全 文 ：珙桐中一个与低温相关基因的克隆及其表达研究*
季红春1,2,苏智先1,2**,杨暋军1,边春象1,
胡进耀2,齐暋刚2
(1西华师范大学生命科学学院,四川 南充暋637002;2绵阳师范学院生命科学与技术学院,四川 绵阳暋621000)
摘要:低温诱导膜蛋白是由低温诱导表达蛋白基因编码的一类疏水性蛋白,在植物抵御寒冷环境时起着一
定的保护作用。从珙桐cDNA文库中获得一个未知基因 (DiRCI),该基因长539bp,其中包括174bp的
开放阅读框,92bp的5曚末端非编码区和273bp的3曚末端非编码区,编码57个氨基酸残基蛋白,在氨基
酸水平上同源性较高的是车前草的低温诱导膜蛋白 (登入号:ACA66247灡1),其相似性为89灡5%。半定
量 RT灢PCR分析发现,25曟以上,该基因在珙桐各器官中基本上均未表达,但经8曟低温处理时,在成熟
叶片、叶柄和成熟未萌发的种子中均有表达,但是在根中基本没有表达,进一步研究发现该基因在24h~
48h内表达量增多并达到最高值,48h后其表达量逐渐减弱直至消失,说明该基因确实与低温诱导相关,
从而初步推测该基因为低温诱导膜蛋白基因。该基因的克隆丰富和保存了珙桐基因资源,并为进一步研究
冷胁迫的分子机制奠定了基础。
关键词:珙桐;基因;克隆;低温诱导;半定量 RT灢PCR
中图分类号:Q785,Q943暋暋 暋暋暋文献标识码:A暋暋暋暋暋文章编号:0253灢2700(2010)02灢151灢07
CloningandExpressionofaCold灢inducedGene(DiRCI)
fromDavidiainvolucrata(Davidiaceae)
JIHong灢Chun1,2,SUZhi灢Xian1,2** ,YANGJun1,BIANChun灢Xiang1,
HUJin灢Yao2,QIGang2
(1ColegeofLifeScience,ChinaWestNormalUniversity,Nanchong637002,China;2ColegeofLife
Science&Biotechnology,MianyangNormalUniversity,Mianyang621000,China)
Abstract:Acold灢inducedplasmamembraneproteinwhichplaysaprotectiveroleinavoidingfreezinginjury
forsomeplantsisoneofhydrophobicproteins.Agene,namedasDiRCI,wasisolatedfromthecDNAli灢
braryofDavidiainvolucrata,containinganopenreadingframeof174bpflankedbya92bp5曚灢untranslated
sequenceandalong273bp3曚灢non灢codingregion.Alignmentanalysisindicatedthatthededucedaminoacid
sequenceof57aminoacidswashighconservedwiththeMpRCIfromPlantagoasiatica,andtheidentitywas
89灡5%.Semi灢quantitativereversetranscription灢polymerasechainreaction(RT灢PCR)analysisshowedthat
DiRCIwasn曚tdetectedinvariousorgansofD灡involucrataabove25曟.Whentheplantgrewunder8曟,the
geneexpressedinleaves,leafstalkandseeds,butnottheroots.Theexpressionlevelgradualyincreasedafter
4hofthecoldtreatmentandreachedamaximumduring24-48h,andthendecreased.Theresultsindicated
thegenedidhavearelationwithcoldstressinplantandwaspredictedtobeoneDiRCIgene.Thepresentda灢
tanotonlyenrichgeneresourcesofD灡involucrata,butalsolaidafoundationfortheresearchonmolecular
云 南 植 物 研 究暋2010,32(2):151~157
ActaBotanicaYunnanica暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋DOI:10灡3724/SP灡J灡1143灡2010灡09190
*
**
Foundationitems:TheKeyChineseNationalNatrualScienceFoundation(30670209),aprojectfromDepartmentofEducationin
SichuanProvince(2003A175)andaProgramforScienceandTechnologyDevelopmentinMianyangCitySichuanProvince
(2005BN001灢1)
Authorforcorrespondence;E灢mail:zxsu@mnu灡edu灡cn
Receiveddate:2009灢09灢28,Accepteddate:2010灢02灢09
作者简介:季红春 (1983-)女,在读硕士研究生,主要从事分子生物学研究。E灢mail:hongchunjiji@163灡com
mechanismofplantacclimationtocoldstress.
Keywords:Davidiainvolucrata;Gene;Cloning;Cold灢induced;Semi灢quantitativeRT灢PCR
暋暋Coldstressisanimportantfactorrestricting
thegeographicdistributionandtheproductionof
manyplants.Acclimationofplantstocoldisan
adaptiveprocessinvolvingphysical,structural
andbiochemicalchanges(WannerandJunttila,
1999;Zhangetal灡,2005).Ingeneral,thecel
membraneisthemostdirecttargetoflowtem灢
peratureinjuryinplants,soplants mustin灢
creasetheircryostabilityoftheplasma mem灢
braneduringcoldacclimationprocessinorderto
minimizefreeze灢inducedirreversiblefusion of
membranes (UemuraandSteponkus,1997).
Coldacclimationprotein (CAP)isonetypeof
proteinstoraiseeffectivelythecryostabilityof
plasmamembraneviaregulatingthesynthesisof
proteinsandthefoldsofmRNAwhenplantsare
stressedbylowtemperature(ColucciandInn灢
iss,1996;Yangetal灡,2003).Agreatnumber
ofgenesencodingCAPhavebeenclonedfrom
bothmonocotyledonanddicotyledonspeciesso
far(Sharmaetal灡,2005;Bretonetal灡,2000).
Studiesonthefunctionsofthegenesandtheir
mechanismsinregulatingplantacclimationto
coldstressusingmolecularbiologicaltechniques
wilbehelpfultobreedstress灢tolerantplants.
DavidiainvolucrataBailonknownasthe
“dovetree暠anda “botaniclivingfossil暠,isa
monotypicgenusbelongingtothefamilyDavidi灢
aceae.Itisarelictfromthetropicalfloraofthe
TertiaryPeriodandendemictoChina,soitis
classifiedasafirst灢gradestateprotectionplant
(Li,2003).Althoughthediverseresearcheson
populationgenetics(SongandBao,2004),taxo灢
nomicposition (He etal灡,2004),chemical
components(LiuandOu曚Yang,2006)andtis灢
sueculture(Luo,2006)ofD灡involucrata,no
functionalgeneshavebeenclonedfromthisen灢
dangeredspeciesuptodate(Qietal灡,2009a,
b).
Tobetterunderstandtheroleofcold灢in灢
ducedgenesinenhancedfreezingtolerancein
D灡involucrata,acold灢inducedgenewascloned
fromthecDNAlibraryoftheplant,thenthe
characteristicsofitsdeducedprotein wasana灢
lyzed,andtheexpressionofthisgenewasin灢
vetigatedbysemi灢quantitativeRT灢PCRinthis
study.
1暋Materialsandmethods
1灡1暋Materialsandgrowthconditions
ThefruitofD灡involucrataandseedlingsusedinthis
studywerecolectedfromtheWo灢LongNationalReserve,
DujiangyanCity,SichuanProvince.Afterremovingthe
exocarp,theseedswereairdriedandstoredat-70曟be灢
foreextractingtotalRNA.Plantsweregrowninagreen灢
houseunderalight/temperatureregimeof16h/25曟and
8hdark/20曟 ,andwatereddailyandirrigatedwithmin灢
eralnutrientsolutiononceeveryfivedays.
1灡2暋GeneclonedfromcDNAlibraryofseeds
BasedoncDNAlibraryconstructedby Qietal灡
(2009a),agenesharinghighsimilaritywiththeMpRCI
from Plantagoasiatica (ACA66247灡1)arousedourin灢
tenseinterest.Thisgene,definedasDiRCI,wascloned
again,sequencedtoconfirmitssequence,anditsexpres灢
sionwasanalyzedbysemi灢quantitativeRT灢PCR.
ThePCRprimersweredesignedbyPrimerPremier
5灡0basedonthecDNAsequence.ForcloningtheORFof
theDiRCI,thespecificprimersareasfolows:1灢F:5曚灢
GATCCATGGCATGAGGCA ACAG灢3曚 (NcoI);1灢
R:5曚灢CGGTCTAGACACTTG GTG ATG ACA TAA
ACAG灢3曚(XbaI).TotalRNAwasextractedfromseeds
ofD灡involucrataforsynthesizingthecDNAusingare灢
versetranscriptaionkitwithOligodTastheprimersfol灢
lowedbyPCRamplificationaccordingtothemanufactur灢
er曚sinstructions(Invitrogen,USA).Afteramplification,
PCRproductswereseparatedbyelectrophoresisin1灡5%
agarosegelwith1暳TAEbuffer,stainedwithethidium
bromideandvisualizedunder UVlight.Theexpected
fragmentsofPCRproductswereharvestedandpurified
fromgelusingaDNAharvestingkit(Omega,China),
andthenligatedintoapDM19灢Tvectorat16曟for1hours.
Therecombinant molecules weretransformedinto E灡coli
completecels(DH5 a),andthenspreadontheLB灢plate
containing50毺g·ml灢1ampicilin,200mg·ml灢1IPTGand20
mg·ml灢1 X灢gal.PlasmidDNAwasisolatedanddigestedby
251暋暋暋暋暋暋暋暋暋暋暋暋 暋暋暋暋暋暋暋云暋南暋植暋物暋研暋究暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋第32卷
NcoIandXbaItoverifytheinsertsize.PlasmidDNA was
sequencedbyTiangenCorporation(Beijing,China).
1灡3暋Semi灢quantitativeRT灢PCR
Matureseedsfreeoftheexocarpandseedlingsof
D灡involucratawereexposedto8曟for0-64hunderthe
samelightandphotoperiodicconditionsasdescribeda灢
bove,thentheleaf,leafstalk,rootandmatureseedwere
harvestedindividualyatdifferentperiodsoftime,frozen
immediatelyinliquidnitrogenandstoredat-80曟 until
theisolationofRNA.
ThetotalRNA wasextractedfrom matureleaves,
leafstalks,rootsand matureseedsforSemi灢quantitative
RT灢PCRsrespectively.TheRT灢PCRswereperformedas
described previously (Meadus,2003;Chen et al灡,
2006).Thegene灢specificprimersforPCRanalysisofthe
cold灢relatedgenetranscript were:2灢F:5曚灢ATC CTC
TTGGCCATCCTTCTGCCT灢3曚;2灢R:5曚灢GTGGGC
TTCACA TCA TCT CCA C灢3曚.ThePCRconditions
were94曟for5min,folowedby30cyclesofamplifica灢
tion(94曟for40s,58曟for30s,72曟for30s),then
72曟for8min.Theactingenewasamplifiedusingthe
primersActin灢F (5曚灢TGT AGG TGA TGA GGC CCA
AT灢3曚)andActin灢R(5曚灢ATACCTGTGGTACGTCCG
CT灢3曚)asacontrol.PCR products wereanalyzedon
1灡5% (w/v)agarosegels.
1灡4暋Dataanalysis
ThesequencedatawereanalyzedbyVecScreen(http://
www灡ncbi灡nlm灡nih灡gov/VecScreen/VecScreen灡html),Gen灢
Scansoftware(http://genes灡mit灡edu/GENSCAN灡html)
andMEGA4灡0(Borland,America).Homologousanaly灢
sisofthegeneclonedfrom D灡involucratawasperformed
usingBlast2灡1(http://www灡ncbi灡nlm灡nih灡gov/blast/)
bycomparingwiththerelativegenesequencesfromother
species.EncodingregionoftheDNAsequencewasana灢
lyzedusingORFfindersoftware(http://www灡ncbi灡nlm灡
nih灡gov/gorf/gorf灡html)andSignalP3灡0 Server (ht灢
tp://www灡cbs灡dtu灡dk/services/SignalP/).Theanalysis
ofhydrophobicityorhydrophilicityofthededucedproteinwas
conductedbyANTHPROT2000.MultipleSequenceAlign灢
mentwasperformedbysoftwareDNAstarLasergeneand
DNAMAN6灡0.Thesecondarystructureoftheproteinwas
predictedbythePHDalgorithmin“PredictProtein暠(http://
cubic灡bioc灡columbia灡edu/predictprotein/)(Rost,1996).
2暋Results
2灡1暋AnalysisofthecDNAoftheDiRCIgene
Afterscreening120effectiveESTsfromthe
cDNAlibraryofD灡involucrataseed,agenewas
selected.BlastanalysisshowedthatthecDNA
sequencesharesahighsimilarity(89灡5%)with
thecold灢inducedplasmamembraneproteingene
(MpRCI)fromPlantagoasiatica(ACA66247灡1),
soitwasdefinedasDiRCIgene.Thelengthof
theDiRCIcDNAis539bplong,containinga92
bp5曚灢untranslatedsequenceandalong273bp3曚
non灢codingregion,asindicatedbythepresence
ofapoly (A)+tail.AnORFof174bpencoding
57aminoacidswasfoundinthisgene(Fig灡1).
Fig灡1暋NucleotidesequenceoftheDiRCIgenefrom D灡involucrataandthededucedaminoacid
sequencefromitsORF.“*暠indicatesthestopcodon
3512期暋暋 暋暋JIHong灢Chunetal灡:CloningandExpressionofaCold灢inducedGene(DiRCI)from ...暋暋暋暋暋
2灡2暋AnalysisoftheORFoftheDiRCIgeneand
thehomologiesofitsdeducedprotein
AnORFoftheDiRCIgeneabout180bp
long was easily cloned from the seeds of
D灡involucrataRT灢PCRandsequenced.There灢
sultindicatedthattheORFwasabsolutelycon灢
sistentwiththesequencefromthecDNAlibrary
ofD灡involucrata.Thededucedproteinfromthe
ORFsequenceshowedthatthemolecularmass
andtheisoelectricpointare6灡34kDaand4灡18,
respectively.Comparingthe DiRCIprotein with
otherreportedproteinsrevealedthattheyarecon灢
servedinmolecularweightandaminoacidsnumber,
butshowsomediferencesinthetheoreticalpI(Ta灢
ble1andFig灡2).Inadditon,theDiRCIwaspre灢
dictedtoshowhighhydrophobicitypossessingasig灢
nalanchorpeptideinitsN灢terminalendandhave
twomembrane灢spanningdomains.
2灡3暋ExpressionprofilesofDiRCIinresponsetocold
Semi灢quantitativeRT灢PCRanalysisrevealed
thatthegenetranscriptscouldbeaccumulatedin
responsetolow灢temperaturetreatment,butthe
temporalandspatialexpressionofthegenevar灢
iedindifferentorgansofD灡involucrataincold
stressconditions.Thegenewasabletoexpress
inleaves,leafstalksandseeds(Fig灡3:A-C),
Table1暋Comparisonthephysicalandchemicalparametersoftheproteinencodedby
DiRCIgeneandassociatedproteinsfromdifferentspecies
ProteinType
GenBankaccession
No.
Species
Theoretical
pI
Molecular
weight/Da
DiRCI ——— Davidiainvolucrata 4灡18 6341灡7
MpRCI ACA66247灡1 Plantagoasiatica 4灡78 6311灡7
stress灢inducedhydrophobicpeptide XP_002329990 Populustrichocarpa 4灡32 6243灡6
plasmamembranceprotein3 BAG54793 Puccineliatenuiflora 4灡32 6355灡7
hydrophobicproteinLTI6A EEF50038 Ricinuscommunis 5灡87 6123灡5
Os07g0635900 NP_001060390 Oryzasativa 4灡56 6229灡6
hydrophobicproteinLTI6B ACG27760 Zeamays 6灡04 6392灡8
plasmamembraneprotein3 BAD34658 Leymuschinensis 6灡49 5945灡3
ProteinofunknownfunctionUPF0057 ABD33207灡2 Medicagotruncatula 6灡49 5946灡3
ProteinofunknownfunctionUPF0057 ABD33189 Medicagotruncatula 4灡68 6050灡4
Fig灡2暋Multiplealignment(a)andphylogenetictree(b)oftheproteindeducedfromtheDiRCIgenefrom D灡involucrata
andassociatedproteins.TheseproteinsareindicatedwithGenBankaccessionNo
451暋暋暋暋暋暋暋暋暋暋暋暋 暋暋暋暋暋暋暋云暋南暋植暋物暋研暋究暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋第32卷
Fig灡3暋Lowtemperature灢inducedaccumulationoftheDiRCIgenetranscriptsduringdifferentperiodsoftime.
RT灢PCRanalysiswasperformedwithtotalRNAisolatedfromleaves(A),leafstalk(B),matureseeds(C)androots
(D)thathadbeenexposedto8曟fortheindicatedtime.Actinwasusedascontrol.Thetoppanelshowsthe
accumulationoftheDiRCIgenefragmentandthebottompanelshowstheaccumulationoftheActinfragment
butnotinroots(Fig灡3:D)by8曟induction.In
leaves,thegeneshowedanincreasingexpression
after4hlow灢temperatureexposure,andreach
themaximalleverafter32hstress,thende灢
creasedquickly.Similartoleaves,theexpres灢
sionofthegeneinleafstalkweredetectedafter4
h,andreachedthemaximumafter40hofcold
stress,andthendisappearedgradualy.Thereg灢
ularityofchangesinmatureseedswassimilarto
thoseofleavesandleastalks(Fig灡3:C).How灢
ever,thisgenecouldnotexpressedinnon灢in灢
duced plantorgansabove25曟.Theresults
demonstratedthisgenecould only expressed
transientlyinreponsetocoldstressinpartialor灢
gansofD灡involucrata.
3暋Discussion
Wheninducedbyapre灢exposuretolowbut
nonfreezingtemperatures(Medinaetal灡,2001;
Sharmaetal灡,2005),theacclimationofplantto
coldisacomplexadaptiveprocess.Theprocessre灢
quiresthesysthesisofnewproteins(Tsengand
Li,1991),theappearanceofnewisozymes,al灢
terationsinlipidandcarbohydratecomposition
(Schraderetal灡,2004;Martzetal灡,2006),the
activationofionchannels(Fuetal灡,2006),the
accmulationofcompatibleosmolytessuchassol灢
ublesugars,prolineandbetaine(StittandHur灢
ry,2002;Uemuraetal灡,2003).Mostlymem灢
branesarefirstlydestroyedbycoldstress,espe灢
cialytheplasmamembranes(MahajanandTu灢
teja,2005).Effortstounderstandthemolecular
mechanismofcoldacclimationhaveledtothei灢
dentificationofmanycold灢inducedgenesinsome
plants,including Arabidopsis,banana (Musa
sp.),maize,cotton,tomatoandsoon(Fowler
and Thomashow,2002;Lynch,1990;Hop灢
kins,1999),butinformationaboutthecold灢in灢
ducedgenesencodingintegralmembranepro灢
teinsisverylimited.
Inthisstudy,wehaveclonedacold灢induced
genefromthecDNAlibraryofD灡involucrata,
DiRCI.Thegeneispredictedtoencodea58a灢
minoacid acidic protein and belongstothe
UPF0057genefamily (uncharacterizedprotein
family).TheDiRCIprotein waspredictedto
havetwomembrane灢spanningdomainswithhigh
hydrophobicity.Whatis more,itshareshigh
similaritywiththeMpRCIfromplantain(Feng
etal灡,2009)and LTI6Afromrice(Morsyet
al灡,2005),two memberintheUPF0057gene
family,which have been confirmed asintegral
membraneproteins.However,theirtheoreticalpI
5512期暋暋 暋暋JIHong灢Chunetal灡:CloningandExpressionofaCold灢inducedGene(DiRCI)from ...暋暋暋暋暋
variedindiferentplants,whichmayhaverelation灢
shipwiththeevolutionarydevelopmentofspecies
andtheirinternalandexternalenvironments.
TheDiRCIgenewasinducedtransientlyby
lowtemperatureinmatureleaves,leafstalksand
seeds,butnotinroots.Thereasonmaybethat
theleaves,leafstalksandmatureseedswereex灢
poseddirectlytotheoutsideenvironmentand
sensitivetocold,butrootswereburiedinthe
soil.Theorganspecificcold灢inducedexpression
ofthe DiRCIgenein D灡involucratasuggested
thatthegene mediatesanearlyprotectivere灢
sponseimmediatelyagainstsuddenstress,be灢
foreanoveralandmorepermanentresponseis
initiated.ConsistentwiththereportbyFenget
al灡 (2009),theresultsdemonstratedthatthe
DiRCIgeneplayedaroleinresistingtocoldand
itmaybeoneoflowtemperaturestressdefense灢
relatedgenesintheplant.
Themechanismsofsomeclod灢inducedgenes
havebeenreported(Gibsonetal灡,1994;Ander灢
sonetal灡,1994;Krishnaetal灡,1995).The
transientexpressionofDiRCIalsomadeahintit
mayperformasaregulatoryproteintoactivate
othercoldrelativegenes,suchasthemitogen灢
activatedprotein(MAP)kinaseandthecalmod灢
ulin灢relatedproteins (Mizoguchietal灡,1996;
PolisenskyandBraam,1996),buttheprecise
functionoftheDiRCIgenerespondingtocold
stessrequirefurtherresearch.
Insummary,aDiRCIgene,thefirstgene
from D灡Involucrata,hasbeenclonedandits
expressionhasbeeninvestigated.Semi灢quantita灢
tiveRT灢PCR resultsshowedthatthe DiRCI
genedidhavearelationwithcoldstress.The
datawilnotonlyenrichandsupplementthein灢
formationaboutthecold灢inducedgenesbutalso
contributetotheprotectionforgeneresources
andthediscussionofthegeneticpolymorphism
ofthisendangeredspecies.However,wegetthe
informationthatmanycold灢responsiveproteins
are alsoinduced by other stresses such as
drought,salinityorABA (Gulicketal灡,1994;
Capeletal灡,1997;Koikeetal灡,2005).There灢
fore,itisofinteresttodeterminetheexpression
patternofthecold灢relatedgeneinresponseto
dehydration,salinityandexogenousABAtreat灢
mentsinfuturetheoreticalstudies.
References:
Anderson,LiQB,HaskelDW etal灡,1994.Structuralorgani灢
zationofthespinachendoplasmicreticulum灢luninal70灢kilo灢
daltonheatshockcognategeneandexpressionof70灢kilodal灢
tonheatshockgenesduringcoldacclimation [J].Plant
Physiology,104:1359—1370
BretonG,DanylukJ,OueletFetal灡,2000.Biotechnological
applicationsofplantfreezingassociatedproteins[J].Bio灢
technologyAnnualReview,6:57—99
CapelJ,JariloJA,SalinasJetal灡,1997.Twohomologouslow灢
temperatureinduciblegenesfrom Arabidopsisencodehighly
hydrophobicproteins[J].PlantPhysiology,115:569—576
ChenX(陈昕),WangBL(王保莉),QuD(曲东)etal灡,2006.
Establishmentofthesemi灢quantitativeRT灢PCRsystemof
sulphurtransportergenesinwheat[J].ActaBotanicaBo灢
reali灢OccidentaliaSinica(西北植物学报),26(2):0309—
0313
ColucciMS,InnissWE,1996.Ethyleneglycolutilization,cold
andethyleneglycolshockandacclimationproteinsinaPsy灢
chrotrophicBacterium [J].CurrentMicrobiology,32(4):
179—182
FengDR,LiuB,LiWYetal灡,2009.Over灢expressionofacold灢
inducedplasma membraneproteingene (MpRCI)from
plantaimenhanceslowtemperature灢resistanceintransgenic
tobacco [J].Environmentaland ExperimentalBotany,
65:395—402
FowlerS,ThomashowM,2002.Arabidopsistranscriptomepro灢
filingindicatesthatmultipleregulatorypathwaysareactiva灢
tedduringcoldacclimationinadditiontotheCBFcoldre灢
sponsepathway[J].ThePlantCel,14:1675—1690
FuXY,ChangJF,AnLZetal灡,2006.Associationofthecold灢
hardinessofChorisporabungeanawiththedistributionand
accumulationofcalciuminthecelsandtissues [J].Envi灢
ronmentalandExperimentalBotany,65:282—293
GibsonS,ArondelV,IbaKetal灡,1994.Cloningofatempera灢
ture灢regulatedgeneencodingachloroplastOmega灢3desatu灢
rasefrom Arabidopsisthaliana[J].PlantPhysiology,106:
1615—1621
GulickPJ,Shen W,AnH,1994.ESI3,astress灢inducedgene
from Lophopyrumelongatum [J].PlantPhysiology,104:
799—800
HeZC,LiJQ,WangHC,2004.KaryomorphologyofDavidia
involucrataandCamptothecaacuminata,withspecialrefer灢
encetotheirsystematicpositions[J].BotanicalJournalof
theLinneanSociety,144:193—198
651暋暋暋暋暋暋暋暋暋暋暋暋 暋暋暋暋暋暋暋云暋南暋植暋物暋研暋究暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋暋第32卷
HopkinsWG,1999.Thephysiologyofplantsunderstress[A].
In:IntroductiontoPlantPhysiology,seconded.[M].
NewYork:Wiley,451—475
KoikeM,SutohK,KawakamiAetal灡,2005.Molecularchar灢
acterizationofacold灢inducedplasmamembraneproteingene
fromwheat[J].MolecularGeneticGenomics,274:445—
453
KrishnaP,SaccoM,CheruttiJFetal灡,1995.Cold灢inducedac灢
cumulationofhsp90transcriptsinBrasscianapus[J].Plant
Physiology,107:915—923
LiYX,2003.Cloning,sequenceanalysis,andprokaryoticexpression
ofcDNAencodingaputativenon灢specificlipid灢transferprotein
fromthebractsofDovetree(Davidiainvolucratabail)[J].
JournalofPlantBiology,46(3):167—172
LiuR(刘荣),Ou曚YangZA(欧阳明安),2006.Structuraliden灢
tificationofpumiloside,aquinolinealkaloidglycoside,isola灢
tedfrom leavesof Davidiainvolucrata [J].Subtropical
PlantScience(亚热带植物科学),35(1):35—38
LuoXJ(罗世家),2006.StudyontissuecultureofDaviaiain灢
volucrata[J].ForestryScienceandTechnology (林业科
技),31(4):4—6
Lynch DV,1990.Chilinginjuryinplants:therelevanceof
membranelipids[A].In:F.Kattermaned.,Environmen灢
talInjurytoPlants[M].NewYork:AcademicPress,17—
34
MahajanS,TutejaN,2005.Cold,salinityanddroughtstresses:
Anoverview [J].ArchivesofBiochemistryandBiophysics,
444:139—158
MartzF,KiviniemiS,PalvaETetal灡,2006.Contributionofo灢
mega灢3fattyaciddesaturaseand3灢ketoacyl灢ACPsynthaseII
(KASII)genesinthemodulationofglycerolipidfattyacid
compositionduringcoldacclimationinbirchleaves [J].
JournalofExperimentalBotany,57:897—909
Meadus WJ,2003.A semi灢quantitative RT灢PCR methodto
measuretheinvivoeffectofdietaryconjugatedlinoleicacid
onporcinemusclePPARgeneexpression [J].Biological
ProceduresOnline,5(1):20—28
MedinaJ,CatalaR,SalinasJ,2001.Developmentalandstress
regulationofRCI2AandRCI2B,twocold灢induciblegenesof
Arabidopsisencodinghighlyconservedhydrophobicproteins
[J].PlantPhysiology,125:1655—1666
MizoguchiT,IrieK,HirayamaTetal灡,1996.Ageneencoding
amitogenactivatedproteinkinasekinasekinaseisinduced
simultaneouslywithgenesforamitogen灢activatedproteinki灢
naseandanS6ribosomalproteinkinasebytouch,coldand
waterstressinArabidopsisthaliana[J].Proceedingsofthe
NationalAcademyofSciencesoftheUnitedStatesofAmeri灢
ca,93:765—769
MorsyMR,AlmutairiAM,GibbonsJetal灡,2005.TheOSLti6
genesencodinglow灢molecular灢weightmembraneproteinsare
differentialyexpressedinricecultivarswithcontrastingsen灢
sitivitytolowtemperature[J].Gene,344:171—180
PolisenskyDH,BraamJ,1996.ColdshockregulationoftheAr灢
abidopsisTCHgenesandtheeVectsofmodulatingintracelu灢
larcalciumlevels[J].PlantPhysiology,111:1271—1279
QiG(齐刚),SuZX(苏智先),LiJT(李劲涛)etal灡,2009a.
ConstructionofcDNAlibraryandanalysisoftheexpressed
sequencedtags(ESTs)propertiesofdormantseedsofDa灢
vidiainvolucrata[J].ScientiaSilvaeSinicae(林业科学),
45(10):69—73
QiG,LiJT,RuanQPetal灡,2009b.Anoptimized,smal灢scale
preparationofhigh灢qualityRNAfromdryseedsofDavidia
involucrata[J].PhytochemicalAnalysis,20(2):139—
142
RostB,1996.PHD:Predictingone灢dimensionalproteinstruc灢
turebyprofile灢basedneuralnetworks[J].MethodsinEn灢
zymology,266:525—539
SchraderJ,MoyleR,BhaleraoRetal灡,2004.Cambialmeri灢
stemdormancyintreesinvolvesextensiveremodelingofthe
transcriptome[J].PlantJournal,40:173—187
SharmaP,SharmaN,DeswalR,2005.Themolecularbiology
ofthelow灢temperatureresponseinplants[J].Bioessays,
27:1048—1059
SongCW (宋丛文),BaoMZ(包满珠),2004.Studyongenetic
diversityofRAPD markfornatural Davidiainvolucrata
population[J].ScientiaSilvaeSinicae (林业科学),40
(4):75—79
StittM,HurryV,2002.Aplantforalseasons:alterationsin
photosyntheticcarbonmetabolismduringcoldacclimationin
Arabidopsis[J].CurrentOpinioninChemicalBiology,5:
199—206
TsengMJ,LiPH,1991.Changesinproteinsynthesisandtrans灢
latablemessengerRNApopulationsassociatedwithABA灢in灢
ducedcoldhardinessinpotato(Solanumcommersoni )[J].
PhysiologiaPlantarum,81:349—358
UemuraM,SteponkusPL,1997.Effectofcoldacclimationon
membranelipidcompositionandfreeze灢inducedmembrane
destabilization[A].In:LiPH,ChenTHH eds.Plant
coldhardiness[M].NewYork:PlenumPress,171—179
UemuraM,WarrenG,SteponkusPL,2003.Freezingsensitivi灢
tyinthesfr4mutantofArabidopsisisduetolowsugar
contentandismanifestedbylossofosmoticresponsiveness
[J].PlantPhysiology,131:1800—1807
WannerLA,JunttilaO,1999.Cold灢inducedfreezingtolerancein
Arabidopsis[J].PlantPhysiology,120:391—399
YangXX,LinXZ,LiGYetal灡,2003.Recentadvanceinpsy灢
chrophiliclipaseandits heterogenousexpression [J].
ShengmingDe Huaxue (ChemistryofLife),23 (3):
204—206
ZhangCK,LangP,DaneFetal灡,2005.Coldacclimationin灢
ducedgenesoftrifoliateorange(Poncirustrifoliata)[J].
PlantCelReport,23:764—769
7512期暋暋 暋暋JIHong灢Chunetal灡:CloningandExpressionofaCold灢inducedGene(DiRCI)from ...暋暋暋暋暋