重点参考不结球白菜CUSOD

Molecularcloningandexpressionanalysisof

aCu/Zn

SODgene(BcCSD1)fromBrassicacampestrisssp.chinensis

LijieCuia,1,QiangHuanga,1,BinYana,YaoWanga,ZhongyinQiana,JingxianPana,GuoyinKaia,

LaboratoryofPlantBiotechnology,DevelopmentCenterofPlantGermplasmResources,CollegeofLifeandEnvironmentSciences,ShanghaiNormalUniversity,Shanghai200234,PRChina

a

articleinfoabstract

Superoxidedismutases(SODs)areafamilyofmetalloproteinsextensivelyexistsineukaryote,whichplaysanessentialroleinstress-toleranceofhigherplants.Afull-lengthcDNAencodingCu/ZnSOD(BcCSD1)wasisolatedfromyoungseedlingsofnon-headingChinesecabbage(Brassicacampestrisssp.chinensis)byrapidampli cationofcDNAends(RACE).BioinformaticsanalysisrevealedthatBcCSD1belongedtotheplantSODsuperfamilyandhadtheclosestrelationshipwithSODfromBrassicanapus.TissueexpressionpatternanalysisrevealedthattheBcCSD1wasconstitutivelyexpressedinallthetestedtissues,andstrongestinleaf,moderateinstem,lowestinroot.Theexpressionpro lesunderdifferentstresstreatmentssuchasdrought,NaCl,hightemperatureandABAwerealsoinvestigated,andtheresultsrevealedthatBcCSD1wasastress-responsivegene,especiallytoABA.TheseresultsprovideusefulinformationforfurtherunderstandingtheroleofBcCSD1resistanttoabioticstressinBrassicacampestrisinthefuture.

Ó2014ElsevierLtd.Allrightsreserved.

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Keywords:

Non-headingChinesecabbage(Brassicacampestrisssp.chinensis)Cu/ZnSODgeneAbioticstress

Geneexpressionanalysis

1.Introduction

Reactiveoxygenspecies(ROS),includingsuperoxide( OÀ),hydrogenperoxide(H2O2),hydroxylradicals(–OH)andsingletoxygen(1O2),areby-productsofcellmetabolismatnormalcondi-tion(Wiseman&Halliwell,1996).ThegenerationandscavengingofROSareinbalanceduringtheplantgrowth(Alscher,Erturk,&Heath,2002).Nevertheless,theROSlevelwillbeenhancedwhenplantisexposedtochemicalandenvironmentalstresses,whichresultintheinactivationofenzymes,breakageofDNAstrandsandmembranelipids(Wu,Wilen,Robertson,&Gusta,1999;Apel&Hirt,2004).Toovercomeoxygentoxicityseveralmechanismshavebeenevolvedinplantcells,forexampletheantioxidativesystemincludingantioxidants(suchasascorbate,a-tocopherol,glutathioneandcarotenoids)andprotectiveenzymes.Thelatteronesoperateinthefollowingcycle:superoxidedismutase(SOD)scavengethesuperoxideradicals,andH2O2,oneofthisenzymaticreaction,isdetoxi edascorbateperoxidaseandglutathionereduc-taseinanascorbate–glutathionemanner(Alscheretal.,2002).SODisakeyenzymeinprocessofdecreasingthedamagecausedbyROS.SODsareafamilyofmetalloproteinsthatcatalyzethedismutationofsuperoxideradicalstohydrogenperoxideand Correspondingauthor.Tel./Fax:+862164321291.

1

E-mailaddresses:gykai@http://wendang.chazidian.com,guoyinkai@http://wendang.chazidian.com(G.Kai).Co- rstauthor.

oxygen.Accordingtothemetalco-factor,SODsareclassi edintothreedistinctgroups:ironSOD(Fe-SOD)inchloroplast,manganeseSOD(Mn-SOD)inmitochondrionandperoxisome,andcopper/zincSOD(Cu/Zn-SOD)inchloroplastandcytosolorintheextracellwhichrepresent90%oftotalSOD(Duetal.,2010).PlantSODsareknowntomakecontributiontodefenceagainsttoxicoxygenspeciesandareimportantforstresstoleranceconsequently.

TheplantSODgenehasbeenclonedfromcorn(Cannon,White,&Scandalios,1987),cassava(Lee,Kim,You,Kwon,&Kwak,1999),rice(Wang,Wang,&Wang,2007),evodia(Wu,Gao,Pan,Luo,&Zhang,2011),camelliaoleifera(Guo&Zhang,2012),andbeenappliedtoimprovetheplantresistanceinrecentyears.Forexam-plethetobaccooverexpressingcytosolicCu/ZnSODwasmoretol-eranttoacutedosesofozonethanthecontrol(Pitcher&Zilinskas,1996).Transgenictobaccoplantsthatoverexpressedchloroplast-localizedpeaCu/ZnSODexhibitedgreaterresistancetophotooxi-dativedamageandmethylviologen-mediatedoxidativestress(Gupta,Webb,Holaday,&Allen,1993).TransgenicpotatothatexpressedtomatoCu/ZnSODalsoenhancedresistancetomethylviologentoxicity,improvedoxidativestressandsalttolerance(Tang,Tang,Kwak,Wang,&Yang,2008).

AlltheformerdataillustratedthattheSODscouldbemanipu-latedtoimprovethestressresistanceinplantcell,howeverlittleisknownaboutSODsinBrassicacampestris.Althoughnon-headingChinesecabbage(B.campestris)‘‘Heiye’’isawidelycultivated

http://wendang.chazidian.com/10.1016/j.foodchem.2014.07.1210308-8146/Ó2014ElsevierLtd.Allrightsreserved.

2L.Cuietal./FoodChemistryxxx(2014)xxx–xxx

varietyinsouthofChinawithstrongresistancetoheat,rainandcold,therehavebeennoreportsonmolecularcloningofSODsfromthisspecies.WhetheritsstrongresistancepropertiesofB.campestristostressesarerelatedorpartlyrelatedtoitsinnerSODsactivitiesisunknown.

Inthiswork,wedescribedthemolecularcloningandcharacter-izationofaCu/ZnSODgenenamedBcCSD1fromB.campestris.Theevolutionalanalysisandtissuespeci cexpressionpatternaswellasstressresponseofBcCSD1wasalsostudied,whichwillbehelp-fultouncoverunderlyingmolecularmechanismforfurtherimprovingstresstoleranceinB.campestrisinthefuture.2.Materialsandmethods2.1.Materials

TheseedsofB.campestris‘‘Heiye’’(purchasedfromChangzhengVegetableSeedsCorporationinShanghai,China)weresowninpotandkeptindarkat25°Cfor2days.Aftergermination,theseed-lingsweretransferredintogreenhouseat25°Cwith14hlight/10hdarkphotoperiodfor7days,thentreatedwithdifferentstres-sesattwo-leafstage.2.2.Stresstreatment

The10-day-oldseedlingsofB.campestrisweresubjectedtosalt(150mMNaCl,irrigatethesoil),heat(40°C),drought(PEG-6000,20%)andABA(0.1mM,sprayonleaves),respectively,whileplantswithwatertreatmentwereperformedascontrolforchemicaltreatmentsandplantswithoutanytreatmentwasusedascontrolforheattreatment.2.3.RNAextraction

Theleavesofseedlingswereharvestedatdifferenttimepointsafterstresstreatment,meanwhile,theroots,stemsandleaveswithouttreatmentwerealsocollectedforexpressionpatterndetection.Allthematerialswerepulverizedwithliquidnitrogen rstly,thentotalRNAwasisolatedfromplantsusingRNApreppureplantKit(TiangenBiotechCo.,Ltd),quali edasdescribedbefore(Kaietal.,2006).

2.4.Molecularcloningofthefull-lengthBcCSD1cDNAbyRACEThe rststrandcDNAwassynthesizedfrom5lgoftotalRNAfromthewholeseedlingaccordingtotheprotocolofthe30RACESystemforRapidAmpli cationofcDNAEnds(GIBCOBRL,USA)usingtheadapterprimer(AP,50-GGCCACGCGTCGACTAGTAC(T)16-30)providedwithintheKit.Fortheampli cationof30endofBcCSD1,aspeci cprimerF2(50-CATGTCCATGCTCTTGGTGACAC-30)wasdesignedaccordingtotheconservedregionofseveralknownCSDgenesequencesfromplantspeciessuchasB.napus,B.rapa,RaphanussativusandArabidopsisthaliana,etc.The30RACEwasperformedasdescribedbeforewithminorrevision(Kaietal.,2007,2013).PCRwasperformedusingthefollowingprocedure:thetemplatewasdenaturedat94°Cfor5minfollowedby35cyclesofampli cation(94°Cfor45s,58°Cfor30s,72°Cfor60s)andextended10minat72°C.ThePCRproductwaspuri edwithGelExtractionKit(CW2302,CWBIO),clonedintothepMD18-Tvector(TaKaRa,Japan)andthensequenced.The50RACEwasperformedasdescribedbefore(Kaietal.,2007,2013),exceptthatadifferentcomplementaryreversegenespeci cprimerR2(50-TAGAGTTTGGTCCAGTAAGAGG-30)http://wendang.chazidian.combiningthenucleotidesequenceofthe30and50RACEproducttogether,thefull-lengthcDNAsequence

ofBcCSD1wassubsequentlyampli edviaPCRusingapairofprim-ersF1(50-CACTTCAGACATTTGATAGCTA-30)andAUAPforthreereplicates.Thecon rmedfull-lengthcDNAofBcCSD1wasusedformolecularcharacterizationandbioinformaticsanalysislater.2.5.Bioinformaticsanalysis

Thenucleotidesequence,deducedaminoacidsequenceandopen-readingframe(ORF)wereanalyzedonline(http://www.ncbi.nlm.nih.govandhttp://wendang.chazidian.com),andthesequencecomparisonwasconductedusingBLASTtoolto ndthehomologyofBcCSD1withotherCSD1s(http://www.ncbi.nlm.nih.gov).SOPMAandSwiss-Modelanalysiswerealsoperformed(http://wendang.chazidian.com/).DNAMANandMEGA4softwarewereusedforsequencealignmentandphylogeneticanalysiswithneighbour-joiningmethod.

2.6.ExpressionanalysisbyquantitativeRT-PCR

Inordertoinvestigatethetissuespeci candstressresponsiveexpressionpatternofBcCSD1,theRNAofroots,stems,leavesandtheseedlingstreatedwithsalt(150mMNaCl,irrigation),heat(40°C),drought(PEG-6000,20%)andABA(0.1mM,spray)wereextracted.The rst-strandcDNAwasreverselytranscribedfrom1ugtotalRNAasdescribedabove.Thereactionwasperformedin20llvolumecontaining10llSYBRGreenPCRmastermix,2llLowROX(PEAppliedBiosystems),0.6lleachprimersolution,1llreversetranscriptionproduct,andthenRNase-freewaterwasaddedtoatotal20llvolume.Thespeci cqRT-PCRprimerswereCSD1-real-F(50-GGAACTGCCACCTTCACAATCA-30)andCSD1-real-R(50-GGTCATCACGTTCTGCATGGA-30),andtheprimersofinternalcontrolBcActinwereActin-real-F(50-GCTTACGTCGCTCTTGACTACG-30)andActin-real-R(50-GATGGTGATGACTTGTCCATCAG-30).SampleswerenormalizedwithBcActin,andtheexpressionlevelswerecalculatedusingthe2ÀDDCtmethod.Eachassaywasrepeatedintriplicate.

3.Resultsanddiscussion

3.1.CloningofBcCSD1andbioinformaticsanalysis

BasedonsequencesoftheconservedregionsofknownCSDgenesfromotherplantspecies,aprimer(F2)wasdesignedandusedfortheampli cationof30endofCSDfromB.campestris.Asin-glefragmentofabout0.54kbwasampli edusingprimersF2andAUAP,anda30untranslatedregion(UTR)of194-bpwasfounddownstreamfromthestopcodonintheampli edsequence.Basedonthe30fragmentsequence,aspeci creverseprimerR2wasdesignedtoamplifythe50endofBcCSD1andthenafragmentabout0.4kbwasobtainedinwhicha50UTRof77-bpwasfoundupstreamofthe rstATGcodon.Byaligningandassemblingthe30RACEand50RACEproducts,thefull-lengthcDNAofBcCSD1wasdeducedandcon rmedbyRT-PCRusingprimerF1andAUAP.Theisolatedfull-lengthcDNAofBcCSD1genewas746bpandcon-taineda459bpopenreadingframe(ORF)encodingadeducedpro-teinof152aminoacidresidueswithanisoelectricpoint(pI)of5.82andacalculatedmolecularmassofabout15.1kDa(Fig.1).BLASTsearchinGenBankdatabaserevealedthatBcCSD1sharedhighhomologywithmanyotherCSD1ssuchasBrassicanapusCSD1(AY970822),BrassicarapaCSD1(AF071112),ArabidopsisthalianaCSD1(NM_100757)andRaphanussativusCSD1(AF009735),sug-gestingthatBcCSD1belongedtotheplantCSD1superfamily.Ataminoacidlevel,BcCSD1was99%,99%,94%and93%identicaltoBnCSD1,BrCSD1,AtCSD1andRsCSD1,respectively(Fig.2),which

L.Cuietal./FoodChemistryxxx(2014)xxx–xxx3

showedthehighconservationofCSDsintheplantkingdomandhintedthatCSD1playedanimportantroleinbioprocess.

Inaddition,the3-DstructuralmodellingofBcCSD1wasperformedonlinewithSwiss-Modelsoftware.TheintricatespatialarchitectureshowedthatBcCSD1containedanoveleightanti-parallelb-strandsforminga attenedcylinder,andthreeexternalloops(Fig.3),whichwassimilartoCSD1inSpinaciaoleracea(Kitagawaetal.,1991).3DstructurepredictionrevealedthatBcCSD1sharedcommonspatialcharacterswithothermembersinCSD1family(Getzoff,Tainer,Stempien,Bell,&Hallewell,1989).Theresultsupportedthehypothesisthattheconservedmotifsmayplayimportantroleinbioprocessandthusareconservedinevolution,whilethevariationsinotherregionsformthemolecularfoundationforthediversity.

WiththeMEGA4program,aneighbour-joining(NJ)treewasconstructedbasedonthededucedaminoacidsequencesofBcCSD1andotherCSD1stoinvestigatetheevolutionaryrelationships.TheresultrevealedthatBcCSD1werederivedfromthesameancestorasotherCSD1sinanimals,plantsandmicroorganismsbutsharedtheclosetrelationshipwiththeCSD1fromB.napus(Fig.4).3.2.TissueexpressionpatternanalysisofBcCSD1

Real-timePCRanalysisshowedthatBcCSD1couldbedetectedinthreetissuesbutthehighestinleaf,theninstemandthelowestinroot(Fig.5).Therefore,BcCSD1wasconsideredtobeaconstitu-tivelyexpressiongene,whichwassimilartoCSD1sinotherspeciessuchasGalegaorientalisL(Lietal.,2012)andMyricarubra(Fang,

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3.3.BcCSD1expressionpro lesundersalt,drought,heatandABAtreatments

InordertoexaminetheexpressionofBcCSD1underdifferentabioticstressesincludingNaCl,PEG,heatandABAinB.campestris,the10-day-seedlingwassubjectedtoNaCl,PEG,hightemperatureandABAandthetotalRNAofleafwasextractedtoperformreal-timeRT-PCR.TheresultsshowedthatunderfourabioticstressestheBcCSD1transcriptwasup-regulated rstlyandthendeclined,whichshowedsimilartendencywithdifferentlevels.

AftersaltstresstreatmenttheexpressionofBcCSD1peakedat24hby3.19-foldincreaseandthendeclined(Fig.6A),whilethein uenceofPEG-inducibledroughtontheexpressionofBcCSD1wascomparativelyslightthanothertreatments,whichonlyreached1.48timesatpeaktimepoint12h(Fig.6B).Theresults

suggestedthatsaltstressanddroughtareeffectiveforinductionofexpressionofBcCSD1,whichisconsistentwithpreviousresultsinplants(Perl-Treves&Galun,1991;Wangetal.,2009).InheattreatmentthehighestexpressionofBcCSD1was2.08timesat90m(Fig.6C),whichindicatedthatheattreatmentat40°CcouldinduceexpressionofCu/Zn-SOD(Wei,Li,Gu,&Liu,2008).Ofallthefourtreatments,theinductioneffectofABAtreatmentwasthemostobviousandthetranscriptofBcCSD1waselevatedto7.31timesatpeaktimepoint12h(Fig.6D),whichiscontrarytothetranscriptofCu/Zn-SODgeneintobaccoBY-2cellsuspensions(Buenoetal.,1998).SowededucedthatBcCSD1wasastressinduc-iblegeneespeciallyforABAtreatment,re ectingthatBcCSD1mayplayanimportantroleinplantstressesprotection.

L.Cuietal./FoodChemistryxxx(2014)xxx–xxx5

Inconclusion,afulllengthcDNAofBcCSD1wasisolatedfromB.campestrisandcharacterizedinthispaper.MultiplealignmentsandbioinformaticsanalysisresultsshowedthatthededucedBcCSD1hadhighsimilaritytootherplantCSDs.Expressionpro lesofBcCSD1underdifferenttreatmentssuggeststhatBcCSD1wasastress-responsivegene,especiallytoABA.Thecloning,character-izationandexpressionanalysisofBcCSD1willbehelpfultounder-standmoreaboutitsroleintheresistancetostressesforplant,whichprovidethebasisforimprovingtheabilitytoanti-stressbygeneticmanipulationinthenearfuture.Acknowledgments

ThisworkwassupportedbyNationalNaturalScienceFund(31270007,31201261,30900110),FokYing-TongEducationFoun-dationforYoungTeachersintheHigherEducationInstitutionsofChina(131041),NewCenturyTalentProject(NECT-13-0902),ShanghaiTalentDevelopmentFund,ShanghaiEducationCommit-teeFund(13ZZ104,09ZZ138,J50401),KeyScienceandTechnologyProjectofYantzeRiverDelta(10140702018)andShanghaiYoungTeacherTrainingProjectofShanghaiEducationCommitteeFund.References

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