miR在豆科植物苜蓿根中对其菌根化和分生组织活性的

2023/1/181miR396affectsmycorrhizationandrootmeristemactivityin

thelegumeMedicagotruncatulaThePlantJournal(2013)doi:10.1111/tpj.121782023/1/182主要内容INTRODUCTIONRESULTS

1、Mtr-miR396aandmtr-miR396bgenesshowdifferenttissue-specificexpressionprofilesinMedicagotruncatularoots2、InM.truncatularoots,miR396regulatessixGRFandtwobHLH79transcriptionfactors3、CharacterizationofthemiR396regulatorynetworkinM.truncatula4、miR396negativelyaffectsrootgrowth,RAMsizeandcellproliferationintherootapex5、SilencingofMtGRFgenesmimicsthemiR396-OErootgrowthphenotype6、HormonalcontrolofmiR396expression7、miR396limitsmycorrhizalcolonizationbutnotnodulation2023/1/183INTRODUCTIONInplants,rootgrowthiscontrolledviacoordinatedcelldivisionandexpansionattherootapex,wherethreeregionsareobserved:(i)themeristematiczone,(ii)theelongationzone,and(iii)thedifferentiationzoneRootdevelopmentisregulatedbycomplexinteractionsofhormonesignallingpathways2023/1/184Inrecentyears,microRNAs(miRNAs)haveemergedasimportantregulatorsofthearchitectureoftherootsysteminArabidopsisthaliana,miR165/166governstheradialpatterningofvascularandpericycletissuesviaquantitativerepressionofHD-ZIPIIItranscriptionfactorsAsetofauxin-relatedmiRNAs,includingmiR160,alsoregulatelateralrootinitiationand/orrootgrowth2023/1/185However,fewmiRNAshavebeenreportedtoplayrolesinRAMfunctionAlthoughlargesetsofmycorrhizationornodulation-responsivemiRNAshavebeenidentifiedinlegumes,fewhavebeenfunctionallylinkedtotheseprocesses.2023/1/186InA.thaliana,miR396isencodedbytwoloci,post-transcriptionallyrepressesseveralmembersofthegrowth-regulatingfactor(GRF)family.Theseplant-specificTFscontrolthegrowthanddevelopmentofleavesandstems.miR396mayhaveevolvedtoplayrolesinvariousregulatorynetworksinplants.Inthispaper,wedescribetherolesofthemiR396regulatorynetworkinM.truncatularoots.2023/1/187RESULTS1、Mtr-miR396aandmtr-miR396bgenesshowdifferenttissue-specificexpressionprofilesinMedicagotruncatularootsMtr-miR396a和mtr-miR396b在根中的表达具有空间特异性2023/1/1882023/1/189miR396expressioninorgansandroottips两种miRNAs在整株植物中都有表达，其中miR396b在根、瘤、叶和豆荚中的表达量较高，miR396a在根尖中较多2023/1/1810两者在根中的表达在根尖横切面中的表达在根尖中的表达在侧根中的表达2023/1/4112、InM.truncatularoots,miR396regulatessixGRFandtwobHLH79transcriptionfactorsWefoundreadscorrespondingtothemiR396-relatedcleavageproductsofsixGRFmRNAs(MtGRF1,MtGi10-TC183867;MtGRF2,MtGi10-TC183494;MtGRF3,MtGi10BG454006;MtGRF4,Medtr5g027250;MtGRF5,Medtr8g020560;MtGRF6,Medtr7g126820).ThesetranscriptspossessaputativemiR396bindingsite,andtheencodedproteinsallcontaintheconserveddomainsofGRFsWealsoobservedabundantdegradomereadscorrespondingtospecificcleavageoftwonon-GRFtranscriptsatmiR396bindingsites,BasedontBLASTXanalysis,the2predictedproteinsshowedhighsimilarity(43and46%respectively)toAtbHLH79/BIGPETAL(At1g59640),aTFinvolvedinthecontrolofpetalgrowthinA.thaliana.2023/1/4122023/1/413InM.truncatula,sixMtGRFandtwobHLH79geneshavemiR396bindingsites3、CharacterizationofthemiR396regulatorynetworkinM.truncatulaToinvestigatemiR396-mediatedgeneregulationinplanta,weover-expressedmtr-miR396aandmtr-miR396bprecursorsinM.truncatularoots(miR396-OE).2023/1/414左图说明OE突变体能成成功过量表表达成熟的的miRNAs2023/1/415LevelsoftranscriptsofmiR396targetsweremeasuredusingquantitativeRT-PCRinmiR396-OErootsMtGRF-mRNA水平和MtbHLH79转录水平平都降低低，说明明miR396可能抑制制这些基基因的表表达水平平Tofurthervalidatetheirtargets,wegeneratedrootsexpressingatargetmimicryconstructthatwassuccessfullydesignedtohindermiR396activityinA.thaliana.Mimicry(MIM)transcriptsspecificallytrapmembersofanmiRNAfamily,andthuspreventtheiractivityonendogenoustargets2023/1/4162023/1/417LevelsoftranscriptsofmiR396targetsweremeasuredusingquantitativeRT-PCRinrootsinactivatedformiR396activity(MIM396)除了GRF6，其他基因表表达水平都上上升，这里还还不能排除有有其他因子对对GRF6进行调节的可可能，本文中中没有深入研研究4、miR396negativelyaffectsrootgrowth,RAMsizeandcellproliferationintherootapexToinvestigatethefunctionofmiR396inroots,wemeasuredthelengthanddryweightofrootsthateitheroverexpressedthemtr-miR396bprecursor(miR396-OE)orwereinactivatedformiR396activity(MIM396).2023/1/4182023/1/419miR396-OE中主主根根长长度度和和干干重重明明显显减减少少，，而而MIM396中根根长长稍稍微微增增加加，，而而干干重重却却大大量量增增加加。。说说明明miR396过表表达达能能抑抑制制根根的的生生长长2023/1/420RootmeristemsizeisreducedinmiR396-OEcompositeplants2023/1/421两者之间间没有大大的变化化，说明明miR396对细胞从从细胞周周期进入入分裂周周期的开开关没有有大的影影响2023/1/422Theexpressionofmitoticcellcycle(CYCB1;1,CYCB1;3,CYCB2;1,CYCD3;1,histoneH4,CDC16)orendocycle(CCS52B)markerswasmeasuredusingquantitativeRT-PCRinmiR396-OEandcontrolrootsThepercentageofcellsreplicating(i.e.EdU-positivenuclei)obtainedfrom20roottipsperconstructinfourindependentexperimentsTogether,thesedatastronglysuggestthatmiR396-dependentmodulationofrootgrowthmayessentiallybeduetoarestrictionofcelldivisionactivity.2023/1/4235、SilencingofMtGRFgenesmimicsthemiR396-OErootgrowthphenotypeTostudytheroleofthemiR396targetsonrootgrowth,weinactivatedthemusinganRNAistrategy.First,wegeneratedcompositeplantsexpressingthreeRNAiconstructstargetingaconservedregionofvariousGRFgenes(MtGRF2,MtGRF4andMtGRF6).Inaddition,wepreparedanRNAivectortargetingbothMtbHLH79genesandconfirmedtheirefficientsilencing2023/1/4242023/1/425SilencingofMtGRFgenesmimicsmiR396over-expressioninroots.iGRF表达减少而而ibHLH79表达不变，，说明很可可能是miR396对GRF基因的负调调控使得miR396-OE和MIM396植株中根的的生长表型型不同6、HormonalcontrolofmiR396expressionWetreatedwild-typeplantsfor1,3or5hwithauxin,cytokinin,gibberellin,abscisicacidorbrassinosteroids(BR),andmeasuredpre-miR396andtargetmRNAlevelsinroots.2023/1/4262023/1/427Gibberellin,cytokininandauxintreatmentsdidnotaffectexpressionofanyofthesegenes2023/1/428miR396b对ABA和BR有明明显显的的反反应应，，并并且且GRF5在BR处理理下下下下调调，，说说明明BR和miR396b,GRF5之间间有有某某种种作作用用关关系系2023/1/429LevelsofGUStranscriptinrootsexpressingtheprom-miR396b:GUSconstruct(greybars)ortheprom-GRF5:GUSconstruct(blackbars)treatedornottreatedwith1nMBRfor3hweremeasuredusingquantitativeRT-PCR.BR可以活活化miR396b的启动动子活活性，，但prom-GRF5:GUS并没有有明显显减少少，说说明BR可能是是在转转录后后水平平抑制制GRF5基因的的表达达，而而且BR和miR396b存在潜潜在联联系，，但本本文中中没有有对其其研究究7、miR396limitsmycorrhizalcolonizationbutnotnodulationWenextdecidedtostudythepotentialroleofmiR396inrootsymbioses.Weanalysedmtr-miR396aandmtr-miR396bexpressionprofilesinrootsinoculatedwiththesymbioticbacteriaSinorhizobiummeliloti.2023/1/4302023/1/431miR396a启动子子首先先在根根瘤导导管组组织中中发现现，miR396b启动子子在维维管组组织，，感染染区（（Ⅱ）和氮氮固定定区（（Ⅲ）含量量很高高2023/1/432虽然然miR396存在在于于根根瘤瘤中中，，但但miR396-OE和MIM396对根根瘤瘤密密度度都都没没有有明明显显影影响响WealsoanalysedtheexpressionofmiR396inresponsetoMyc-LCOs,thelipochitooligosaccharidesignalmoleculesproducedbyAMfungi.TheinvolvementofmiR396duringmycorrhizationwasthenanalysedat5weeksaftercolonizationwithR.irregularis(formerlyGlomusintraradices)inmiR396-OEandMIM396roots.2023/1/4332023/1/434miR396affectsfungalcolonizationduringAMsymbiosis.菌根根的的丛丛枝枝量量和和形形态态没没变变化化，，但但AM定植在miR396-OE根中的量量减少，，而在MIM396中的量增增加Hence,theactionofmiR396limitsmycorrhizalcolonization.Here,weshowedthat,inthemodellegumeM.truncatula,miR396repressesnotonlyMtGRFbutalsotwoMtbHLH79TF-encodinggenes,andthatitsde-regulationaffectsrootgrowthandtheabilitytobecolonizedbymycorrhizalfungi.2023/1/4352023/1/436Thankyou!9、静夜四无邻邻，荒居旧业业贫。。1月-231月-23Wednesday,January4,202310、雨中黄黄叶树，，灯下白白头人。。。21:26:0021:26:0021:261/4/20239:26:00PM11、以我独沈沈久，愧君君相见频。。。1月-2321:26:0021:26Jan-2304-Jan-2312、故人江江海别，，几度隔隔山川。。。21:26:0021:26:0021:26Wednesday,January4,202313、乍乍见见翻翻疑疑梦梦，，相相悲悲各各问问年年。。。。1月月-231月月-2321:26:0021:26:00January4,202314、他乡生白发发，旧国见青青山。。04一月20239:26:00下午21:26:001月-2315、比不了得得就不比，，得不到的的就不要。。。。一月239:26下下午1月-2321:26January4,202316、行行动动出出成成果果，，工工作作出出财财富富。。。。2023/1/421:26:0021:26:0004January202317、做前，能够够环视四周；；做时，你只只能或者最好好沿着以脚为为起点的射线线向前。。9:26:00下午9:26下下午21:26:001月-239、没有失败败，只有暂暂时停止成成功！。1月-231月-23Wednesday,January4,202310、很多事情情努力了未未必有结果果，但是不不努力却什什么改变也也没有。。。21:26:0021:26:0021:261/4/20239:26:00PM11、成成功功就就是是日日复复一一日日那那一一点点点点小小小小努努力力的的积积累累。。。。1月月-2321:26:0121:26Jan-2304-Jan-2312、世世间间成成事事，，不不求求其其绝绝对对圆圆满满，，留留一一份份不不足足，，可可得得无无限限完完美美。。。。21:26:0121:26:0121:26Wednesday,January4,202313、不知香积寺寺，数里入云云峰。。1月-231月-2321:26:0121:26:01January4,202314、意意志志坚坚强强的的人人能能把把世世界界放放在在手手中中像像泥泥块块一一样样任任意意揉揉捏捏。。04一一月月20239:26:01下下午午21:26:011月月-2315、楚塞塞三湘湘接，，荆门门九派派通。。。。。一月239:26下下午午1月-2321:26January4,202316、少年年十五五二十十时，，步行行夺得得胡马马骑。。。2023/1/421:26:0121:26:0104January202317、空