全 文 ：广 西 植 物 Guihaia　Sept．２０１４,３４(５):６７５－６８０　　　　　　　　　　 http://journal．gxzw．gxib．cn　
DOI:１０．３９６９/j．issn．１０００Ｇ３１４２．２０１４．０５．０１７
王继玥,汪生庆,曹墨菊．玉米CMSＧC不育系和保持系mads３基因克隆与表达分析[J]．广西植物,２０１４,３４(５):６７５－６８０
WangJY,WangSQ,CaoMJ．Cloningandexpressionanalysisofmads３inmaizeCMSＧCsterilelineanditsmaintainerline[J]．Guihaia,２０１４,３４(５):
６７５－６８０
Cloningandexpressionanalysisofmads３inmaize
CMSＧCsterilelineanditsmaintainerline
WANGJiＧYue,WANGShengＧQing,CAOMoＧJu∗
(MaizeResearchInstituteofSichuanAgriculturalUniversity/KeyLaboratoryofCropGeneticResourceandImprovement,
MinistryofEducation/KeyLaboratoryofMaizeBiologyandGeneticBreedingonSouthwest,
MinistryofAgriculture,Chengdu６１１１３０,China)
Abstract:MADSＧboxgenesinvolveinvariousdevelopmentalprocessesinplant,andplayanimportantroleintheregＧ
ulationoffloraldevelopment．Inthispaper,mads３,amemberofMADSboxgenefamilywasclonedbyRTＧPCRin
maize．Sequenceanalysisshowedthat８sitssingleＧbasesubstitutionmutationsinCDSregionwerefoundinCMSＧC
lineincomparisontomaintainerline,whichmayresultin５individualaminoacideschange．Furthermore,realＧtime
qPCRanalysisshowedthatmads３wassignificantlydownＧregulatedinCMSＧClineattetradstage,andwassignifiＧ
cantlyupＧregulatedatuninucleatestage．TheseresultslaidthefoundationforfurtherstudyingtherelationshipbeＧ
tweenMADSＧboxgenesandCMSＧCinmaize．
Keywords:maize;CMSＧC;anther;mads３
CLCnumber:Q９４３．２　　Documentcode:A　　ArticleID:１０００Ｇ３１４２(２０１４)０５Ｇ０６７５Ｇ０６
玉米CMSＧC不育系和保持系mads３基因克隆与表达分析
王继玥,汪生庆,曹墨菊∗
(四川农业大学 玉米研究所/教育部作物基因资源与遗传改良重点实验室/农业部
西南玉米生物学及遗传育种重点实验室,成都６１１１３０)
摘　要:MADSＧbox基因参与植物多种发育过程,尤其在花发育过程中发挥着重要的调控作用.该研究以玉
米不育系C４８Ｇ２及保持系N４８Ｇ２为材料,采用RTＧPCR克隆mads３基因.序列分析发现,在不育系C４８Ｇ２中
该基因编码区发生单碱基突变,导致其推定的蛋白质中５个氨基酸序列发生改变.荧光定量PCR显示,相对
于保持系,花粉母细胞时期和四分体时期mads３基因在C４８Ｇ２下调表达,而单核期和双核期上调表达.这些
结果为进一步了解玉米C型细胞质雄性不育的发生机制提供参考.
关键词:玉米;CMSＧC;花药;mads３基因
　　MADSboxgenesencodingMADSＧboxtranscripＧ
tionfactorsareveryimportantforplantdevelopment,
andtheyarepromisingcandidatesforresearchingcruＧ
cialregulatorynetworksinplant(Yuanetal．,２０１３)．
MIKCＧtypeMADSdomainproteinsalsharethesame
structure(Fig．１),containingaMADSＧbox(DNAbindＧ
ing),Iintervening,Kkeratinlike(proteininteraction)
andCＧterminal(transactivation)．MIKCＧtypegenes
havebeenclassifiedintotwotypes:MIKCCＧtypeand
MIKC∗Ｇtype(Henscheletal．,２００２)．MIKCCＧtype
收稿日期:２０１３Ｇ１２Ｇ２８　　修回日期:２０１４Ｇ０３Ｇ３１
基金项目:国家自然科学基金(３０９７１７９４)
作者简介:王继玥(１９８４Ｇ),男,四川南充人,博士研究生,主要研究方向为玉米细胞质雄性不育,(EＧmail)acute２８０３７６４＠１６３．com.
∗通讯作者:曹墨菊,博士,教授,主要研究方向为玉米生物技术育种,(EＧmail)caomj＠sicau．edu．cn.
MADSboxgenesmayinvolveintheregulationofthe
developmentofsporophyte(Gramzowetal．,２０１０;
Smaczniaketal．,２０１２),andMIKCCＧtypegenesplay
keyrolesinpolendevelopmentofplant．(Verelstet
al．,２００７;Adamczyketal．,２００９;Yuanetal．,２０１３)．
IncreasingnumbersofMADSboxgeneswerecharacＧ
terizedinrice,Arabidopsisthaliana,tomatoandmaize．
Thesestudymaycontributetounderstandingofthe
mechanismoffloralarchitecture(Coenetal．,１９９１;
Nagasawaetal．,２００３;Yamaguchietal．,２００６;Dreni
etal．,２００７;Yaoetal．,２００８)．Forinstance,CFO１,a
MIKC∗ＧtypeMADSboxgene,playsakeyroleinthe
regulationoffloralorganidentityinrice(Xianchunet
al．,２０１２)．ThelossoffunctionalCFO１generatedchiＧ
mericfloralorgans,defectivemarginalregionsofthe
paleaandectopicorgansphenotype．
AlargeamountofMADSＧboxgeneswereidentiＧ
fiedinmaize,mostofthemwereinvolvedinfloraldeＧ
velopment,polentubegrowth,antherdehiscenceand
polenmaturation(Schreiberetal．,２００４;Danilevskaya
etal．,２００８;Thompsonetal．,２００９)．Someofthemare
tissuespecificexpresssedgenes,suchasZmMADS２
whichisexpressedrestrictivelyinpolenandroots(SiＧ
gridetal．,２０００)．AndsomeMADSboxgeneswere
regulatedbyabioticstress,suchasZMM７ＧLinmaize
(Zhangetal．,２０１２)．Theobjectiveofthisstudyisto
clonemads３,aMADSＧboxgeneinmaize,andanalyze
theexpressionpatternsofmads３geneinantheratdifＧ
ferentstages．Whichwillaythefoundationforfurther
understandingthemechanismofCMSＧCinmaize．
Fig．１　ThestructureofMADSＧbox
１　Materialsandmethods
１．１Plantmaterials
CtypeCMSlineofmaize(C４８Ｇ２)anditsmainＧ
tainerline(N４８Ｇ２)wereplantedintheresearchfieldof
SichuanAgriculturalUniversity．CMSＧCline(C４８Ｇ２)
wasgeneratedbysuccessivebackcrossingwithN４８Ｇ２．
Fig．２　TheRTＧPCRamplificationofmads３
Fig．３　Expressionofmads３revealedbyqRTＧPCR１Ｇ４:
atthepolenmothercels(PMCs)stage,thetetradstage,
theuninucleatestageandthebinuclearstage,respectively
ThenuclearbackgroundofC４８Ｇ２isconsistentwith
N４８Ｇ２．Theanthersofmaizetasselswerecolectedat
thepolen mothercels (PMCs)stage,thetetrad
stage,theuninucleatestageandthebinuclearstage,reＧ
spectively．
１．２RNAextractionandcDNAfirststrandsynthesis
TotalRNAofantherwasextractedfromN４８Ｇ２
andC４８Ｇ２atfourstagesusingTrizolkitaccordingto
themanufacturer′sinstruction．RNAwassubjectedto
reversetranscriptionusingPrimeScript􀆿RTreagent
Kit(TaKaRa)．ThegenomicDNAwascompletelyreＧ
movedfromtotalRNAbeforereversetranscription．
１．３RTＧPCRofmads３
RTＧPCRamplificationofmads３wasperformed
withTAR􀆿 HSDNAPolymerase(TaKaRa)using
specificprimer．PCRreactionsystem(２０μL):cDNA１
μL,DNTPMixture１．６μL,PCRForwardPrimer(１０
μmol/L)０．４μL,PCRReversePrimer(１０μmol/L)
６７６ 广　西　植　物　　　 　　　　　　　　　　　　　　３４卷
７７６５期　　　　　　王继玥等:玉米CMSＧC不育系和保持系mads３基因克隆与表达分析
Fig．４　Alignmentofmads３nucleotide(A)andaminoacid(B)sequencebetweenC４８Ｇ２andN４８Ｇ２
Redboxisinitiationcodon,yelowboxisterminationcodon．
０􀆰４μL,PrimeSTAR Bufer(Mg２＋)４μL,Prime
STAR HS０􀆰２μL,ddH２O１２􀆰４μL．PCRreaction
condition:Initialdenaturationat９５℃５min,denaturＧ
ationat９８℃for１０s,annealat５８℃for５s,extenＧ
sionat７２℃for１min,３５cycles,reＧextensionat７２℃
for８min．TherecoveredPCRproductswereconnected
withpEASYＧzero,andthentransformedintoE．coli．
Threepositivecloneweresequenced．Thesequence
wasanalyzedbyDNAMANsoftware．Transmembrane
helicesinproteinswasanalyzedusingTMHMMServＧ
erv．２􀆰０ (http://www．cbs．dtu．dk/services/TMＧ
HMM/)．Spatialstructureofproteinwasestimatedby
SWISSＧMODELhomologousmodeling(http://swissＧ
model．expasy．org/)TheprimersequenceswereasfolＧ
８７６ 广　西　植　物　　　 　　　　　　　　　　　　　　３４卷
lows:F５′CACGCACGCTCATCACACAGAC３′and
R５′CCCTCATTCAACCCCTACACA３′
１．４RealＧTimeqPCR
Todetecttheexpressionlevelsofmads３between
C４８Ｇ２andN４８Ｇ２atdiferentdevelopmentstages,realＧ
timeqPCRwasperformedusingacommercialkit(AcＧ
cuPower􀆿２XGreenstarqPCRMasterMix,Bioneer)．
SpecificprimersweredesignedintheconservativereＧ
gion．１８sandβＧActinwereassignedasinternalcontrol
genesforrealtimeqPCR．Theprotocolofreactionwas
asfolows:denaturationprogramat９５℃for１０min,
９５℃for５s,５８℃for２０s,７２℃for１０swithasinＧ
glefluorescencemeasurement,４４cycles．Alreactions
wereperformedintriplicate．Theprimersequences
wereasfolows:
１８s:F５′CTGAGAAACGGCTACCACA ３′and
R５′TCTGAGAAGGAAGTATTGGCTATGC３′
βＧActin:F５′GTCCCTCACCCTCCCAAAAG３′
andR５′GCTGCCTCAACACCTCAACCC３′
mads３:F５′GAAGCACATCAGATCAAGAAAＧ
GA３′andR５′GAAGCAGATGTTTTGTGGAGGT３′
２　Resultsanddiscussion
２．１Sequencesanalysesofmads３
Themads３inantheratuninucleatestagewas
evaluated．Twotranscriptswereclonedandsequencing
(Fig．２),thefirstis９９７bp,thesecondis３５０bp．SeＧ
quencesanalysesofthefirsttranscriptshowedthat８
sitssinglebasesubstitutionmutationsinCDSregion
werefoundinC４８Ｇ２comparedtoN４８Ｇ２,whichmay
resultin５individualaminoacideschange(Fig．４)．
Thephysicalandchemicalparametersofitsproducts
(MADS３)mightbediferentfromthenormalMADS３
inN４８Ｇ２basedonprimaryproteinstructureprediction．
thespaceconformationalofC４８Ｇ２putativeproteinis
slightlydiferentformN４８Ｇ２(Fig．５)．However,the
predictedtransmembranedomainsandconservative
structuredomainofMADS３inC４８Ｇ２wasthesame
withN４８Ｇ２．ThefunctionofalteredMADS３remains
tobeseenandfurtherexploredbyreversegeneticsapＧ
proach．Inaddition,thesecondtranscriptwasidentified
asanoveltranscriptofmads３bymeansofsequences
sequencealignment,whichunderwentalternativespliＧ
cing．ThesequenceofnoveltranscriptinC４８Ｇ２isthe
samewithinN４８Ｇ２．
Fig．５　Tertiarystructurepredictionof
firsttranscriptinmads３
　　TheAGhomologsMADS３,aCＧclassMADSbox
geneinrice,regulatefloralorganidentityduringearly
flowerdevelopment．OSMADS３wasnecessaryforstaＧ
menspecificationinrice,developmentaldefectswere
observedifthefunctionmads３wasimpairedorlost
(Yamaguchietal．,２００６)．MADS３mutantdisplayed
defectiveantherwals,abortedmicrospores,andcomＧ
pletemalesterility(Huetal．,２０１１)．Interestingly,a
specificamplifiedbandwasfoundinCMSＧCline(Fig．
２)．ThespecificfragmentinCMSＧClinewasidentified
asanonspecificamplificationbymeansofsequence
analysis．Whythenonspecificsegmentonlypresentin
CMSＧClinewasambiguousforus．Onemorespecial
productofmads１wasfoundinriceCMSZhenshan
９７AcomparedtoitsmaintainerlineZhenshan９７Bat
thesamedevelopmentalstage(Yuanetal．,２０００)．ItilＧ
lustratedthattheexpressionofMADSＧboxgeneinrice
CMSlinewasdiferentfromitsmaintainerline．
２．２Theexpressionprofilingofmads３
Theexpressionpatternsofmads３atthepolen
mothercels(PMCs)stage,thetetradstage,theuniＧ
nucleatestageandthebinuclearstageweredetermined
(Fig．３)．ItwasupＧregulatedattheuninucleatestage
andthebinuclearstage,whilewasdownregulatedat
thepolenmothercels(PMCs)stageandthetetrad
stageintheCMSＧClineincomparisontomaintainer
line．Exceptbinuclearstage,thediferencesofmads３
expressionlevelbetweenCMSＧClineandmaintainer
lineareremarkable．Especialy,theexpressionlevelof
９７６５期　　　　　　王继玥等:玉米CMSＧC不育系和保持系mads３基因克隆与表达分析
CMSＧClineatuninucleatestagewasnearlytenfoldas
comparedwithmaintainerline．Thisphenomenonjust
wasgoodfitwithpolenabortionstageinC４８Ｇ２．
Therefore,therelationshipbetweenmads３andanther
polenabortionisamusing,andthisresultslaidafounＧ
dationforfurtherresearch．
MADS３isakeytranscriptionalregulatorformale
reproductivedevelopmentinrice,whichishighlyexＧ
pressedinthetapetumandmicrosporesduringlateanＧ
therdevelopment(Huetal．,２０１１)．ZMM４,aMADSＧ
boxgeneinmaize,wassignificantlyupＧregulatedin
shootapicesafterthefloraltransition,andoverＧexＧ
pressionofZMM４havechangedthefloweringtimein
transgenicmaize(Danilevskayaetal．,２００８)．CytoＧ
plasmicmalesterilitywascausedbynucleargenes
whichwasmediatedbyretrogradesignalingemitted
frommitochondria(Yangetal．,２００７)．Theimmense
amountsofstudieshavedemonstratedthatpolenaＧ
bortionisresponsibleforcytoplasmicnuclearincomＧ
patibility．Asaconsequence,theexpressionofMADSＧ
boxgeneswouldberegulatedbyadetrimentalinteracＧ
tionbetweennuclearandmitochondria．
３　Conclusion
RNAsequenceanalysisofmads３showedthat８
sitssinglebasediferenceinCDSregionweredetected
inCMSＧClineincomparisontomaintainerline．qRTＧ
PCRanalysisshowedthatmads３wassignificantlyupＧ
regulatedinmaizeCMSＧClineatuninucleatestage．
Thediferencedexpressionofmads３geneduringanＧ
therdevelopmentmaycontributestounderstandingof
themechanismofCMSＧCinmaize．
Acknowledgments　Thisworkwasfinancedbythe
NationalNaturalScienceFoundationofChina (no．
３０９７１７９４)．
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