肌醇磷酸合成酶 [Planta]

Planta(2010)231:1211–1227DOI10.1007/s00425-010-1127-8123ORIGINALARTICLEIdentiWcationandorganizationofchloroplasticandcytosolicL-myo-inositol1-phosphatesynthasecodinggene(s)inOryzasativa:comparisonwiththewildhalophyticrice,PorteresiacoarctataSudiptaRay·BarunavaPatra·AparajitaDas-Chatterjee·ArnabGanguli·ArunLahiriMajumderReceived:28November2009/Accepted:11February2010/Publishedonline:6March2010©Springer-Verlag2010AbstractThegenecodingforricechloroplasticL-myo-inositol-1-phosphatesynthase(MIPS;EC5.5.1.4)hasbeenidentiWedbymatrix-assistedlaserdesorptiontime-of-XightmassspectrometryanalysisofthepuriWedandimmunolog-icallycross-reactive»60kDachloroplasticproteinfollow-ingtwo-dimensionalpolyacrylamidegelelectrophoresis,whichexhibitedsequenceidentitywiththecytosolicMIPScodedbyOsINO1-1gene.ApossiblechloroplastictransitpeptidesequencewasidentiWedupstreamoftheOsINO1-1geneuponanalysisofricegenome.RT-PCRandconfocalmicroscopestudiesconWrmedtranscription,eVectivetrans-lationanditsfunctioningasachloroplasttransitpeptide.BioinformaticanalysismappedthechloroplasticMIPS(OsINO1-1)geneonchromosome3,andasecondMIPSgene(OsINO1-2)onchromosome10whichlacksconven-tionalchloroplasttransitpeptidesequenceasinOsINO1-1.TwonewPcINO1genes,withcharacteristicpromoteractiv-ityandupstreamcis-elementswereidentiWedandcloned,butwhethertheseproteinscanbetranslocatedtothechloroplastornotisyettobeascertained.ElectrophoreticmobilityshiftassaycarriedoutwithnuclearextractofPorteresiacoarctataleavesgrownunderbothcontrolandstressedconditionshowsbindingofnuclearproteinswiththeupstreamele-ments.NucleotidedivergenceamongthediVerentOryzaandPorteresiaINO1geneswerecalculatedandcompared.KeywordsChloroplasticL-myo-inositol-1-phosphatesynthase·MALDI-TOFMS·Transitpeptide·GFPXuorescence·Genomewalking·PorteresiacoarctataAbbreviationsMIPSL-myo-Inositol-1-phosphatesynthaseMALDI-TOFMSMatrix-assistedlaserdesorptionioniza-tiontime-of-XightmassspectrometryCaMVCauliXowermosaicvirusGFPGreenXuorescentprotein2DPAGETwo-dimensionalpolyacrylamidegelelectrophoresisORFOpenreadingframeGUS
-GlucuronidaseIntroductionL-myo-Inositol-1-phosphatesynthase(MIPS,EC5.5.1.4)catalyzestheenzymaticconversionfromglucose6-phosphateS.Ray·B.Patra·A.Das-Chatterjee·A.Ganguli·A.L.Majumder(&)PlantMolecularandCellularGenetics,BoseInstitute(CentenaryCampus),P1/12CITSchemeVIIM,Kolkata700054,Indiae-mail:[e-mail]:S.RayDepartmentofBotany,CentreofAdvancedStudies,UniversityofCalcutta,35,BallygungeCircularRoad,Kolkata700019,IndiaPresentAddress:B.PatraKentuckyTobaccoResearchandDevelopmentCentre,UniversityofKentucky,Lexington,KY,USAPresentAddress:A.Das-ChatterjeeMolecularandCellBiologyDepartment,GoldmanSchoolofDentalMedicine,BostonUniversityMedicalCenter,Boston,MA02118,USAPresentAddress:A.GanguliChembiotek,BlockBN,Plot7,SectorV,SaltlakeElectronicComplex,Kolkata700091,India1212Planta(2010)231:1211–1227123toL-myo-inositol1-phosphate,theimmediateprecursoroffreeinositol.ThereactionconsistsofacoupledoxidationandreductioncatalyzedbyL-myo-inositol-1-phosphatesynthase(MIPS)(Shermanetal.1969;LoewusandLoewus1983).TheoccurrencesofMIPSindiverseorgan-ismssuggestthatthepathwayisofancientevolutionaryorigin(Majumderetal.1997,2003;BachhawatandMande2000).MIPSwasshowntobepresentbothinthecytosolicfrac-tionandinorganelles(Lackeyetal.2003).TheorganellarformwasearliershowntobepresentinthechloroplastsofPea(ImhoVandBourdu1973),Vignaradiata,Euglenagracilis(Adhikarietal.1987),andOryzasativa(RayChaudhurietal.1997;Haitetal.2002).Thechloroplasticformoftheenzymewasfoundtobethesimilartothecyto-solicformwithrespecttobiochemicalandimmunologicalpropertiesandshowntoberegulatedbylightandsalt(RayChaudhuriandMajumder1996;RayChaudhurietal.1997;Haitetal.2002).ThechloroplasticMIPSprotein/genemayhavecomplexevolutionaryhistoryasMIPShasbeenreportedfromhigherplantsandalsofromcyanobacteria,e.g.Spirulinasp,thepre-sumedchloroplastprogenitor(RayChaudhurietal.1997).ThenativeproteinfromSpirulinashowedsimilarenzymaticandimmunologicalpropertiestootherMIPSproteins.However,nouniquestructuralgenecodingforMIPSfromSpirulinaoranyothercyanobacteriumwasreporteduntil,twounidentiWedopenreadingframes(ORFs),sll1722andsll1981fromSynechocystisspPCC6803werereportedasMIPScodinggenes(Chatterjeeetal.2004,2006).Outofthe65genespresentinthechloroplastgenomeofO.sativa(Accessionno.NC_008155),nocodinggeneforchloroplasticMIPScouldbedeterminedbyacomprehen-sivebioinformaticsanalysissuggestingthatthechloroplas-ticMIPSmightbenuclearencoded.Moreover,theMIPScodingORFssll1722andsll1981fromSynechocystisspPCC6803,amemberofthepresumptiveprogenitorgroupofhigherplantchloroplasts,showednosimilaritywithanyofthegenesencodedbythechloroplastgenome.HenceitwaspresumedthatthechloroplasticMIPSproteinmightbecodedbythenucleargenomeandpost-translationallytar-getedtothechloroplastbyachloroplast-speciWctransitpeptide(JarvisandSoll2001).Thoughsuchtransitpep-tidespossesscharacteristicfeatures,thereissofarnoknownmotifoftheseandthedegeneracyofthesequenceprecludesaPCR-basedapproachtoidentifyc