Molecular Cloning, Expression Patterns and Characterization of a New Spermidine Synthase Gene in Oryza sativa

Abstract

Spermidine synthase is an enzyme that catalyzes the transfer of the propylamine group from S-adenosylmethionine to putrescine in the biosynthesis of spermidine, and which plays a critical role in a wide range of growth and developmental process. In this research, a new spermidine synthase gene, designated as OsSPDS3, was isolated by 3' RACE method in Oryza sativa. The OsSPDS3 contained an 1 194 bp open reading frame encoding 397 amino acids. The OsSPDS3 included a complete spermine_synth domain, two putative low complexity regions and several binding sites of S-adenosylmethionine. The predicted 3-D structure indicated that OsSPDS3 contained alpha-helices, bate-sheets and coils. The phylogenetic analyses revealed that OsSPDS3 belonged to plant spermidine synthase family, in which AtSODS3, OsSPDS3 and OsSPDS2 were clustered into one clade. OsSPDS3 transcripts were significantly up-regulated under high salinity stress. In contrast, no such induction of the OsSPDS1 and OsSPDS2 were observed under high salinity stress. In addition, the expressions of three rice spermidine synthase genes did not change under drought and cold treatments. The results suggested that OsSPDS3 indicated a distinct function in high salinity stress.

Full text

Thecellularpolyaminesputrescine(Put),spermidine
(Spd),andspermine(Spm)areubiquitousinnature
and have been implicated inawide range of
growthanddevelopmentalprocessofbothprokaryotes
andeukaryotes
[1]
.Inhigherplants,severalresearches
indicatedthatpolyamineswereassociatedwith a
numberofdevelopmentalprocessesincludingresponse
to stress,celldivision,stabilization ofthylakoid
membranes, retardation of senescence, somatic
embryogenesis,rootgrowthandfloralinitiation
[2-13]
.
热带作物学报 2013袁34渊2冤院 285-291
ChineseJournalofTropicalCrops
收稿日期 2012-11-15 修回日期 2013-01-15
基金项目 国家自然科学基金项目渊No.31071173)曰教育部重点项目渊No.210173冤遥
作者简介 郭会娜渊1988年要冤袁 女袁硕士曰研究方向院植物生物化学与分子生物学曰*为并列第一作者遥**通讯作者院李德军袁E-mail院
djli.rricatas@gmail.com遥
MolecularCloning,ExpressionPatternsandCharacterizationof
aNewSpermidineSynthaseGenein Oryzasativa
GUOHuina
1,2
,XIAZhihui
1
,LIDejun
2
1InstituteofBiologicalScienceandTechnology,CollegeofAgriculture,HainanUniversity,Haikou,570228,China
2KeyLaboratoryofBiologyandGeneticResourcesofRubberTree,MinistryofAgriculture,
RubberResearchInstitute,CATAS,Danzhou,Hainan571737,China
Abstract SpermidinesynthaseisanenzymethatcatalyzesthetransferofthepropylaminegroupfromS-adenosylmethionine
toputrescineinthebiosynthesisofspermidine,andwhichplaysacriticalroleinawiderangeofgrowthand
developmentalprocess.Inthisresearch,anewspermidinesynthasegene,designatedas OsSPDS3,wasisolatedby
3'RACEmethodin Oryzasativa.The OsSPDS3 containedan1194bpopenreadingframeencoding397amino
acids.TheOsSPDS3includedacompletespermine_synthdomain,twoputativelowcomplexityregionsandseveral
bindingsitesofS-adenosylmethionine.Thepredicted3-DstructureindicatedthatOsSPDS3contained 琢-helices,
茁-sheetsandcoils.ThephylogeneticanalysesrevealedthatOsSPDS3belongedtoplantspermidinesynthase
family,inwhichAtSODS3,OsSPDS3andOsSPDS2wereclusteredintooneclade. OsSPDS3 transcriptswere
significantlyup-regulatedunderhighsalinitystress.Incontrast,nosuchinductionofthe OsSPDS1 and OsSPDS2
wereobservedunderhighsalinitystress.Inaddition,theexpressionsofthreericespermidinesynthasegenesdid
notchangeunderdroughtandcoldtreatments.Theresultssuggestedthat OsSPDS3 indicatedadistinctfunctionin
highsalinitystress.
Keywords Abioticstress; OsSPDS3;Oryzasativa;Spermidinesynthase
doi 10.3969/j.issn.1000-2561.2013.02.016
一个新的水稻亚精胺合成酶基因克隆尧
表达及特性分析
郭会娜 1袁2袁夏志辉 1*袁李德军 2**
1海南大学农学院生物科学技术研究所袁海南海口 570228
2中国热带农业科学院橡胶研究所
农业部橡胶树生物学与遗传资源利用重点实验室 海南儋州 571737
摘 要 在亚精胺生物合成中袁亚精胺合成酶负责从 S-腺苷甲硫氨酸到腐胺过程中催化转移丙胺袁其在生物生
长发育中发挥重要作用遥本研究利用 3末端 cDNA 快速扩增技术从水稻中获得一个新的亚精胺合成酶基因袁命
名为OsSPDS3遥OsSPDS3 包含一个 1194bp 的开放阅读框袁该阅读框编码 397 个氨基酸遥结构域预测结果表明
亚精胺合成酶含有一个完整的精胺合成酶结构域袁2个推测的低复杂性区段和多个S-腺苷甲硫氨酸结合位点遥
三维结构预测显示 OsSPDS3 含有 琢螺旋袁茁
折叠和卷曲结构遥进化分析表明 OsSPDS3 属于植物类亚精胺合成酶
基因家族袁与拟南芥亚精胺合成酶 3和水稻亚精胺合成酶 2划分为一个亚类遥盐处理能显著上调 OsSPDS3 表
达袁但水稻亚精胺合成酶 1和2对盐处理没有响应遥此外袁干旱和低温处理都不能改变 3个水稻亚精胺合成酶
基因表达袁以上结果暗示 OsSPDS3 可能在水稻盐反应中发挥独特作用遥
关键词 非生物逆境曰OsSPDS3曰水稻曰亚精胺合成酶
中图分类号 Q343 文献标识码 A

第34 卷
热带作物学报
Inpolyaminebiosyntheticpathway,thediamine
putrescine issynthesized by eitherOrn or Arg
decarboxylase(ODC andADC,respectively) and
convertedtospermidineandsperminethroughthe
sequential addition ofaminopropyl residues by
spermidine and spermine synthases
[1]
.ThecDNAs
encoding spermidine synthase have been cloned
fromvariousplantspecies
[8,14-20]
.Hanzawa etal.
[16]
clonedthree genesencoding spermidine synthase
andanalyzedtheircharacterizationin Arabidopsis.
Imai etal.
[18]
furtherreportedthatspermidinesynthase
geneswere essentialforsurvivalof Arabidopsis.
Three spermidine synthase geneswere found by
searchingtheconservedspermine_synthdomainin
NCBI, butonly two spermidine synthaseshave
beenreported. OsSPDS2 mRNA wasaccumulated
in rootsduring long term exposure to chilling
temperature.Incontrast,nosuchinductionofthe
OsSPDS1 wasobservedduringthechillingtreatment.
Theresultssuggestedadistinctfunctionof OsSPDS2
inchillingresponseinrice
[19]
.
In thispaper,we reportthe isolation and
characterizationofaspermidinesynthasegenefrom
rice(OsSPDS3,GenBankAccessionNumber:EU714031).
Inaddition,theexpressionprofilesof OsSPDS3 were
alsoinvestigatedbysemi-quantitativeRT-PCRanalysis.
Thestudynotonlycontributestounderstandingthe
molecularcharacterization of OsSPDS3,butalso
providesnewinsightsinto OsSPDS3 inrice.
1MaterialsandMethods
1.1 Plantmaterials
Riceseeds(Oryzasativa var.Nipponbare)were
growninapaddyfieldundernaturalconditions.
Afterthreeweeks,theseedlingsweretreatedwith
drought(15%PEG8000),cold(8 益)andhighsalinity
(0.5mol/L NaCl). After all the treatments,the
leaveswereharvestedat0,2,5,8and24hours,
frozeninliquidnitrogen,andstoredat-80 益for
furtheranalysis. The control samples were also
harvestedatthesametimewiththetreatedplants.
1.2 Molecularcloningof OsSPDS3 cDNA
TotalRNA was extracted from rice leaves
according to theprotocolofRNeasyPlantMini
Kits(Qiagen).UsingtotalRNAasthe template,
reverse transcriptions offirststrand cDNA were
performedwithSuperScriptIIReverseTranscriptase
(Invitrogen).The OsSPDS3 wasisolated by 3忆
RACEstrategywith3忆-fullRACEkit(TaKaRa).
Thegene-specificforwardprimerandnestedprimer
are5忆-ggaacttcgaggaggagcag-3忆and5忆-ggtggaggcgca
agaaatgg-3忆,respectively.ThePCRproductswerepurified,
clonedintothepGEM-TEasyVectorandsequenced.
Afteraligningandassemblingthesequencesof
theinternalESTsequence,3忆-RACEproducts,the
OsSPDS3cDNAwasgotandtestifiedbysequencing
thePCRproducts.Theforwardandreverseprimers
were5忆-ggaacttcgaggaggagca-3忆and5忆-aaacaaagctgt
aacaacaagc-3忆,respectively.ThePCRreactionwas
performed with the following thermal cycling
parameters:94 益for4min,followedby32cycles
of94 益for30s,58 益for30sand72 益for2min,
andthefinalextensionwasperformedat72 益for
10min. ThePCR productwascloned into the
pGEM-TEasyVectorandsequenced.
1.3 RT-PCRanalysis
AllRT-PCR reactions in this paper were
reproducedatleastthreetimeswithindependent
cDNA preparations.In each PCR reaction, the
gene-specificprimerswereused,and actinwas
utilizedastheinternalreference.Thegene-specific
forward(F)andreverse(R)primersusedforRT-PCR
areasfollows: actin (F5忆-agcaactgggatgatatgga-3忆
andR5忆-cagggcgatgtaggaaagc-3忆), OsSPDS1 (F5忆-
cgaagagagtcattggatcc-3忆andR5忆-gtaaatggtaagggagat
gcg-3忆), OsSPDS2 (F 5忆-catatgcgacaaccatgttc-3忆
andR5忆-catgcgattcactccacatc-3忆), OsSPDS3 (F5忆-
cagggtttttgcctgctcac-3忆andR5忆-gcctcactcatttcaacagt
c-3忆).ThePCRconditionswereasfollows:5min
at94 益,22to28cyclesof30sat94 益,1min
at55or60 益and1minat72 益,and10minat
72 益.The PCR products were analyzed by
electrophoresisin1.2%agarosegels.
1.4 Multiplealignmentsandbioinformaticanalyses
Nineteenspermidinesynthaseand2spermine
synthase proteins were downloaded from NCBI
databaseandalignedwithMultipleAlignmentTool
programinNCBI(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
ThecharacterizationsofOsSPDS3proteinwereanalyzed
bytheconserveddomaindatabase
[21]
andsimplemodular
286- -

第2期
ThecDNAfragmentof OsSPDS3 wasencoded
apolypeptideof397aminoacidswithacalculated
molecularmassof42.9kuandanisolectricpoint
of5.33.Thesearch in NCBIconserved domain
databaseshowedthatOsSPDS3containedaconserved
spermine_synthdomain.Thequeryresultalsoindicated
thattheOsSPDS3proteinpossessedseveralpredicted
bindingsitesofS-adenosylmethionine(Fig.1).In
addition,tworegionsoflowcomplexitywerealso
predictedwithinOsSPDS3proteinbysimplemodular
architectureresearchtool(Fig.1).The predicted
3DstructureofOsSPDS3,whichwassimulatedwith
Swiss-Model,contained 琢-helices, 茁-sheetsand
coils(Fig.2).Thisstructurewassimilartothe3D
structureofAtSPDS3determinedbyresolutionX-ray
(http://swissmodel.expasy.org/).
architectureresearchtool(http://smart.embl-heidelberg.
de/).Thephylogenetictreewasconstructedbythe
neighbor-joiningmethod(1000bootstrapreplicates)
usingtheMEGAprogram4.1
[22]
.The3Dstructures
wereconstructedwithanonlineprotein3Dstructure
predictiontool(http://swissmodel.expasy.org/).
2Results
2.1 Isolationandcharacterizationof OsSPDS3
AccordingtoariceESTfragmentofspermidine
synthasegene(GenbankAccession:CB660525),the
genewasamplifiedby3忆RACEstrategy,andthe
genewestudiedwasnamedasOsSPDS3.ThePCR
productsof3忆-endwereclonedintothepGEM-TEasy
Vectorandsequenced;Afteraligningandassembling
withCB660525sequence,atotalof1546bpcDNA
wasdeduced,andthesequenceswereconfirmedby
amplifyingandsequencingthewholecDNAfragment.
ThecDNAfragmentcontainedan1194bpORFencoding
397aminoacids,with25bp5忆UTRand309bp
3忆UTR(Fig.1).ThesequencewassubmittedtoGenebank,
andtheaccessionnumberwasEU714031.
Theconversedspermine_synthdomain,lowcomplexityregionsandbindingsitesof
S-adenosylmethioninewerehighlightedwithdottedline,boxandline,respectively.
Fig.1 Nucleotidesequenceof OsSPDS3 anditsdeducedamino
acidsequence(GenBankAccessionNo.EU714031)
茁-sheets
琢-helices
coils
ThestructureofOsSPDS3proteinwasmodeledwiththe
knownstructureofAtSPDS3(ParentPDB:1xj5Chain:C,http:
//www.pdb.org)asatemplateontheSwiss-Modelserver(http://
swissmodel.expasy.org/).Theconservedregions(琢-helices, 茁-
sheetsandcoils)weremarkedinthefigure.
Fig.2 The3DstructuresofOsSPDS3
(generatedbySwiss-Model)
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第34 卷
热带作物学报
2.2 Phylogeneticanalysesofspermidinesynthase-
relatedproteins
ThededucedaminoacidsequencesofOsSPDS3
arealigned with 18 spermidine synthase and 2
sperminesynthaseproteins.AsshowninFig.3A,
the aligning resultsindicated that the OsSPDS3
was highly conserved among the spermidine
synthaseproteins,especiallyinthespermine_synth
domainregion.OsSPDS3proteinsshowed52%~83%
identities and64%~89%positivestootherplant
spermidine synthase proteins,whereasonly with
36%~50% identitiesand 56%~68% positivesto
non-plantspermidinesynthaseproteins.Toevaluate
theOsSPDS3evolutionaryrelationships,thephylogenetic
treewasconstructedamong19spermidinesynthase
and2sperminesynthaseproteinsbytheneighbor-
joiningmethodusingtheMEGA4.1program
[22]
.As
showninFig.3B,sperminesynthaseproteinswere
apparently diverged from spermidine synthase
proteins,andtheywereformedonegroup.The19
spermidine synthase proteins were divided into
anothergroup,andthegroupwasfurtherorganized
intothreesubgroups.OsSPDS3proteinsbelongedto
plantspermidinesynthase proteins,in which the
OsSPDS3,OsSPDS2andAtSPDS3wereclustered
inoneclade.
21aminoacidsequencesofspermidinesynthase-relatedproteinswerealignedwithClustalW2
(http://www.ebi.ac.uk/Tools/msa/clustalw2/).Theperfectlymatchedresidues,conservedresidues,and
lessconservedresiduesareindicatedbyanasterisk(*),acolon(:),andaperiod(.),respectively.
Fig.3A SequencealignmentofdeducedOsSPDS3andspermidinesynthase-relatedproteins
288- -

第2期
2.3 Expressionanalysisesof OsSPDS3 underabiotic
stresses
Someresearchesindicatedthatpolyamineswere
involvedinstressresponses
[22-28]
,sotheexpression
patternsofthreespermidinesynthasegenesinrice
wereanalyzedunderabioticstress.Asshownin
Fig.4,the OsSPDS3 transcriptsweremarkedlyup-
regulatedbyhighsalinitystress.Incontrast,there
was no significant change in mRNA levels of
OsSPDS1 and OsSPDS2 duringthecourseofhigh
salinitytreatments.Undernormalgrowingcondition,
theexpressionprofilesofthreespermidinesynthase
genessuchas OsSPDS1,OsSPDS2 and OsSPDS3,
did not show distinct change. Compared with
normalgrowingcondition,threespermidinesynthase
genesinricearenotobviouslyresponsivetocold
anddroughttreatments(datawasnotshown).These
resultssuggestedthat OsSPDS3 mighthaveadistinct
functioninhighsalinitystress amongthree rice
spermidinesynthasegenes.
Fig.3B PhylogeneticanalysesofOsSPDS3andspermidinesynthase-relatedproteins
ThetreewasconstructedbytheMEGAprogramwiththeneighbor-joiningmethod.Numbersonnodesindicatethebootstrapvaluesafter
1000replicates.Thescalebarindicatestheestimatednumberofaminoacidsubstitutionspersite.Theaccessionnumbersofspermidine
synthase-relatedproteinareasfollows:AtSPDS1(AJ251296),AtSPDS2(AJ251297),AtSPDS3(AY040013),AtSPDM(AF184093),CaSPDS
(AB015599),DsSPDS1(Y08252),DsSPDS2(Y08253),HnSPDS1(AB006690),HnSPDS2(AB006691),LeSPDS(AJ006414),NsSPDS(AB006692),
OsSPDS1(AJ251298),OsSPDS2(AB098063),OsSPDS3(EU714031),OsSPDM(NP_001046395),PsSPDS1(AF043108),PsSPDS2(AF043109),
ScSPE3(U27519),HsSPDS(M34338),EcSPDS(YP_001742248)andAnSPDS(XP_658291).
OsSPMS
AtSPMS
EcSPDS
AnSPDS
ScSPDS
HsSPDS
OsSPDS3
OsSPDS2
AtSPDS3
AtSPDS2
AtSPDS1
OsSPDS1
PsSPDS2
PsSPDS1
CaSPDS
HnSPDS
DsSPDS1
NsSPDS
DsSPDS2
LeSPDS
HnSPDS1
49
45
99
98
23 97
41
55
88
100
100
100
99
0.05
100
100
100
100 100
Fig.4 Expressionpatternsofthreericespermidinesynthasegenesunder
highsalinitytreatmentandnormalgrowthcondition
OsSPDS3
OsSPDS2
OsSPDS1
Actin
Theleavesfromhighsalinitytreatmentandnormalconditionwereharvested
at0,2,5,8and24h.Actinwasusedasaninternalcontrol.
highsalinitytreatment normalgrowthcondition
0 2 5 8 24h 0 2 5 8 24h
郭会娜等院一个新的水稻亚精胺合成酶基因克隆尧表达及特性分析 289- -

第34 卷
热带作物学报
3Discussions
Thericegenomeprojectprovideduswithan
opportunitytosearchforanentireplantgenome
forspermidinesynthase genes.Thequeryresults
revealed that the rice genome contained three
geneswithahighsimilaritytoknownspermidine
synthasegenesequences. OsSPDS1 and OsSPDS2
(accession numbers: AK099606 and AB098063)
havebeenreportedbyImai etal.
[19]
.Anadditional
gene,namedas OsSPDS3,wasfoundtobelocated
onchromosome2andwasonecopyinricegenome
inthisresearch.Differentfromtheinformationon
KOME(AccessionNO:AK065153),the OsSPDS3
encodes397 aminoacids,not 128 amino acids
provided by KOME(http://cdna01.dna.affrc.go.jp/
cDNA/report/KOME_J013002B15.html).
Beingsimilartoallthespermidinesynthases,
OsSPDS3containedconversedspermine_synthdomain
andbindingsitesofS-adenosylmethionine.Although
sperminesynthasealsobelongedtospermidinesynthase-
relatedgenesandpossessedspermine_synthdomain
andbindingsitesofS-adenosylmethionine,itsignificantly
diverged from spermidine synthase,which also
supported bythephylogenetic analysis.Different
fromplantandnon-plantspermidinesynthaseproteins,
sperminesynthaseproteinsformedanindependent
class, suggesting that spermidine and spermine
synthases might carry out different functions.
Consistentwith the previousresults
[28]
,spermidine
synthaseproteinsweremainlydividedintoplantand
non-plantsubclasses.Phylogeneticanalysisrevealed
thatOsSPDS3wasmostcloselyrelatedtoaputative
OsSPDS2.Together,OsSPDS3,OsSPDS2andAtSPDS3
wereorganized into one cladewithin the plant
spermidinesynthasefamily.Besideshighlypositives
toAtSPDS3onamino acid level,OsSPDS3 and
AtSPDS3 sharedsimilar3D structures,suggesting
thattheymightcarryoutsimilarfunctions.
Besidestherolesingrowthanddevelopment
process,polyamine is also related with stress
response
[22-28]
.Asakeygenefamilyinpolyamine
biosynthetic pathway,spermidine synthase might
playanimportantroleinstressresponse.Inrice,
OsSPDS2 mRNAinrootswasupregulatedduring
thechillingtreatment
[19]
.Inthisresearch,theexpression
profilesofthreespermidinesynthasewereanalyzed
inriceleavesundercold,droughtandhighsalinity
treatments. The results indicated that only the
expressionof OsSPDS3 increasedmarkedlyunder
highsalinitytreatment,whereastheexpressionsof
OsSPDS1 and OsSPDS2 werenotsignificantlychanged.
Wu etal.
[29]
reported thatthe ABRELATERD1
(ACGTG)andABRERATCAL(MACGYGB)were
cis-elementsofsalinityresponse.Inthisstudy,the
expression patterns ofthree spermidine synthase
underhighsalinitytreatmentmightberelatedto
their promoters,therefore wesearched the cis-
elementsofsalinityresponselocatedinpromoter
region ofthree spermidinesynthase.Theresults
showedthattwocis-elementsofsalinityresponse
werepredicted in promoterregion of OsSPDS3,
whereasnocis-elementsofsalinityresponsewere
predicted in promoterregions of OsSPDS1 and
OsSPDS2,suggestingthattheresultswereconsistent
withourspeculation.Inaddition,theexpressionsof
all three rice spermidine synthase were not
regulated under cold and drought treatmentsin
leaves.Imai etal.
[19]
alsoshowedthat OsSPDS1 and
OsSPDS2 mRNAlevelsinshootswerenotresponsive
tocoldstress.Accordingtotheresultsmentioned
above,threericespermidinesynthasemightplay
differentrolesinstressresponse,whereas OsSPDS3
mightberelatedtosalinitystress,notcoldand
droughtstresses.
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责任编辑院黄 艳
郭会娜等院一个新的水稻亚精胺合成酶基因克隆尧表达及特性分析 291- -