张荣娟差异基因课件

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