全 文 ：FinancialsupportedbyChinaHighTechnology(863)Project(2001AA241191)，FundofNationNaturalScience（30370901）andfundofdoctoral
ofuniversityfromministryofeducationP.R.C（20040389009）
*Authorforcorespondengce，E-mail：zmuqing@163.com，Tel：0591-83789177
收稿日期：2006-10-27 修回日期：2006-11-06
热 带 作 物 学 报
CHINESEJOURNALOFTROPICALCROPS
Vol.28No.2
Jun.2007
第 28卷 第 2期
2007年 6月
CloningandExpressionofacofromErianthus
arundinaceusExposedtoWater-Stress
ZhangJisen1,2 LiWei1 RuanMiaohong1 QueYouxiong1 ChenRukai1 ZhangMuqing1*
1MinistryofAgricultureKeyLaboratoryofEcophysiology&GeneticImprovementforSugarcane,
FujianAgricultureandForestryUniversity Fuzhou 350002 China
2ColegeofLifeScience,FujianNormalUniversity Fuzhou 350108 China
Abstract l-aminocyclopropane-1-carboxylateoxidasegene(aco)wasisolatedfromErianthusarundinaceusbyRT-
PCR.SequencingandBLASTXanalysisinNCBIrevealedthattheclonedacois1123bplongandcontainstheORF
(openreadingframe)of966bpwhichencodes322aminoacids.Conserveddomainsweresearchedandthreedomains
werefound:onePcbCdomainthatpossessesoxidationability,andbivalentFe2+andactivecentreofCO2inPcbC
domain.Real-timefluorescentquantitativePCRanalysisshowedthattheexpressionprofileofacounder4stagesofPEG
stresswasiregularattheearlystage,andlowattheconsequentstages.Atthestageof2hoursofPEGstress,the
expressionwasobviouslyhigher,whichwas5.4timesasthatofthecontrol,andsincethenbecamelower,butwasstil
positivelyhigherthanthatofthecontrol,nomaterat4hoursor12hoursofstresses.Itisinterestingtonotethatthe
expressionofacoandcontrolgeneswerealmostthesamewithslightdiscrepancy.Thepresentstudyconfirmedthe
sensitivityofacotoexternalfactors,andprimarilyitsrelationshipwithwaterstress.
Keywords Erianthusarundinaceus l-aminocyclopropane-1-carboxylateoxidasegene(aco) Reverstranscription
polymerasechainreaction(RT-PCR) realtimePCR PEG-stress
ErianthusarundinaceusReiz.,thewildrelativeofSaccharumoficinarumL.,isakinspecieswithdesirable
charactersincludingexcelentvigorandratooning,adaptabilitytoenvironmentalstressessuchasmoisture
deficitsandexcesses,andresistancetoPachymetrarootrot[1~3].SocloningofcertainfunctionalgenesfromE.
arundinaceuswilnodoubtbroadentherelativelynarowgeneticbaseandimprovetheeficiencyofsugarcane
breeding.Ethyleneisasimplegasthatactsasaplanthormone;itcontrolsvariousprocessesintheplantlife
cycle,includingseedgermination,roothairdevelopment,rootnodulation,flowersenescence,abscission,and
fruitripening[4].ThesynthesispathwayofethyleneinplantswasMet-SAM-ACC-Ethylene[5].ACO (l-
aminocyclopropane-1-carboxylateoxidase),termedasethylenesynthase,cancatalyzetheconversionofACC
toethylene.Also,theACOwoulddirectlycontrolthecontentinvivobecauseofitsefectinthefinalstepofthe
pathway.WhileitisinconvenienttoisolateACOgene(aco)throughnormalbiochemicalmeasures,the
molecularcloningmethod,whichcanovercomethedificultyinstraightforwardextraction,wasappliedto
facilitatethecorespondingresearch.Uptopresent,theacofromdiverseplantspecieshasbeencloned[6~11].Its
non-constitutiveexpressionandmediationattranscriptlevelmaybethecontrolingfactorsinethylene
producingvelocity.ItisindicatedthattheacomayinhibitorinspiretheexpressionofendogenousACOtosome
extent,andhencereduceorincreasetheethylenecontentanddelaythecaducityperiodorevenchangethe
resistance.Therefore,itsisolationandsubsequentapplicationcanalwaysbethehotspotingeneticengineering[11].
Ethyleneissynthesizedinresponsetostressessuchaspathogenatack,wounding,hypoxia,ozone,chiling,and
2期 张积森等：斑茅 ACO基因(aco)的克隆及其在水分胁迫下的表达（英文）
freezing[12].Thesignificanceofacocanbedemonstratedbyitsorgan-specificexpressionanditsdiference
expressioninvariousstagesorspaces[14~18].Investigationofitsresponsivenesstoenvironmentalstimulationwas
conductedtoprobeintothemolecularmechanism andfurthertoexplorethegenefunctionmore
comprehensively.Noneofanyefortswasmadetorevealtheresponsivenesstowaterstressaccordingtothe
publisheddata.Asaneficientandefectivemethodusedinnucleotidequantification,real-timefluorescent
quantitativePCR techniquewasestablishedaccordingtotheprincipleofconventionalPCR andsome
breakthroughsinitsdetectionextentandsensitivity[19].Sinceamplificationoftheproduct,detectionandquantification
takeplacewithinasingletube,real-timePCRcanfacilitatethepost-PCRmanipulationandreducetheriskof
contamination.RT-PCRisapowerfultoolforthedetectionandquantificationofmRNA.Real-timePCRis
widelyandincreasinglyused,becauseofitshighsensitivity,goodreproducibilityandwidequantificationrange.
Itisthemostsensitivemethodfordetectionandquantificationofgeneexpressionlevels,inparticularforlow
abundancemRNA[20，21].Inthepresentstudy,acowasisolatedfrom E.arundinaceusthroughhomology-based
cloningtechnique.Inordertosetthefoundationforitsgeneticengineeringresearchandprovideprimary
theoreticexplanation,areal-timePCRmethodwasappliedtoinvestigatetheexpressionofacoofE.
arundinaceussubjectedtoPEGstress.
1 MATERIALS&METHODS
1.1 Plantmaterials
E.arundinaceusReizwasplantedinthesandheldintheporcelainpotwith50cmhighnessand35cmin
wide.Undersimilarwaterandnutrientconditions,thewelgrownplantsweretreatedwith30%PEG,andwith
waterascontrol.ThetreatedandcontrolleaveswerecolectedandkeptinfluidnitrogenforRNAextraction
fromthePEGtreatmentsfor2hours,4hours,12hoursand20hoursrespectively.
1.2 Chemicalsandreagent
TaKaRaRNAPCRKit(AMV)Ver.3.0;restrictionendonuclease;pMD18-Tvector(TaKaRa)；TRIZOL
reagents（Intrivogen）；SYBGREENI(TaKaRa),ExscriptTMRT-PCRKit(TaKaRa).Primersweresynthesized
bySangonBiotechCompanyinShanghai,andfluorescentquantificationPCRamplificationprimerswere
composedbyTaKaRaCo.LtdinDalian.TheremainingmolecularreagentswerepurchasedfromSangonin
Shanghai,TaijinginXiamenandTaKaRainDalian.
1.3 TotalRNAExtraction
TotalRNAofleaveswasextractedwithTRIZOLreagents(Invitrogen)andoperatedintermsofthekit
protocol.Absorptionvaluewascalculatedbyaspectrometerand700ngRNAwasdetectedtoevaluatethequality.
1.4 RT-PCRamplification
Twospecificprimers,(acoF1:AGACACGCACAAGGAAGAAACandacoR1:CAGAACGCCGCAAATA
CA),weredesignedfrom theinformationoftheaccessionAY521566inNCBI.Thereversetranscription
amplificationwasperformedina10μLreactionvolumecontainingthefolowingreagents:2μL10×RTPCR
bufer(250mmol/LTris-HCl,pH8.0,0.1375mmol/LKCl),2μLMgCl2 (1.5mmol/L),1μL10mmol/L
dNTPs,0.5μLoligodT-AdaptorPrimer(10pmol/μL),1μL(200UAMV)reversetranscriptase(TaKaRa),
0.25μLRnaseInhibitorand1μL (about300ng)oftemplatetotalRNAandfinaly2.25μLRnasefreeH2O.
After30minreactionat42℃,itwasconductedfor5minat99℃andthenheldat5℃.With10μLreaction
volume,eachPCRreactionincluded5μLcDNA,1×PCRbufer,20pmolreverseprimer(acoR1),20pmol
forwardprimer(acoF1),when5mindenaturationat94℃wasfinished,0.25μL(3Units)TaqDNApolymerase
wasadded，andcomplementedto10μLwithddH2O.Theinitialdenaturationwascariedoutat94℃for4min,
61
热 带 作 物 学 报 28卷
folowedby35cyclesofdenaturationat94℃ for1min,annealingat58℃ for1min,andextensionat72℃
for1minwithfinalextensionstepof72℃for5min.Then5μLPCRproductswereseparatedon1%agarosegel.
ThefragmentderivedfromcorespondingprimerswasexcisedfromagarosegelsunderUVlightandthen
purified.ThepurifiedfragmentwasclonedintopMD18-TvectorandtransformedwithcompetentE.coliDH5α.
RecombinantswereidentifiedaswhitecoloniesonLBplateswithX-galandIPTG.Usingthebacterialstrainas
template,thesecolonieswerefurtherscreenedbyPCRaccordingtotheprotocoldescribedabovewiththe
originalprimer.TheplasmidDNAwasisolatedfolowingthemethodofAlkalidecomposition,andtheputative
positiverecombinantsverifiedbyPCRwerefurthercharacterizedbyenzymedigestion.Onepositiveclonewas
senttoShanghaiBioAsiaBiotechCompanyforsequencing.Sequenceoftheclonewassubmitedintonucleotide
databaseofNCBIforblastinganalysis.
1.5 Real-timefluorescentquantitativePCR
Eachreversetranscriptionmixtureconsistedofthefolowingcomponents:2μL5×RTbufer,0.5μL10mmol/L
dNTPs,10μLRamdom6mers,50UExscriptTMRTase,0.25μLRNaseinhibitor,and1μLofRNA(500ng)as
templatesupplementedto10μLwithRNasefreeH2O.Whenreactedfor10minat42℃ andthendenaturedfor
2minat95℃,theRTproductswaspreservedat5℃ for5min.Accordingtothesequencesoftheacocloned
byotherresearchersandtheprincipleofstandardfluorescentquantitativePCR,onesetofprimersweredesigned
toamplifytheacoinE.arundinaceus.Thesequencesofforwardandreverseprimerwereasfelows:
acoF2—GTAGATGTTCAGCATGGAAGGAGA;
acoR2—GACAGCAAGGGAGAAAACAAATAGA
ToquantificationhostcDNAandtocorectthepresenceofpotentialPCR-inhibitingcompoundsinthe
cDNAsamples,thegapdhgenewasselected.ToamplifyaPCRproductof146bpofgapdh,thesequencesof
forwardandthereverseprimerusedwere5′-AAGGGTGGTGCCAAGAAGG-3′(GAPDHF)and5′-
CAAGGGGAGCAAGGCAGTT-3′(GAPDHR),respectively.
IntwoquantitativePCRs,theacoandgapdhprimerswereusedinseparatereactionwithcDNAas
templates.Inbothcases,thereactionwasperformedina25μLvolumewith12.5μL2×SYBRPrimixExTaqTM
(TaKaRa),0.5μLreverseandforwardprimer,2.5μLtemplatecDNAand9μLofddH2O,respectively.Three
duplicatesweresetforbothsamples.Amplificationwasconductedbyafeasibleprotocol(denaturationat95℃
for30s,andthen40cyclesofdenaturationat94℃ for10s,annealingat54℃ for25s,andextensionat
72℃ for30s,finaly5minadditionalextensionat72℃).Aprogramme,whichalowstheautomated
memorizationofaveragefluorescencevalueatthefinal10s,wassetforeachextensionprocess.SYBRGreen
(TaKaRa)waschosenandfiltrationlensselectedintermsofsetupinspirationandemissionspectrum.The
procedurewasrunafteralsetupsweresaved.ModeofPCRbaselinesubtractionwasperformedfordata
analysisandamendment.Thecyclethreshold(Ct)canonlybeacquiredwhenbaselinecyclesadjustmentand
thresholdvaluecalculationarecariedthrough.
1.6 Standardcurveexecutionanddataanalysis
Whilethesamplewithdiferenttreatmentbeingamplified,cDNAtemplatemixedwitheachsamplewas
dilutedtodiferentconcentrationstoproduceastandardcurve[22].Inthisstudy,non-PEG-treatmentsamplesand
4gradientPEG-treatmentsampleswere4timesdilutedandusedastemplatesforamplificationofacoand
gapdh,withtheirrespectivespecificprimers.Thestandardcurvewasestablishedwiththethresholdvaluesas
verticalcoordinatesandthelogofdilutedconcentrationasabscissa.Therelativeconcentrationofeachsamplewas
foundaccordingtothelinearformulaacquiredfromthestandardcurve.Theexpressionleveloftargetgenewasin
accordancewiththeratiobetweenconcentrationsoftargetgeneandhouse-keepinggeneofthesametemplate.
62
2期 张积森等：斑茅 ACO基因(aco)的克隆及其在水分胁迫下的表达（英文）
1 50 100 150 200 250 300 322
AGACACGCACAAGGAAGAAACAACAGCCGGAGAGAGCTCCTATCTAGCCAGTCCAGTAGTCCCGCGAGCTCGTCGAGAGAAACAGAGACAGTAGAC
94atggcgcctgcattgtcattcccgatcatcgacatggggctgctcggcggggaggagaggccggccgcgatggagctgctgcgcgatgcg
MAPALSFPIIDMGLLGGEERPAAMELLRDA
184tgcgagaactggggcttcttcgagattctgaaccacggcatctcgacggagctgatggacgaggtagaaaagctgaccaaggaccactac
CENWGFFEILNHGISTELMDEVEKLTKDHY
274aagcgggtgcgcgagcagaggttcctcgagttcgccagcaagacgctcaaggacgcggagggcgtgaaggcggaaaacctggactgggag
KRVREQRFLEFASKTLKDAEGVKAENLDWE
364agcaccttcttcgtccgccacctcccggaatccaacatcgccgagataccggacctcgacgacgagtaccggcgcgtcatgaagcggttc
STFFVRHLPESNIAEIPDLDDEYRRVMKRF
454gccggcgagctggaggcgcttgcggagcggctgctggacctgctgtgcgagaacctcggcctcgacaagggctacctcgcgcgggcgttc
AGELEALAERLLDLLCENLGLDKGYLARAF
544cgcgggcccagcaagggcaccccgacgttcggcaccaaggtgagcagctacccgccgtgcccgcgcccggacctcgtgaacggcctccgt
RGPSKGTPTFGTKVSSYPPCPRPDLVNGLR
634gcgcacaccgacgctggcggcatcatcctgctgttccaggacgaccgagtgggcgggctccagctgctcaaggacggcgagtgggtggat
AHTDAGGIILLFQDDRVGGLQLLKDGEWVD
724gtgccgcccatgcgccactccatcgtggtcaacctgggcgaccagctggaggtgatcaccaacggcaggtacaagagcgtgatgcaccgg
VPPMRHSIVVNLGDQLEVITNGRYKSVMHR
814gtggtggcgcagcccgacgggaacaggatgtccatcgcgtccttttacaacccgggtagcgacgcggtcatcttccccgcgcccgagctg
VVAQPDGNRMSIASFYNPGSDAVIFPAPEL
904gtgaaggtcgaggaggcggccgcggagacgtaccccaggttcgtgttcgaggactacatgaagctgtatgtgcggcacaagttcgaggcc
VKVEEAAAETYPRFVFEDYMKLYVRHKFEA
994aaggagccacggtttgaggccttcaagtccatggagacgagcagctccaattgcatagccatcgcgtaa1062
KEPRFEAFKSMETSSSNCIAIA*
AAGTTGGGCCCTGCGCCTTGCTCTGGTTTACTGTTCGAGATGTATTTGCGGCGTTCTGAAT
Fig.2 Ea-acosequenceandItsConserveddomains
bp
2000
1000
750
500
250
1123bp
Fig.1 RT-PCRforacofrom
E.arundinaceus
2 Results&Analysis
2.1 RT-PCRamplificationofacoinE.arundinaceusanditssequence
analysis
ThetotalRNAwasisolatedfromtheE.arundinaceussubjectedtoPEG
stressfor12hoursastemplate,andprimersacoF1andacoR1wereusedto
amplifythetargetaco.Thebandofourexpectationwaschosenforfurther
cloningandsequencing(Fig.1).BLASTXanalysisinNCBIrevealedthatthe
clonedacowas1123bpinsizeandcontainedtheORF(openreadingframe)
of966bpwhichencoded322amino (Fig.2).Conserveddomainswere
searchedandfoundwerethefolowingthreedomains:onePcbCdomainwith
oxidationability,andbivalentFe2+andactivecentreofCO2.Throughmultiple
alignmentbetweenaminosequenceofORFintheacoclonedinE.arundi-
naceusandotherspecies(Fig.3),2highlyconservedACOaminoacidse-
quenceswerefound.
2.2 Phylogeneticanalysisoftheaminoacidssequencesdeducedfromaco
The322aminosequencesdeducedfromthe966bpORFdomainofacoinE.arundinaceusweresubmited
intoNCBIforalignment,andaphylogenetictreewasestablished.Fromthephylogenetictree,E.arundinaceus
andS.oficinarumwerefirstclustered,andsubsequentlyO.sativa,A.thaliana,PhylostachysandZeamays.The
mostremoterelationshipwasfoundtoexistinV.radiataandC.papaya,bothofwhichbelongedtodicotyledon
(Fig.4).
63
热 带 作 物 学 报 28卷64
2.3 Expression of aco from E. arundinaceus
exposed to PEG -stress through Real -time
fluorescent quantitativePCR
Through melting curves analysis of the
product of PCR for aco and gapdh the single
sharp apex was found with Tm=79℃ and Tm=
83℃, respectively(Fig.5), which suggested that
the product ofPCR was specific enough and was
separatedon1.0%agarosegel.
Equation for gapdh standard curves was y=
-1.423 x+ 26.726(Fig.6), and that of acowas y
= -1.753 3 x+ 32.21.The data in Fig.6 showed
that theyexhibited good linear relationship at the
0.05
E.arundinaceus
S.officinarum
O.sative
A.thaliana
P.pubescens
Z._mays
Phalaenopsis_cv.
x_Doritaenopsis_
Cattleya_bicolor
M._acuminata
Tulipa_gesnerian
Diospyros_kaki
P._x_hortorum
L._esculentum
S._tuberosum
H_brasiliensis
F._x_ananassa
Gossypium_hirsut
B._pendula
C._papaya
V._radiata
Fig.4 Phylogenetic tree of ACC oxidase protein sequences
Fig.3 Blast for the aco amino acid sequences from E.arundinaceus with the reference sequences from Arabidopsis
thaliana(AAN12929), Oryza sativa (japonica cultivar-group)(BAD38208), Phal enopsis cv.(AAR00506)
and Diospyros kaki(BAB89352)

2期 张积森等：斑茅 ACO基因(aco)的克隆及其在水分胁迫下的表达（英文）
five gradients with somewhat wide linearityextent.There was a close connection between Ct value and the dilu-
tionratiooftemplates(R2=0.9964and0.9921).
Threerepeatswereperformedforamplificationofbothacoandgapdh,theirproductsweredetectedbyreal-
timePCR.Thevariationrateofeachrepeatwassmallerthan2.6%,alsoindicatinggood stability.
2.4 acoexpression under PEG-stress
Throughreal-timefluorescentquantitativePCR,theexpressionprofilesofacogenefromE.arundinaceusex-
posedtoPEGstressfor4stageswere asfollows: irregular at the earlystage, and since then became low(Fig.7).
At the stage of2 hours ofPEG stress, the expression reached to the highest, which was 5.4 times ofthe control,
then descended with the stages, but wasstill positivelyhigher than that ofthe control, no matter at 4 hoursor 12
hours stress. It is interesting to note that the expression of aco and control gapdh were almost the same with
slightdiscrepancy(Fig.8).
3 Discussion
Relatively high conservation was found to
exist in aco from various plant species,and the
closerthekindred,thehigherthe homology. So it
is feasible to clone these genes using degenerate
primers designed from the conserved sequences
among the cloned aco.Therefore, the regions at
both sides of the ORF were aligned to find the
conservedsequences,andonesetofprimerswere
5 10 15 20 25 30 35 40
0.15
0.125
0.1
0.075
0.05
0.025
0
S-curve for aco S-curve for gapdh
cycle cycle
Fig.7 Real-timefluorescencequantitativeamplificationcurvewithflatbaseline,distinctexponen alarea,largeandstableslope
5 10 15 20 25 30 35 40
0.1
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
50 55 60 65 70 75 80 85 90
temperature/℃
Fig.5 Melting curves of aco and gapdh
F
l
u
o
r
e
s
c
e
n
c
e
gapdhaco
Log4
Fig.6 Standard curves for cDNAof aco and gapdh
20
22
24
26
28
30
32
34
0 1 2 3 4
y=-1.753 3 x+32.21
R2=0.966 4
y=-1.423 x+26.726
R2=0.992 1
C
t
0
1
2
3
4
5
6
c k 2 4 1 2 2 4
t/h
Fig.8 Relative expression amount of aco under
different PEG-stress
aco gapdh
F
l
u
o
r
e
s
c
e
n
c
e
R
e
l
a
t
i
v
e
e
x
p
r
e
s
s
i
o
n
a
m
o
u
n
t
65
热 带 作 物 学 报 28卷
designeddirectlyaccordingtothealignmentresult.ThewholeORFwasamplifiedimmediatelybyRT-PCR.It
wasprovedtobeasimpleandeficientmethod.Inthisstudy,ful-lengthORFwasisolatedfromE.arundinaceus
forthefirsttime.Furtherinvestigationshouldbeaimedtoconstructsenseandanti-senseplantexpressionvec-
torsforsugarcanegenetictransformation.
Throughthealignmentofaminoacidsequencesfromfiveplantspecies,2highlyconservedregionswere
founddistributedinenzymaticactivitycenter(Fig.2).Thisagreeswiththegeneralpointthatcodingsequencesof
enzymaticactivitycenterandtheircorespondingaminoacidsequencesarealwaysconserved.Phylogeneticanal-
ysiswasperformed,whichcanwelexhibitthephylogeneticrelationshipamongdiferentspecies(Fig.4).From
thephylogenetictree,E.arundinaceusandSaccharumsinensisRoxbwereclusteredfirst,andsubsequentlyO.
sativa,A.thalianaandPhylostachys,Z.mays.ThemostremoterelationshipwasfoundtoexistinV.radiataand
C.papaya,bothofwhichbelongedtodicotyledon.Itwascontradictedtofindthattherewasarelativelyfarrelation
betweenACOofZeamaysandE.arundinaceus,bothofwhichbelongtodicotyledon,andthatA.thalianaexhib-
itedcloserconnectiontoE.arundinaceusthanZ.mays.Maybeitistheresultofbeteradaptationtoenvironment
duringevolutionprocess.Inthepreviouswork,mostofACOwerederivedfromclimacticorgansinplants[7~10].
TheacowasisolatedfromtheleavesofE.arundinaceus,whichsuggestedthattheacowasalsoexpressedin
leavesofnon-climacticplants.ItwasfurtherconfirmedbytherelativelylowerCtvalue(<28cycles)inreal-
timePCRanalysis.
Inrecentyears,real-timefluorescentquantitativePCRhasjustbeenbeingappliedinplantresearchand
canproducereliableinformationatmRNAexpressionlevelwithhighcomparabilityandrepetition[24].Real-time
fluorescentquantitativePCRhasadvantagesoverconventionalPCR,insituhybridizationandNorthernblot,and
itismuchsimplerandquicker.Inaword,real-timefluorescentquantitativePCRhasagoodapplication
prospectandresearchvalueandisanidealapproachthatcanbeusedininvestigationofgeneexpression.
Ethylenealsoissynthesizedinresponsetostressessuchaspathogenatack,wounding,hypoxia,ozone,
chiling,andfreezing[13].Inthispaper,real-timefluorescentquantitativePCRwasusedforanalysis,andtheex-
pressionprofileofacounder4stagesofPEGstresswasfoundtobeiregularattheearlystage,andthenlowat
thelaterstages(Fig.8).Atthestageof2hoursofPEGstress,theexpressionreachedtothehighest,whichwas
5.4timesthecontrol,andthendescendedwithanincreaseatthelaterstages,butwasfoundstilpositivelyhigher
thanthatofthecontrol,nomaterat4hoursor12hoursstress.Itisinterestingtonotethattheexpressionofaco
andcontrolgeneswerealmostthesamewithslightdiscrepancy(Fig.5).Itcanbededucedfromtheaboveinfor-
mationthattheleavesofE.arundinaceuswereteriblysensitivetowaterstressandthattheacoisonememberof
thegenefamilywhichistermedasaresponsivegenefamilytowaterstress,characteristicwithincreaseatthe
earlystageanddecreasesubsequently.ThisphenomenonwassimilartotheexpressionofMA-ACO1inbananas
attheclimacticstage,whichistheexpressionlevelofmRNA:increasequicklyandthendecreasefleetly.More-
over,theincreasedexpressionbeforematurityandsubsequentmaintainingofhighenzymaticactivitywerede-
tectedduringalthematuritystages[24].Besides,incauliflower,itresemblestheexpressionoftwoacoinorgans
beforeandafterharvest.Noexpressionofthesetwoacowasdetectedatthestageofharvest,andtheexpression
wouldascendrapidlyin2hoursandthehighexpressioncanlastforrelativelyalongtime,withoutanyinfluence
fromhormoneorinjuryafterharvest.Incontrary,amountsofethylenewereinspired[25].Fromaloverabove,the
presentstudyconfirmedthesensitivityofacotoexternalfactors,anditswaterstresscharacterprimarily.Deter-
minationofethylenecontentandtheefectof2ethylenesynthasegenesonwaterstressandeventheaccumula-
tionofethyleneshouldbeapromisingtaskinthenearfuture.
66
2期 张积森等：斑茅 ACO基因(aco)的克隆及其在水分胁迫下的表达（英文）
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热 带 作 物 学 报 28卷
斑茅ACO基因(aco)的克隆及其在水分胁迫下的表达
张积森 1,2 李 伟 1 阮妙鸿 1 阙友雄 1 陈如凯 1 张木清 1
1福建农林大学农业部甘蔗生理生态与遗传改良重点实验室 福州 350002
2福 建 师 范 大 学 生 命 科 学 学 院 福州 350108
摘 要 利用 RT-PCR技术，首次克隆了斑茅 1-氨基环丙烷-1-羧酸氧化酶基因序列.该片段长度为 1123bp
的片段，包括 1个编码 322个氨基酸的完整开放读码框，包括具有与异青霉素合成酶功能区类似的氧化功能
PcbC区，和1个 Fe2+和 CO2活性中心。应用实时荧光 PCR技术分析了 1-氨基环丙烷-1-羧酸氧化酶基因在聚
乙二醇胁迫下4个时段的表达。结果表明，聚乙二醇胁迫 2h时，该基因在叶片明显上调表达，4h后的表达趋
向平缓。本研究为 1-氨基环丙烷-1-羧酸氧化酶基因进一步用于基因工程研究奠定基础，同时也初步揭示 1-
氨基环丙烷-1-羧酸氧化酶基因的表达与水分胁迫相互关系，为探讨乙烯对水分胁迫响应的分子机制提供了
初步依据。
关键词 斑茅 1-氨基环丙烷-1-羧酸氧化酶基因（ACO基因，aco） RT-PCR 实时荧光定量 PCR
聚乙二醇胁迫
中图分类号 Q78 S566.103.53
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