miR在豆科植物苜蓿根中對其菌根化和分生組織活性的

2023/1/181miR396affectsmycorrhizationandrootmeristemactivityin

thelegumeMedicagotruncatulaThePlantJournal(2013)doi:10.1111/tpj.121782023/1/182主要內(nèi)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在根中的表達具有空間特異性2023/1/1882023/1/189miR396expressioninorgansandroottips兩種miRNAs在整株植物中都有表達，其中miR396b在根、瘤、葉和豆莢中的表達量較高，miR396a在根尖中較多2023/1/1810兩者在根中的表達在根尖橫切面中的表達在根尖中的表達在側(cè)根中的表達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左圖說明OE突變體能成成功過量表表達成熟的的miRNAs2023/1/415LevelsoftranscriptsofmiR396targetsweremeasuredusingquantitativeRT-PCRinmiR396-OErootsMtGRF-mRNA水平和MtbHLH79轉(zhuǎn)錄水平平都降低低，說明明miR396可能抑制制這些基基因的表表達水平平Tofurthervalidatetheirtargets,wegeneratedrootsexpressingatargetmimicryconstructthatwassuccessfullydesignedtohindermiR396activityinA.thaliana.Mimicry(MIM)transcriptsspecificallytrapmembersofanmiRNAfamily,andthuspreventtheiractivityonendogenoustargets2023/1/4162023/1/417LevelsoftranscriptsofmiR396targetsweremeasuredusingquantitativeRT-PCRinrootsinactivatedformiR396activity(MIM396)除了GRF6，其他基因表表達水平都上上升，這里還還不能排除有有其他因子對對GRF6進行調(diào)節(jié)的可可能，本文中中沒有深入研研究4、miR396negativelyaffectsrootgrowth,RAMsizeandcellproliferationintherootapexToinvestigatethefunctionofmiR396inroots,wemeasuredthelengthanddryweightofrootsthateitheroverexpressedthemtr-miR396bprecursor(miR396-OE)orwereinactivatedformiR396activity(MIM396).2023/1/4182023/1/419miR396-OE中主主根根長長度度和和干干重重明明顯顯減減少少，，而而MIM396中根根長長稍稍微微增增加加，，而而干干重重卻卻大大量量增增加加。。說說明明miR396過表表達達能能抑抑制制根根的的生生長長2023/1/420RootmeristemsizeisreducedinmiR396-OEcompositeplants2023/1/421兩者之間間沒有大大的變化化，說明明miR396對細胞從從細胞周周期進入入分裂周周期的開開關沒有有大的影影響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表達減少而而ibHLH79表達不變，，說明很可可能是miR396對GRF基因的負調(diào)調(diào)控使得miR396-OE和MIM396植株中根的的生長表型型不同6、HormonalcontrolofmiR396expressionWetreatedwild-typeplantsfor1,3or5hwithauxin,cytokinin,gibberellin,abscisicacidorbrassinosteroids(BR),andmeasuredpre-miR396andtargetmRNAlevelsinroots.2023/1/4262023/1/427Gibberellin,cytokininandauxintreatmentsdidnotaffectexpressionofanyofthesegenes2023/1/428miR396b對ABA和BR有明明顯顯的的反反應應，，并并且且GRF5在BR處理理下下下下調(diào)調(diào)，，說說明明BR和miR396b,GRF5之間間有有某某種種作作用用關關系系2023/1/429LevelsofGUStranscriptinrootsexpressingtheprom-miR396b:GUSconstruct(greybars)ortheprom-GRF5:GUSconstruct(blackbars)treatedornottreatedwith1nMBRfor3hweremeasuredusingquantitativeRT-PCR.BR可以活活化miR396b的啟動動子活活性，，但prom-GRF5:GUS并沒有有明顯顯減少少，說說明BR可能是是在轉(zhuǎn)轉(zhuǎn)錄后后水平平抑制制GRF5基因的的表達達，而而且BR和miR396b存在潛潛在聯(lián)聯(lián)系，，但本本文中中沒有有對其其研究究7、miR396limitsmycorrhizalcolonizationbutnotnodulationWenextdecidedtostudythepotentialroleofmiR396inrootsymbioses.Weanalysedmtr-miR396aandmtr-miR396bexpressionprofilesinrootsinoculatedwiththesymbioticbacteriaSinorhizobiummeliloti.2023/1/4302023/1/431miR396a啟動子子首先先在根根瘤導導管組組織中中發(fā)現(xiàn)現(xiàn)，miR396b啟動子子在維維管組組織，，感染染區(qū)（（Ⅱ）和氮氮固定定區(qū)（（Ⅲ）含量量很高高2023/1/432雖然然miR396存在在于于根根瘤瘤中中，，但但miR396-OE和MIM396對根根瘤瘤密密度度都都沒沒有有明明顯顯影影響響WealsoanalysedtheexpressionofmiR396inresponsetoMyc-LCOs,thelipochitooligosaccharidesignalmoleculesproducedbyAMfungi.TheinvolvementofmiR396duringmycorrhizationwasthenanalysedat5weeksaftercolonizationwithR.irregularis(formerlyGlomusintraradices)inmiR396-OEandMIM396roots.2023/1/4332023/1/434miR396affectsfungalcolonizationduringAMsymbiosis.菌根根的的叢叢枝枝量量和和形形態(tài)態(tài)沒沒變變化化，，但但AM定植在miR396-OE根中的量量減少，，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、靜夜四無鄰鄰，荒居舊業(yè)業(yè)貧。。1月-231月-23Wednesday,January4,202310、雨中黃黃葉樹，，燈下白白頭人。。。21:26:0021:26:0021:261/4/20239:26:00PM11、以我獨沈沈久，愧君君相見頻。。。1月-2321:26:0021:26Jan-2304-Jan-2312、故人江江海別，，幾度隔隔山川。。。21:26:0021:26:0021:26Wednesday,January4,202313、乍乍見見翻翻疑疑夢夢，，相相悲悲各各問問年年。。。。1月月-231月月-2321:26:0021:26:00January4,202314、他鄉(xiāng)生白發(fā)發(fā)，舊國見青青山。。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、行行動動出出成成果果，，工工作作出出財財富富。。。。2023/1/421:26:0021:26:0004January202317、做前，能夠夠環(huán)視四周；；做時，你只只能或者最好好沿著以腳為為起點的射線線向前。。9:26:00下午9:26下下午21:26:001月-239、沒有失敗敗，只有暫暫時停止成成功！。1月-231月-23Wednesday,January4,202310、很多事情情努力了未未必有結(jié)果果，但是不不努力卻什什么改變也也沒有。。。21:26:0021:26:0021:261/4/20239:26:00PM11、成成功功就就是是日日復復一一日日那那一一點點點點小小小小努努力力的的積積累累。。。。1月月-2321:26:0121:26Jan-2304-Jan-2312、世世間間成成事事，，不不求求其其絕絕對對圓圓滿滿，，留留一一份份不不足足，，可可得得無無限限完完美美。。。。21:26:0121:26:0121:26Wednesday,January4,202313、不知香積寺寺，數(shù)里入云云峰。。1月-231月-2321:26:0121:26:01January4,202314、意意志志堅堅強強的的人人能能把把世世界界放放在在手手中中像像泥泥塊塊一一樣樣任任意意揉揉捏捏。。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，，荊門門九派派通。。。。。一月239:26下下午午1月-2321:26January4,202316、少年年十五五二十十時，，步行行奪得得胡馬馬騎。。。2023/1/421:26:0121:26:0104January202317、空