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cDNA-AFLP-------差异基因(所有挑选的差异均为间作处理中特异存在)代号基因推断功能同源性RJ2Conservedprotein(氧化还原酶位于同一个操纵元中,还没有关于此基因的研究)98%RJ4secAProbablepreproteintranslocaseSecAsubunitfunctionsinproteinexport;74%RJ6CgtA和ProB1PutativeGTP-bindingprotein和Probableglutamate5-kinase98%RJ7prkAPrkA;PutativeSerproteinkinase(Signaltransductionmechanisms)76%RJ9gltAtypeIIcitratesynthase:(Citratesynthaseactivitywasessentialfornodulemaintenance)70%RJ14thiCputativethiaminebiosynthesisprotein68%RJ5fix
RJ12
Transcriptionalregulator,LysRfamily与1021相似性低但与其他物种比对也是这个蛋白
总结----6个差异基因Q-PCR验证部分差异基因RJ4 secARJ6 CgtARJ6bProB1RJ7prkARJ9gltARJ14thiC总结abaabcbacbabaaaabbbbbacccJY9(gltA)文献报道:根瘤菌突变体中:不能结瘤或形成无效瘤,类菌体减少,胞外多糖比野生型多三倍,推测破坏了侵染线,缺乏能量,对结瘤固氮必须;大肠杆菌中:突变体生长速度减慢。abcJY14---thiC文献报道:Putativethiaminebiosynthesisprotein;突变体会推迟结瘤;Thiamineisaroleduringthetransitionfromaerobictofermentative-likemetabolismbaJY4(secA)文献报道:细菌特有的一种蛋白质,具有ATP酶活性,是Sec蛋白质转运途径中的“动力泵”,通过ATP的水解循环驱使多肽穿过通道。cbaJY7(prkA)文献报道:丝氨酸蛋白激酶;仅在芽孢杆菌中有报到;是一种cAMP依赖的蛋白激酶作用。cbaJY6B和JY6(下调)DisruptionofthecgtAgenewaslethal,demonstratingthatthisgeneisessentialforcellgrowth.secAE.coliandBacillus
功能已经明确,hydrolyzesATPandanessentialcomponentoftheproteintranslocationATPase;SecAProtein的ATP-bindingsite已经确定;SecA识别并结合preprotein-SecBcomplex,结合到inner-membraneanionicphospholipids,插入themembranebilayer以后,再与thepreproteintranslocator,SecY/SecE结合,从而将蛋白运输过膜.putativetransportproteintransmembranehypotheticalproteincbaSecBthiC文献分布:Sinorhizobiummeliloti(2)R.etli(2)E.coliandelse(15)Sinorhizobium突变体库中有关于这个突变体的信息;Rhizobiumetli:在其中表达thiCOGE,将增加其固氮效率;Thiaminbiosynthesisinprokaryotes:其中涉及12个基因(thiFSGH,I,anddxs,thiC,thiE,thiD,thiM,thiL,andpdxK)突变体库查询内容prkA(1)hypotheticalproteinSMc01266Bacillussubtilis
:prkAgeneencodinganovelserineproteinkinasecbaSMc00367(假定蛋白)Protein:putativeoxidoreductaseproteinputativeoxidoreductaseproteinRJ2360bpSMc00367Conservedprotein(氧化还原酶位于同一个操纵元中,同源性98%,还没有关于此基因的研究)98%JY6B和JY6(下调)/obgEobgE/proB150SribosomalproteinL21hypotheticalproteingamma-glutamylkinasegamma-glutamylphosphatereductasenicotinicacidmononucleotideadenylyltransferaseobgE(15篇—E.colimost)
(cgtA,yhbZ,obgE)codingforanessentialGTP-bindingprotein,染色体复制、分裂等过程中起调节作用,可能作用于核糖体合成的最后一步。proB1(Escherichiacoli
)Glutamate-5-kinase:
proline(脯氨酸)由glutamate(谷氨酸盐)合成需要三步:catalyzedbyglutamate5-kinase(G5K),glutamyl5-phosphatereductase(G5PR)andpyrroline5-carboxylatereductase;脯氨酸playsanimportantroleasanosmoprotectantinmicroorganismsGlutamate-5-kinase的activesiteresidues已确定TheEMBOJournalvol.8no.3pp.961-966,1989SecAproteinhydrolyzesATPandisanessentialcomponentoftheproteintranslocationATPaseofEscherichiacoliCharacterizationofaBacillussubtilisSecAmutantproteindeficientintranslocationATPaseandreleasefromthemembraneHistidineResiduesAreInvolvedinTranslocation-CoupledATPHydrolysisbytheSecAProtein
MolMicrobiol.1993Apr;8(1):31-42.CharacterizationofaBacillussubtilisSecAmutantproteindeficientintranslocationATPaseandreleasefromthemembrane-------ItisconcludedthattheGKTmotifintheamino-terminaldomainofSecAispartofthecatalyticATP-bindingsite.ThissitemaybeinvolvedintheATP-drivenproteinrecyclingfunctionofSecAwhichallowsthereleaseofSecAfromitsassociationwithprecursorproteins,andthephospholipidbilayer.
secASecAprotein:autoregulatedATPasecatalysingpreproteininsertionandtranslocationacrosstheEscherichiacoliinnermembran
RecentinsightintothebiochemicalmechanismsofproteintranslocationinEscherichiacoliindicatesthatSecAATPaseisrequiredbothfortheinitialbindingofpreproteinstotheinnermembraneaswellassubsequenttranslocationacrossthisstructure.SecAappearstopromotetheseeventsbydirectrecognitionofthepreproteinorpreprotein-SecBcomplex,bindingtoinner-membraneanionicphospholipids,insertionintothemembranebilayerandassociationwiththepreproteintranslocator,SecY/SecE.ATPbindingappearstocontroltheaffinityofSecAforthevariouscomponentsofthesystemandATPhydrolysispromotescyclingbetweenitsdifferentbiochemicalstates.Asacomponentlikelytocatalysearate-determiningstepinproteinsecretion,SecAsynthesisisco-ordinatedwiththeactivityoftheproteinexportpathway.ThisformofnegativeregulationappearstorelyonSecAproteinbindingtoitsmRNAandrepressingtranslationifconditionsofrapidproteinsecretionprevailwithinthecell.AprecisebiochemicalschemeforSecA-dependentcatalysisofproteinexportandthedetailsofsecAregulationappeartobecloseathand.TheevolutionaryconservationofSecAproteinamongeubacteriaaswellasthegeneralrequirementfortranslocationATPasesinotherproteinsecretionsystemsarguesforamechanisticcommonalityofallprokaryoticproteinexportpathways.prkACloningandcharacterizationoftheBacillussubtilisprkAgeneencodinganovelserineproteinkinase.Cloning,expression,purificationandcharacterizationofthestresskinaseYeaGfromEscherichiacoli.CharacterizationoftheBacillussubtilisthiCOperonInvolvedinThiamineBiosynthesisExpressionofThiaminBiosyntheticGenes(thiCOGE)andProductionofSymbioticTerminalOxidasecbb3inRhizobiumetli
ThesedatashowadirectrelationshipbetweenexpressionofthiCandproductionofthecbb3terminaloxidase.Thisisconsistentwiththepropositionthatapurine-relatedmetabolite,5-aminoimidazole-4-carbox-amideribonucleotide,isanegativeeffectoroftheproductionofthesymbioticterminaloxidasecbb3inR.etli.ThiaminbiosynthesisinprokaryotesTwelvegenesinvolvedinthiaminbiosynthesisinprokaryoteshavebeenidentifiedandoverexpressed.Ofthese,sixarerequiredforthethiazolebiosynthesis(thiF-SGH,I,anddxs),oneisinvolvedinthepyrimidinebiosynthesis(thiC),oneisrequiredforthelinkingofthethiazoleandthepyrimidine(thiE),andfourarekinases(thiD,thiM,thiL,andpdxK).AconservedRNAstructure(thibox)isinvolvedinregulationofthiaminbiosyntheticgeneexpressioninbacteria(R.etlistrainCE3)thiCThiaminelimitationdeterminesthetransitionfromaerobictofermentative-likemetabolisminRhizobiumetliCE3ThiCIsan[Fe-S]ClusterProteinThatRequiresAdoMetToGeneratethe-Amino-5-hydroxymethyl-2-methylpyrimidineMoietyinThiaminSynthesisReactionofAdoMetwithThiCgeneratesabackbonefreeradicalRoleofthecgtAgenefunctioninDNAreplicationofextrachromosomalelementsinEscherichiacoli(2003)ItseemsthatDNAsynthesisperseisnotaffectedbyCgtA,andthatthisproteinmightcontrolreplicationinitiationindirectly,byregulationoffunction(s)orproductionofoneormorereplicationfactors.Infact,wefoundthatlevelofthehost-encodedreplicationproteinDnaAissignificantlydecreasedinthecgtAmutant.ThisindicatesthatCgtAisinvolvedintheregulationofdnaAgeneexpression.OverexpressionofthecgtA(yhbZ,obgE)gene,codingforanessentialGTP-bindingprotein,impairstheregulationofchromosomalfunctionsinEscherichiacoli.(2002)DNAreplicationdefectintheEscherichiacolicgtA(ts)mutantarisingfromreducedDnaAlevels(2006)Obg/CtgA,aSignalingProteinThatControlsReplication,Translation,andMorphologicalDevelopment?(2006)TherecentfindingthattheObgEGTPaseactsasaatedwithObg:theeffectsonbacterialreplication(FotireplicationcheckpointproteininEscherichiacolihasetal.,2005).AnewE.coliobgEmutantwasisolatedimportantimplications.InvolvementofthecgtAgenefunctioninstimulationofDNArepairinEscherichiacoliandVibrioharveyi(2003)TheEscherichiacoliGTPaseCgtAEIsInvolvedinLateStepsofLargeRibosomeAssembly†(2006)MutationsinCgtAEcausebothpolysomeandrRNAprocessingdefects;small-andlarge-subunitprecursorrRNAsaccu-mulateinacgtAEmutant.TheObgE/CgtAGTPaseinfluencesthestringentresponsetoaminoacidstarvationinEscherichiacoli(2009)FunctionalanalysisoftheGTPasesEngAandYhbZencodedbySalmonellatyphimurium(2007)AbstractTheS.typhimuriumgenomeencodesproteins,designatedEngAandYhbZ,whichhaveahighsequenceidentitywiththeGTPasesEngA/DerandObgE/CgtAEofEscherichiacoli.Thewild-typeactivityoftheE.coliproteinsisessentialfornormalribosomematurationandcellviability.InordertocharacterizethepotentialinvolvementoftheSalmonellatyphimuriumEngAandYhbZproteinsinribosomebiology,weusedhighstringencyaffinitychromatographyexperimentstoidentifystronglybindingribosomalpartnerproteins.Acombinationofbiochemicalandmicrocalorimetricanalysiswasthenusedtocharacterizetheseprotein:proteininteractionsandquantifynucleotidebindingaffinities.TheseexperimentsshowthatYhbZspecificallyinteractswiththepseudouridinesynthaseRluD(KD¼2mMand1:1stoichiometry),andweshowforthefirsttimethatEngAcaninteractwiththeribosomalstructuralproteinS7.ThermodynamicanalysisshowsbothEngAandYhbZbindGDPwithahigheraffinitythan
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