斯氏假單胞菌固氮調(diào)節(jié)基因nifLA的表達(dá)調(diào)控機(jī)制研究.pdf

1、中國(guó)農(nóng)業(yè)大學(xué)博士學(xué)位論文斯氏假單胞菌固氮調(diào)節(jié)基因nifLA的表達(dá)調(diào)控機(jī)制研究姓名：解志紅申請(qǐng)學(xué)位級(jí)別：博士專業(yè)：遺傳學(xué)指導(dǎo)教師：林敏王國(guó)英20050601AbstractPseudomonasstutzeriA1501fformerlyAlcaligenesfaecal掃A1501)，isanassociativenitrogenfixingbacteria，whichcolonizestherhizosphereofriceThiss

2、traincanfixnitrogenundermicroaexobicconditionsinthefreelivingstate。Inthisstudy，wepresentdataonthemechanismofregulationofnitrogenfLxafionbyNifLAinpseudomonasstutzeriAl501AcosmidlibraryofA1501wasconslructedusingSuperCoslCo

3、smidVectorkit，andtwotransformantscontaining岫Z“l(fā)ikeweTescreenedbycolonyPCRandSouthernhybridizationThe77kbDNAfragmentcontainingnijL4iikewassubaloned(EMBL／GenBenk：AJ297529)，Thesequenceanalysisrevealedthatthehighestidentityo

4、f72％WaSobservedwithNiILofAvinelandiiend82％withNifAofAvinelandiiH牡geneissinglecopyinPstutzeriAl501只J船￡m—A1501niffIptacZfusionvectorwastransferedintothewildtypestrainA1501bytriparentalmatingassaythen13galactosidaseaetivi|y

5、WaS矗ss玨y舞afterinduced鑫tdifferentammoniumandoxygenconcentrationTheresultsuggestedthattheexpressionofn／fi1wasrepressedbyammoniumandoxygen，Inordertostudytheregulationfunctionofn／JZ—ofPseudomonasstutzer／A1501，月拉or聹弘mutantsan

6、drecombinantplasmidsofnifAorHl江(pVA3andpvL)wereconstructed，respectivelyThen／fAmutantand硇曼polar。mutant蝴NifnegativewhereasnonpolarH寥￡mutantdisplayedsomeresidualnitregenaseactivityevenunderhighammoniumandoxygenconcentration

7、Byassayingacetylenereductionactivityoftransconjugants，theresultsshowedthatplasmidwhichexpressed訂￡翻fromtheKmpromoterrestored30％nitrogenaseactivitytothe”0疆mutantswain，andthisplasmidalsoincreasednitrogenaseactivityofthe硒醚ty

8、peA1501andthe囂穢互nonpolarmutantTheplasmidwhichconstitutivelyexpressedn／fLdescreasednitrogenaseactivityofA1501andthen犯nonpolarmutantAlsooverexpressedNi￡^increasedexpressionofnifHplacZendoverexpressedNitLcouldn’tworkonn／fge

9、neexpression。Inordert。studytheinteractionbetweenNifLandNifA，fulllengthm瓣andn濰maddifferentregionsencodingtheopenreadingfral／leofHi724inPstutzeriA1501WereamplifiedbyPCR謝1hspecificprimersandclonedintoplasmidpGADC(I)andpGBDC

10、(1)RecombinantplasmidswerecotransformedintotheexpressionhostScereviMaePJ694Abythelithiumacetatemetho是andselectedWansfomumtswe球grown0nSDplateslackingHis／Ade，Len。andTipAninteractionbetweenNi凡andNifAinvivoWasdetectedbyyeast

11、twohybridsyst鋤anditwasshownthatonlytheCterminaldomainofNiff—displayedbindingactivitytoNifA，ContmctionofpET28anifAandpGEX2TngLcfusionexpressionvectorw∽performedandtransformeditinto曼c(diǎn)otiBL2I(DE31cellsOverexpressioninsolubl

12、eformwasachievedwithisopropylBDthingalactoside(IPTG)InteractionbetweenNi協(xié)andNiILinvitroWaSprovedusingpurifiedNifAandNiiI_cbybindingassayTheresultsuggestedthatthereexistedaregulationmodethatn／fganes％㈣inactivatedbyinteract