固氮斯氏假單胞菌A1501鞭毛調(diào)控基因fleQ的功能研究.pdf

1、中國(guó)農(nóng)業(yè)科學(xué)院碩士學(xué)位論文固氮斯氏假單胞菌A1501鞭毛調(diào)控基因fleQ的功能研究姓名：張秋磊申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：生物化學(xué)與分子生物學(xué)指導(dǎo)教師：平淑珍20080601AbstractStableandhighefficiencymicroplantinteractionisessentialtotheoptimalnitrogenaseactivityofrootassociatednitrogenfixationbacteriaT

2、hereareseveralfactorsthatparticipateinthisprocessincludingtherecognitionofoutsidesignal，thechemotaxis，colonizationontherootsandtheinvasionintothesuperficiallayersoftherootcortexDiazotrophicbacteriacallbeinducedbysonlecer

3、tainsignalsfromenvironmentandmovetothesurfaceofrootsdirectionallyDuringthisprocess，theflagellumplaysallimportantrole，PseudomonasstutzeriA1501，isolatedfromricepaddysoilsinsouthChina，exhibitsgoodnitrogen—fixingactivityunde

4、rtheconditionoflowoxygentensionandammoniaconcentrationAtpresent，themainresearchregionsofA1501arefocusedontheregulationofnitrogen—fixation(n／y)genesandtheuptakesystemofcarbonsource，whichhavebeenmadegreatprogressHoweveriti

5、sstillunclearonhowtheassociativemicroplantinteractionwasestablishedAnalysisoftherecentlypublishedA1501genomesequence，atleast100genesrelatedtotheflagellabiosynthesisandchemotaxiswereidentifiedWefurtherselecttheputativereg

6、ulatorgenefleQasthetargetgeneformutationandfunctionidentificationSequenceanalysisofPstutzeriAI501genomeshowedthatthefleQandthetwodownstreamgenes，tieSandtierencodedregulatorarecloselyrelatedtoeachotherTheRTPCRresultsshowe

7、dthatfleQbelongedtoatranscribedunit，whilefleSandfieRweresharedinanoperonAminoacidsequenceswerecomparedusingtheNCBI—BLASTsearchsystemA1501FIeQwasfoundtoshareproteinsequencehomologywithknownFIeQregulatorproteinsbacterialsp

8、ecieswhichshowedhighesthomologyto只aeruginosaFleQ(identity283％)ToanalyzethefunctionofA1501regulatorgenefleQ，weconstructedthesuicideplasmidvectorpfleQ，containingthefleQgenefragmentThen，pfleQwastransferedintothewildtypestra

9、inA1501bytri—parentalmatingassayandthemutationstrain△FIeQwasobtainedbyhomologousrecombinationselectionnecomplementarystrain(FleQComp)carryingtheexpressingpeQgeneplasmidwasalsoconstructedThemutantoffleQleadstothedisappear

10、ingofflagellumbutitcanberecoveredbythecomplementationstrainFleQCompThisresultwasobservedandconlrurnedbythetransmissionelectronmicroscopyfTEM)DisruptionoffleQdoesn’taffectthegrowthandnitrogenaseabilityofthemutant，butinflu

11、encestheabilitytomoveandaffecttheformationofthebiofilmsReal—timeRTPCRassaysshowthattheexpressionsofsomekeystructuregenesencodingoftheflagellumbiosynthesis，suchasfi98，flgE，∥iCandfliG，weredescenddown—regulateddramaticallyb

12、yRTPCRinthefleQmutantTheresultsshowedindicatesthatFleQcanbeapositiveregulateoronpositivelytheexpressionoftheflagellagene，andthemutantwillstopthethesynthesisofflagellumwhichwillinfluencetheassemblyofflagellumTheresultsfro

13、mexperimentofricebreedingexperimentwithbyinoculatethemutantandWTA1501strainsfoundthattherootsandheightofriceweresignificantlyweakerinmutantthaninⅥ叮A1501Thissuggeststhatthegrowthofcropswasinfluencedbecausethesystemofthede