ta sirna途径调控百脉根根瘤发育的研究-study on ta sirna pathway regulating the development of root nodule of pulse.docx

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ta sirna途径调控百脉根根瘤发育的研究-study on ta sirna pathway regulating the development of root nodule of pulse.docx

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ta sirna途径调控百脉根根瘤发育的研究-study on ta sirna pathway regulating the development of root nodule of pulse

附录AbstractRhizobiaarecapableofinfectingtherootsofleguminousplants,leadingtotheformationnitrogen-fixingnodules.Whilenitrogen-fixingnodulecanprovidenitrogenforhostplants,toomanynodulesbecomeundesirable.Thenumberofnodulesistightlyregulatedbyenvironmentalfactorsandautoregulationofnodulation.Inthisstudy,weanalyzedphenotypesandmolecularmechanismsofLotusjaponicusrel3symbioticmutantwithreducednodules.Insymbioticandnon-symbioticconditions,thelengthofthemainrootisshorterandthenumberoflateralrootsislessinrel3thaninwildtype.Moreover,therel3mutantdevelopedapproximatelyhalfnumberofnodulesfoundinthewildtype.Andtheratioofnodulationzonelengthtothemainrootlengthintherel3plantswaslessthanthatinthewildtype.However,thereisnodifferenceinnitrogenaseactivitybetweenrel3andwildtype.Thephenotypicanalysisindicatedthatrel3showedfewernodulesduetonarrownodulationzone.WedeterminedtheinfectionfrequencyusingNZP2235/HemA::LacZthatconstitutivelyexpressestheβ-galactosidasereportergene.Afterinoculation,infectionthreadformationwasrepressedinrel3comparedtothatinthewildtype.Thissuggestthatthedecreasedinfectionfrequencyinrel3producenarrownodulationzoneinrel3,leadingtothereducednumberofnodulesformedinrel3.Inordertoinvestigatemolecularmechanismsofrel3withreducednodules,wefirstanalyzedthesensitivityofrel3tonegativeregulatorsofthenodulationincludingnitrogenandethylene.Theresultsshowthatthemutationdoesnotaffectthenegativeregulationofnoduledevelopmentbynitrateandethylene.Inaddition,thegraftingexperimentsofrel3andwildtypeindicatedthatthephenotypeofreducednodulenumberinrel3mutantisincontrolofshoot-derivedsignals.ByAgrobacteriumrhizogenes-mediatedroottransformation,weanalyzedREL3promoter-GUSactivityininoculatedrootsandnodules.TheGUSactivityshowedthatREL3genespecificallyexpressedinrootvascularbundleandnodulevascularstrands,particularlyincellsthatwerederivedfromthepericycle.AndREL3alsoexpressedinvascularbundleofdevelopingnodules.ThisindicatedthatREL3playsanimportantrolefornoduleinitiation.REL3mutationsblockedthesynthe