全 文 ：分子植物育种，2006年，第4卷，第2期，第209-215页
MolecularPlantBreeding,2006,Vol.4,No.2,209-215
ResearchReport
研究报告
MolecularCloningandCharacterizationofaBetaine-aldehydeDehydroge-
naseGeneinDescurainiaSophia
DongHaibin GuanRongzhan* ZhangHongsheng HuangJi
NationalKeyLabofCropGeneticsandGermpalsmEnhancement,NanjingAgriculturalUniversity,Nanjing,210095
*Corespondingauthor,guanrzh@njau.edu.cn
Abstract AnovelcDNAencodingbetainealdehydedehydrogenase (BADH)wasclonedfromDescurainia
sophiabyRT-PCRanddesignatedasDsBADH.Theful-lengthcDNAwas1653bpinlengthwithacomplete
openreadingframeof1506bp,encoding501aminoacidwithapredictedmolecularmassof54kDandapIof5.5.
Thededucedproductscontainedtheconserveddomainsequenceofthealdehydedehydrogenaseandcysteineasso-
ciatedwithaldehydedehydrogenasefunction.N-terminalsignalpeptiderevealedthatDsBADHwastargetedinto
chloroplast.SequencealignmentshowedthatDsBADHpresentedhighersimilaritywithotherBADHsatthelevel
ofbothnucleicacidandaminoacid.PhylogenetictreeanalysisshowedthatDsBADHfelintoasmalgroupwith
Cruciferousspecies.Semi-quantitativeRT-PCRresultsrevealedthatDsBADHwasexpressedinroots,stems,
leavesandsiliques,buttheexpressinglevelwasthehighestinsiliques.Inaddition,theexpressionofDsBADH
genewasinducedbysaltstressinaltissuesdetected.
Keywords DescurainiaSophiaL.Webb,DsBADHgene,Cloning,Expression
播娘蒿甜菜碱醛脱氢酶基因的克隆和表达分析
董海滨 管荣展 * 张红生 黄骥
南京农业大学作物遗传与种质创新国家重点实验室,南京,210095
*通讯作者,guanrzh@njau.edu.cn
摘 要 采用RT-PCR技术克隆了播娘蒿的甜菜碱醛脱氢酶基因全长cDNA序列(DsBADH)。DsBADHcD-
NA序列全长1653bp，其中开放阅读框长1503bp，编码一个由501个氨基酸残基组成的蛋白质，推测的蛋
白质相对分子质量为54kD，pI为5.5。序列比对结果表明DsBADH与其它物种的BADHs无论在核酸水平还
是在蛋白质水平上都表现较高的同源性，表明BADH基因家族具有较高的保守性。DsBADH基因的氨基酸
序列在进化上与同属十字花科植物BADH基因距离较近。蛋白质序列存在一个编码十肽的高度保守序列，
该结构在醛脱氢酶中是高度保守的，这些残基可能包含NAD+结合位点及酶催化位点，而且含有与酶功能有
关的醛脱氢酶高度保守的氨基酸残基Cys，表明DsBADH可编码活性蛋白。N端存在信号肽，初步将该酶定
位于叶绿体。半定量RT-PCR分析表明，DsBADH在根、茎、叶以及角果中均表达，但在角果中的表达显著高
于其它组织，而且表明DsBADH受盐诱导正调节表达。
关键词 播娘蒿,播娘蒿甜菜碱醛脱氢酶基因,克隆,表达
Manyplantsaccumulatebetaineasanontoxicor
protectiveosmolyteundersalineanddryconditions
(WeretilnykandHanson,1990).Inhigherplants,
glycinebetaineissynthesizedviathetwo-stepoxida-
tionofcholine.Inthefirststep,cholinemonooxygenase
(CMO)catalyzescholinetobetainealdehyde;Inthe
secondstep,betainealdehydedehydrogenase(BADH)
catalyzesbetainealdehydetobetaine,andsynthesisof
thesetwoenzymeswereinducedbysalinityand
drought(Handsonetal.,1994;Arakawaetal.,1987).
Uptonow,BADHgeneshasbeenisolatedfromS.
olercea(WeretilnykandHanson,1990;Maetal.,1996;
分子植物育种
MolecularPlantBreeding
Shuetal.,1997),A.hortensis(Xiaoetal.,1995),B.
vulgaris(McCueandHanson,1992),H.vulgare(Ishi-
tanietal.,1995;Nakamuraetal.,2001),S.bicolor
(Woodetal.,1996),O.sativa(Nakamuraetal.,1997),
A.marina(Hibinoetal.,2001),A.triangularis(Heet
al.,2004),A.hypochondriacus(Legariaetal.,1998),B.
napus,A.centralasiatica(Chenetal.,2001),T.aes-
tivum(LiandZou,2002),zeamays(Yuetal.,2004),
P.sativum,S.liaotungensi(Lietal.,2000;2002),A.
thaliana,G.hirsutum.Furthermore,thesalt-toleranceof
transgenicplantsweremuchhigherthanthecontrol,
BADH activityofthesetransgenicplantshavein-
creasedapparently,andwaspositivecorelatedtothe
salt-tolerance(Guoetal.,1997;Liuetal.,1997;Guoet
al.,2000;Lietal.,2000).Descurainiasophiabelongs
toDescurainiaspeciesofCruciferae,isanoilplantof
specifiedfunction.Descurainiasophiaisadaptableto
extremlyadversenaturalconditions,possessmacha-
nismofgoodresistancetostress(Gaoetal.,1998).
Herewereportedtheisolationandcharacterizationof
DsBADH encodingbetainealdehydedehydrogenase
fromDescurainiasophiaandshoweditsexpressionwas
salt-inducible.
1MaterialsandMethods
1.1PlantMaterials
Descurainiasophiawasplantedinsoilundernatu-
ralconditions.Siliqueswereharvestedfromtheendof
matureDescurainiasophiaplants.Fortheanalysisof
BADHexpression,theDescurainiasophiaplantswere
treatedwith200mmol/LNaClattheendofflowering，
thenleaves,stems,footsandsiliqueswereharvested
fromtheplantsatdiferenttimeundersaltstress.
1.2cDNALibraryConstructionofDescurainiasop-
hiasiliqueandSequencing
DescurainiasophiasiliquecDNAlibrarywascon-
structedbyusingSMARTTM technology(Clontech).
Onehundredpositiveplaqueswerechosenrandomly
forsequencingafterconvertingthe!TriplEx2cloneto
TriplEx2plasmid.Oneofcloneswithincompletestruc-
turewashighlyhomogouswithA.thalianabe-
taine-aldehydedehydrogenasegene(GenBankacces-
sionNo.NM_106150).
1.3IsolationofDsBADHGene
TotalRNAwasextractedfromDescurainiasophia
leavesusingRNApurekits(Tianwei).M-MLVReverse
transcriptsystem (Promega)wasusedtoreversetran-
scribethefirststrandofDescurainiasophiabe-
taine-aldehydedehydrogenasecDNA.DsBADHspecif-
icprimerP1 (5-GCACCACGAATCCACGATCCAG
-3)andP2(5-AGCACAAAGATGGGACATAC-3)
wasdesignedaccordingtoA.thalianabetaine-aldehyde
dehydrogenaseand3-UTRofDsBADHcDNAclone,
whichwereusedtoamplifiedtheful-lengthDsBADH
cDNA.ThePCRconditionswereasfolows:50sat
94℃,90sat56℃,and2minat72℃ for35cyclesfol-
lowedby10minat72℃.PCRproductswereclonedin-
topGEM-Easyvector(Promega)andsequenced.For
furtherverificationoftherecombinantclones,thermo-
cycleconditionswereasabovebutcyclenumberwas
reducedto28.DsBADHcDNAsequencewereanalyzed
byDNAssist.Themultipleproteinsequencesalignment
andphylogenetictreeweregeneratedusingClustalW
andClustalX,respectively.
1.4ExpressionAnalysisbySemi-quantitativeRT-
PCR
Forfurtherunderstandingtheexpressionofthe
DsBADHgenes,weperformedSemi-quantitativeRT-
PCRusingRNAsamplesatdiferenttimeundersalt
stressof0h,1h,6h,12h,24hand72h.Atthesametime,
theexpressionanalysisfromdiferenttissueswasalso
performed.PCRamplificationwasperformedusingthe
primersP1andP2,whichamplifytheful-lengthDs-
BADHcDNA.ThePCRconditionswerethesameas
describedaboveforisolationofDsBADHgene.Anoth-
erPCR was performed using the primers P3
(5-TGGGATACCTGCCAGTAGTCAT-3) and P4
(5-CTGGATCCAATTACCAGACTCAA-3)of18S
rRNA.Foramplifyingthe18SrRNA,thermocyclecon-
ditionswere:30cyclesof94℃(30s),at53℃(50s),and
72℃(80s),folowedby10minat72℃.
2Results
2.1IsolationandDsBADH
210
MolecularCloningandCharacterizationofaBetaine-aldehydeDehydrogenaseGeneinDescurainiaSophia
播娘蒿甜菜碱醛脱氢酶基因的克隆和表达分析
AccordingtoAtBADHand3-UTRofDsBADH
cDNAsequences,specificprimersweredesignedtoob-
tainDsBADHful-lengthcDNAsequence.Descurainia
sophiabetainealdehydedehydrogenase,DsBADH,was
clonedbyRT-PCR.ElectrophoresisofPCRproducts
showedthatthereisonespecificandbrightbandatthe
1.7Kb(Figure1).InordertoverifyingRT-PCRprod-
uctshasinsertedintopGEM-Easyvector,anotherPCR
wasperformed.Electrophoresisresultsissimilarto
RT-PCRelectrophoresisresultsofDsBADH(Figure2),
whichindicatedthatDsBADHwereclonedintopGEM-
Easyvectorsuccessfuly.
orientatedtochloroplastduetothesignalpeptideinthe
N-terminalregion(WeretilnykandHanson,1990;Mc-
CueandHanson,1992;Ishitanetal.,1995;Woodetal.,
1996;Nakamuraetal.,1997).
2.3AlignmentofthePredictedAnimoAcidSe-
quenceofdiferentBADH
SequencealignmentrevealedthatDsBADHpre-
sentedhighersimilaritywiththoseofotherBADHsre-
portedinGenbank(htp:/www.ncbi.nlm.nih.gov).In
addition,thelengthofalofBADHaminoacidse-
quenceswerealmostthesameandincludedthecon-
serveddomainsequenceofthealdehydedehydrogenase
andhighlyconservedcysteineassociatedwithaldehyde
dehydrogenasefunction.Theconserveddecapeptideis
“ VTLELGGKSP”undermanyinstances,butitis
“ VTLELGGKSP”intheO.sativa,H.vulgare2,G.hir-
sutum,S.bicolor,B.napus,P.sativum,namelythreo-
nineofthesecondwasreplacedbyserine.However,
theconserveddecapeptideis“ VSMELGGKSP”atAt-
BADH andDsBADH,threonineofthesecondand
leucineofthethirdwasreplacedbyserineandmethio-
nine,respectively.Theaboveresultsindicatedthatthe
BADHfamilywerehighlyconservativeatthelevelof
evolution,butdiferentBADHsarediverse.According-
ly,althoughtheirrelativerelationarenotclose,dueto
selectionpresstheirproteinsequenceareinclinedto
conservedduringtheprocessofevolution.
2.4PhylogeneticTreeAnalysisofBetaineAldehyde
Dehydrogenase
AccordingtoBADHsanminoacidsequence,phy-
logenetictreewasconstructedusingneighbour-joining
methods(Figure4).Theresultshowedthattheywas
plotouttwomajorgroups,corespondingtotheclassi-
calbotanicaldividionofplants.P.sativum,G.hirsu-
tum,A.thalianaandD.sophiabelongtoasmalgroup,
T.aestivum,S.bicolor,H.vulgare1,H.vulgare2andO.
sativaconstituteasmalgroup,thesebothandB.napus
comeposeofagrouptogether.BADHsoftwoA.mari-
na,twoB.vulgaris,S.liaotungensis,A.centralasiatica,
A.hortensis,A.triangularis,S.olerceaandA.hypocho
ndriaccomposeofagroup.
2.5ExpressionAnalysisofDsBADHGene
Figure1PCRamplificationofDsBADHgene
Note:M:DL-2000marker;1and2:PCRproducts
Figure2PCRamplificationofrecombinantclones
Note:M:DL-2000marker;1and2:Recombinantclones
2.2AnalysisofDsBADHcDNASequence
TheDsBADHful-lengthcDNAis1653bpwith
anopenreadingframeof1506bp(Figure3).Thede-
ducedproteinencoded501aminoacidwithapredicted
molecularmassof54kDandanisoelectricpointof5.5.
Theproteincontainedonesequenceencodingten-pep-
tide“ VSMELGGKSP”,whichwashighlyconserved
inthealdehydedehydrogenaseandwaslikelytocon-
tainNAD+combinedsiteandenzymiccatalysesite
(Ishitanietal.,1995).Also,DsBADHcontainedhighly
conservedcysteineassociatedwithaldehydedehydro-
genasefunction.DsBADH-encodedactiveproteinwas
211
分子植物育种
MolecularPlantBreeding
Figure3ThenucleotideacidsequenceanddeducedaminoacidsequenceofDsBADH
Note:Boxedregionindicatesthehighlyconservativeten-peptide;Underlinesequenceindicatethesignalpeptide;Thehighlycon-
servedcysteineisshowninbold
TheexpressionanalysisofDsBADHindiferent
tissuesanddiferenttissuesatdiferenttimeunder
salinestressusingsemi-quantitativeRT-PCRrevealed
thatDsBADHwasexpressedinroots,stems,leavesand
siliques,butthelevelofexpressionwashigherin
siliquesthaninothertissues(Figure5A).DsBADHex
212
MolecularCloningandCharacterizationofaBetaine-aldehydeDehydrogenaseGeneinDescurainiaSophia
播娘蒿甜菜碱醛脱氢酶基因的克隆和表达分析
pressionwasalup-regulatedby200mmol/LNaClstess
treatmentinleaves(Figure5B),stems(Figure5C)and
roots(Figure5D),whichisconsistentwithformerre-
searchresultsinspinach (Handsonetal.,1994;
Arakawaetal.,1987).
3Discussion
Betainealdehydedehydrogenase (BADH)isan
importantenzymeforGlybetainesynthesis.It’simpor-
tanttoknowaboutthelocalizationofBADHinthecel
forunderstandingthemechanism ofinfiltrationand
regulation.Accordingtotheformerresearchresults,the
localizationofdiferentBADHswerevariousinthecel
(Weigeletal.,1986;Luoetal.,1995),whichisproba-
blyrelatedtothelackoftypicalsignalpeptidetransit
sequences,almonocotyledonousBADHsarelocalized
inperoxisomes,dicotyledonousBADHsmightbelocal-
izedinchloroplasts.IntheBADH aminoacidse-
quencesreported,therearetwotypesofsignalse-
quences.OneisN-terminalsequence(QLFIDGE)that
isasignalfortargetingpreproteinstochloroplasts,but
thesignalsequencelacktypicaltransitpeptidese-
quencesneededforentryintochloroplasts.Theotheris
C-terminaltripeptideSKL,asignalknowntotarget
preproteinstoperoxisomes.Atpresent,thereisnodis-
putethatBADHcontainedthesignalsequence-SKLis
localizedinperoxisomes.BecauseBADH prefers
NAD+ratherthanNADP+asacofactorforitsenzymat-
icactivity(Arakawaetal.,1990),NAD+existsinper-
oxisomesathigherlevelsthaninchloroplasts,however
localizationofBADHlackedtypicaltransitpeptidese-
quencesisworthyofdiscussing.DsBADHwasorientat-
edtochloroplastsduetothesignalpeptideintheN-ter-
minus,butitwaslocalizedinchloroplasts,peroxisomes
orcytosolfinaly,whichneedbefutherexaminedby
experiment.
DsBADH encodingproteincontainedthecon-
serveddecapeptidesequenceofthealdehydedehydro-
genaseandcysteineassociatedwithaldehydedehydro-
genasefunction.Therefore,theaboveresultsshowed
thatDsBADHbelongstooneofBADHfamilies.Isola-
tionofDsBADHgeneisnotonlyforestablishingthe
foundationforfurtherstudyingmechanismofplantsin
tolerancetostress,butalsoprovidingforgeneticengi
Figure4Phylogenetictreeofbetainealdehydedehydrogenase
Note:P.sativum(AJ315853);G.hirsutum(AY461804);A.marina1
(AB043540);A.marina2(AB043539);T.aestivum(AY050316);
A.thaliana(NM_106150);B.napus(AY351634);S.liaotungensis
(AF359282);A.centralasiatica (AY093682);A.hortensis(X69
770);A.triangularis(AY256971);S.olercea(M31480);A.hypoch
ondriacus(AF017150);S.bicolor(U12196);H.vulgare1(AB063
178);H.vulgare2(AB063179);O.sativa(AB096083);B.vulgaris1
(X58463);B.vulgaris2(X58462);D.sophia
Figure5AnalysisofDsBADHexpression
Note:A:Theexpressionanalysisfrom diferenttissue;18S
rRNAwasusedasloadingcontrol;B:DsBADHexpressionin
leavesofDescurainiasophiaatdiferenttimeundersaltstress;C:
DsBADHexpressioninstemsofDescurainiasophiaatdiferent
timeundersaltstress;D:DsBADHexpressioninrootsofDescu-
rainiasophiaatdiferenttimeundersaltstress
213
分子植物育种
MolecularPlantBreeding
neeringofsalt-stresstolerance.
Acknowledgement
TheresearchwassupportedbyNationalNatural
ScienceFoundationofChina(010600317).
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■分子植物育种原理与方法
■植物遗传标记与DNA标记技术
■QTL定位的原理和方法
■植物遗传群体构建与应用
■植物遗传图谱的构建与应用
■植物突变体建立的理论与方法
■植物突变体发掘-以水稻为例
■国际农业生物技术现状与展望
■植物应用基因组学与分子育种研究策略
■应用GITS改良水稻外观品质——一个分子选择的例子
■利用公共图谱定位复杂性状QTL——以SCN抗性QTL为例
■杂交水稻第四遗传因子精细定位和分离——图位法克隆
基因的例子
■若干专题讲座
报名条件及办法
●报名条件：中级职称以上从事常规育种的科研人员。
截止时间2006年7月15日。研究生学员需有导师推荐。
●请报名学员打电话或发邮件索取报名表
海南省生物工程协会秘书处：
0898-68966415,hnabe@hitar.org,吴海兰
分子植物育种编辑部：
010-62556198,mpbbj@vip.sina.com,李迪
培训地点
海南省三亚市（ 正式录取时通知具体地点）
培训费用
（ 一）培训费（ 注册费）：1600元/人
（ 二）住宿费自理。会议住宿为三星级标准，标准间（ 两人一间）
每日房费为120-140元，如果两人住宿每人每日60-70元。
注：会务费包括资料费、讲座费和餐费。
培训费用优惠申请办法
为了鼓励一些边缘地区的育种家参加本培训班，海南省
生物工程协会和分子植物育种编辑部联合设立了分子育种奖
学金，有意参加培训的育种家可向海南省生物工程协会申请
奖学金，本期培训班的奖学金金额在600~1000元，奖励人数
20名。具体事宜请与海南省生物工程协会秘书处联系。
培训费用缴纳方法
欢迎通过email注册。注册费通过银行，请寄：
开户单位：海南省生物工程协会
帐号：21-160001040023776
开户银行：中国农业银行海口市海秀支行
用途：培训费
欢迎通过email注册。注册费通过邮局，请寄：
汇款地址：海南省海秀中路107号北岸青年公寓507室，
邮编：570206
收款人：海南省生物工程协会
用途：培训费
收到注册费后，我们将用email予以确认。
关注会议进展情况,请登录:
南亚遗传资源会务网
htp:/meeting.shangwusou.com
分子植物育种理论与实践研讨班
2006年8月7-11日在海南省三亚市举行
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