巴西固氮螺菌Sp7中與NifA相互作用的陽(yáng)性因子的基因克隆和功能分析.pdf

1、中國(guó)農(nóng)業(yè)大學(xué)博士學(xué)位論文巴西固氮螺菌Sp7中與NifA相互作用的陽(yáng)性因子的基因克隆和功能分析姓名：涂然申請(qǐng)學(xué)位級(jí)別：博士專(zhuān)業(yè)：微生物學(xué)指導(dǎo)教師：陳三鳳20060601AbstraetAzospit‘iliumbrasilenaeisanitrogenfixingrhizobacteriumlivingincloseassociationwithplantsrootsLikeotherdiazotrophs，theexpressionof

2、thenitrogenfixation(H∞genesnthisbacteriumisdependentontheUanscriptional8ctiV8靜rNifANifAplaysakeyroleinregulatingthesynthesisofnitrogenaseTheexpressionandactivityofNifAisregulatedinresponsetotheenvironmentalsignalsofmolec

3、ularoxygenandfixednitrogenHowevernoFixL／JorNifL—likeproteinhasbeendetectedinAbrasilenseThusthemechanismsinvolvedinNifAexpressionandactivityinresponsetooxygenandammoniastillremainunclearInrecentstudy，weconstructedanAbrasi

4、lenseSp7libraryforyeasttwohybridstudiesandscreenedfourdifferentgeneswhosepredictedproteinsinteractwithNifA。ButthestopcodonswerenotfoundinthesegenesSo，inthisstudytheopenreadingframesofthefourpositivecloneswereobtainedbyth

5、ehomologousrecombinationfirstlyThedifferentsequencesofthepositivecloneswereasfollowss4clone(I4kb)，$35clone(106kb)，$64clone(137kb)and$65clone(157kb)TheirfunctionswereexploredlnthisstudythenThemultiplealigmnentsofthededuce

6、damino—acidsequencessuggestedthatFhuEwasaspecificoutermembranereceptorforcoprogenandOr935wasatwo—componentsensorkinase／responseregulatorhybridThisisthefirstreportthatthetwocomponentsensor／responseregulatorhybridexistsinA

7、brasilenseStructuralanalysesshowedthatOr935comprisedthreediscreteconserveddomains：theN—terminalPAS，thecentralhistidineproteinkinase(HPK)andtheC—terminalresponseregulatmrRRlWetestedtheinteractionbetweenthedifferentdomains

8、ofOr935andthedifierentdomainsoiNifA，Theyeasttwo—hybridanalysisindicatedthattheinteractionbetweenOr935andNifAproteinswasmediatedbyPASdomainofOr935andtheNterminalGAFdomainofNifAInfurtherstudy，or935mutantwasconstructedbydel

9、etingPASdomainandreplacingitwitha20kbnfragmentTheor935mutantdisplayedasignificantlyhighernitrogenaseactivitythanwildtypebutthemutantwasn’treleasedfromtheinhibitoryeffectofoxygenandfixednitrogenTheexpressionlevelsoin(／H1c

10、Zwassignificantlyhigherin。，935mutantthaninwildtypeindicatedthatOr935mighthaveallinhibitoryeffectOnNifAactivityintilepresenceot’ammoniumandhigh()，concentrationsWepostulatedthatOr935mightinhibitetheNifAactivitythroughthefo

11、rmationofproteinproteincomplexunderconditionsinappropriateforffitrogenfixation，F(xiàn)inalllyweatsoconstructed$64mutantbydeletingitsPASdomainsandreplacingthemwithanfragmentandthencharacterizedthemutantThefunctionsofthesepositi