydrolyzetheliberated,water-solublechitinoligomerstoGlcNAc.TheresultingGlcNAcwasdeterminedaccordingtoReissigetal.(1995).Absorbanceoftheresultingsupernatantwasmeasuredspectrophotometricallyat585nm.Chitinaseactivitywasdeterminedfromacalibrationcurve.Oneunit(U)isdefinedastheamountofenzymerequiredtocatalyzetheformationof1nmolproduct(GlcNAc)perminute.TheactivityofPALwasassayedaspreviouslydescribedbyMozzettietal.(1995):onegramofroottissuewashomogenizedin6mLof100mMboricacidbuffer,pH8.8,1%PVPand15mM2-mercaptoethanol.Thehomogenatewascentrifuged(15000g,15min)at4℃.1mLsupernatantwasaddedto3mLreactionmixturecontaining100mMboricacidbufferand25mML-phenylalanine.Themixturewasincubatedat40℃for1h,andPALactivityestimatedbymeasuringtheconversionofL-phenylalaninetotranscinnamicacidasthechangeinabsorbanceat290nm.ControlreactionlackedeitherL-phenylalanineorthesupernatantofthecrudehomogenate.PALactivitywasexpressedasthechangesofOD1.4pcr和wragmenb3SoybeanclassⅢacidicchitinaseChib1cDNAwasamplifiedbyreversetranscriptionpolymerasechainreaction(RT-PCR),thesenseandantisenseprimerswere5′ACACACCAACCCACTAGAAACAT3′and5′GTAGCGTTCACAGCACATTCAAT3′,respectively(Watanabeetal.,1999).PCRwasperformedtoamplifythePAL5DNAfragmentfromleafofbean(PhaseolusvulgarisL.)usingthesenseprimer5′ATTGCTGGTTTGCTCACTGGC3′andantisenseprimer5′AAGGTCCTATCTGACATGGAG3′,whichwerepreparedonthebasisofbeanPAL5cDNAsequence(Edwardsetal.,1985).TheresultingcDNAandDNAproductswerethenclonedintopGEM-Tvectorandsequenced.TheChib1chitinasecDNAandPAL5DNAfragmentswerelabeledrespectivelywith[α-p1.5rex-n-bine-swi探索RelativequantitativeRT-PCRwasperformedaccordingtothemethodofBurleigh(2001).OneµgeachofthetotalRNAsampleswasreverse-transcribedusingmurinereversetranscriptase(Promega)accordingtothemanufacturer’sprotocol.TheprimerOligo(dT)1.6mebaci开发的必要性ThirtyµgoftotalRNAweretransferredontonylonmembranesasdescribedpreviously(Wang&Fang,2002)Hybridizationwasperformedassection1.5.2-d-音频接口2.1InteractionsbetweentheAMfungusG.fasciculatumandH.glycinesToinvestigatethemutualinterferenceofthegrowthoftheAMfungusG.fasciculatumandtheSCNthatwerecolonizedonthesamerootsofsoybeanseedlingsof25-d-old,thinsectionsoftypicalappressoria,intracellularhyphae,arbusculesandvesiclesfromrootswereobservedundermicroscope(datanotshown).WhenmycorrhizalsoybeanrootswereinfectedbytheSCNandincubatedforupto13d,nosignificanteffectoftheSCNwasfoundonthecolonizationofG.fasciculatuminsoybeanroots(Fig.1).Ontheotherhand,theinfectionrateofSCNdisplayedaremarkablereductioninmycorrhizalsoybeanrootswithG.fasciculatumwhencomparedwiththatinnon-mycorrhizalsoybeanrootsinfectedbytheSCN(Fig.2).TheseresultsshowedthattheAMfungusG.fasciculatumconferredeffectiveprotectionagainsttheSCNuponsoybeanroots.2.2soybeanrepaloffig..3inrelactingsix对于scn的保护作用.InordertoevaluatethepossibleroleofchitinaseandPALinthedefenseresponsesinducedbytheAMfungusagainsttheSCN,thedynamicchangesinactivitiesofthesetwoenzymeswereanalyzed.Theresultsshowedthat,oneachtimepoint,thelevelsoftheactivitiesofbothchitinaseandPALinrootinoculatedwithG.fasciculatumalonewerehigherthanthatinrootsinfectedbytheSCNalone.Furthermore,inmycorrhizalsoybeanrootsinfectionbytheSCN,theactivitiesofthesetwoenzymesweresignificantlyenhancedascomparedwiththatinnon-mycorrhizalsoybeanrootsinfectedbytheSCN(Figs.3,4).Moreover,thepeaklevelofactivitiesofchitinaseandPALoccurred3dafterinfectionbytheSCN,whentheinfectionrateofSCNwasremarkablyreducedrelativetothatinnon-mycorrhizalsoybeanrootschallengedbytheSCN(Fig.2).Therefore,itwassuggestedthatAMsymbiosespredisposedsoybeantoamorerapidresponsetotheSCNattackthroughtheinductionofchitinaseandPALbytheAMfungusG.fasciculatum,andthesetwoenzymesmayplayanimportantroleinmycorrhizalsoybeanrootswiththeAMfungusagainsttheSCN.2.3civititatort-pcrsiphedTodetermineifthesymbiosiswiththeAMfungusG.fasciculatumcouldaffectexpressionprofilesofchitinasegenesinsoybeanroots,thetranscriptlevelswereexaminedbyRNAblotanalysisusingprobesspecifictothefourchitinasegenes.Todate,soybeanhasatleastfourchitinasegenes(Yeboahetal.,1998;Watanabeetal.,1999;Gijzenetal.,2001).However,thedetectionofanyofthefourchitinasetranscriptswasunsuccessful(datanotshown).Accordingly,themethodofBurleigh(2001),arelativequantitativeRT-PCR(RQRT-PCR)wasperformed.Theresultshowedthatonlyasinglebandofapproximately1.0kbforthetranscriptsChib1cDNAwasdetectedinmycorrhizalsoybeanrootswiththeAMfungusG.fasciculatum(datanotshown).Then,thedynamicchangesofChib1expressionindifferentsoybeanrootsinoculatedwiththeAMfungus,SCNorAMfungusplusSCNwereanalyzedbyRQRT-PCRusingsenseandantisenseprimers5′ATTCGGCACGAGTGCAAGAAT3′and5′TCCATACATTCATCGAGGATG3′(seeMaterialsandMethods).TheresultsshowedthataccumulationofChib1transcriptsoccurredonlyinsoybeanrootsinoculatedwiththeAMfungus,SCNorAMfungusplusSCN(Fig.5),anddisappearedintherootsofuninoculatedcontrolsoybean.However,ateachtimepointthelevelofChib1transcriptsappearedtoaccumulateathigherlevelintheorderofG.f+SCN>G.f>SCN,themaximumlevelofChib1transcriptswasachievedinmycorrhizalsoybeanroots3dafterinfectionbytheSCN,whentheinfectionrateofSCNshowedaremarkablereductionrelativetothatinnon-mycorrhizalsoybeanrootsinfectedbytheSCN(Fig.2).TheseresultssuggestedthattheAMfungusG.fasciculatuminducedtheexpressionofchitinasegeneChib1atatranscriptionallevel,whichcouldbeoneofthedefensiveresponsesinducedbytheAMfungusagainsttheSCN.2.4保证治疗pts自由基因子phity4.3.4indictory,whichinciphenge+3.4esiphincidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidicidiph+治疗whichcib3.4ssicidicidicidicidicidicidicidicidicidiph+治疗前后治疗前后sicidiph.sicidicidicidicidiph+治疗反应UsingPAL5DNAfragmentisolatedfromleavesofPhaseolusvulgarisasprobe,weexaminedthedifferentialexpressionofPAL5geneinsoybeanrootsofdifferenttreatmentsbyNorthernblothybridizationanalysis.ThePAL5transcriptsaccumulationwasobservedinthesoybeanrootsofallthetreatmentsincludinguninoculatedcontrol(Fig.6),whichindicatedthatthePAL5genewasconstitutivelyexpressedinsoybeanroot.However,oneachtimepoint,especially3dafterinoculationwiththeSCN,PAL5transcriptswasmostabundantintherootspre-inoculatedwiththeAMfungusandpost-inoculatedwiththeSCN,whichwasinaccordancewiththedynamicchangesofthePALactivity(Fig.4).Thisresult,togetherwiththedifferentialexpressionofchitinasegeneChib1demonstratedthatChib1andPAL5playedcriticalroleontranscriptionallevelindefenseresponsesinducedbytheAMfungusagainsttheSCN.3选择目标函数AMfungusGlomusfasiculatumconferredabioprotectiveeffectagainstsoybeancystnematode(SCN)Heteroderaglycinesrace4onsoybeancv‘Ludou4’whichsusceptibletothepathogen,i.e.,nosignificanteffectoftheSCNwasfoundonthecolonizationofG.fasciculatuminsoybeanroots,buttheinfectionrateofSCNdisplayedaremarkablereductioninmycorrhizalsoybeanrootswithG.fasciculatumwhencomparedwiththatinnon-mycorrhizalsoybeanrootsinfectedbytheSCN(Figures1,2).Theseresultsareinaccordancewiththosedescribedfordifferentmacorrhiza-nematodeinteractionsintherootsofotherplants(Jothietal.,1998;Diedhiouetal.,2003).PlantchitinaseandPALisconsideredtobetheimportantcomponentsoftheplantdefenseresponses,theirtransientactivationaswellastheinductionanddifferentialexpressionhavebeenreportedindifferentmycorrhizalplants(Pozoetal.,1996,1998,2002;Liuetal.,2003).However,todate,theexactrolesofthesetwoenzymesremainunclear.Inourpresentstudies,weanalyzedthetime-courseactivitiesofchitinaseandPAL,andexaminedthedifferentialexpressionofChib1andPAL5genesinsoybeanrootsinoculatedwiththeAMfungus,SCNorAMfungusplusSCN.AlthoughtheactivitiesofchitinaseandPALinrootinoculatedwithG.fasciculatumalonewerealittlehighertha