全 文 ：植物病理学报
ACTAPHYTOPATHOLOGICASINICA　38(3):258-262(2008)
Receiveddate: 2007-05-28;Reviseddate: 2008-02-19
Foundationitem:ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(30471137)
Correspondingauthor:ZHOUXue-ping, Ph.D., Professor, engaginginmolecularplantvirology;E-mail:zzhou@zju.edu.cn
Biography:RoyB.Mugiira(1969 -), male, KenyanPh.D.student, engagedinthestudyofgeminivirusplantdiseases;E-mail:
rmugira@yahoo.com.
MolecularCharacterizationofMalvastrum
leafcurlGuangdongvirusIsolated
fromFujian, China
RoyB.Mugira1 , WUJian-bing1 , WUZu-jian2 ,
LIGui-xin1 , ZHOUXue-ping1＊
(1InstituteofBiotechnology, ZhejiangUniversity, Hangzhou310029, China;
2InstituteofPlantVirology, FujianAgricultureandForestUniversity, Fuzhou350002, China)
Abstract:Sixvirusisolates(FJ1-FJ6)wereobtainedfromMalvastrumcoromandelianumplantsshowing
leafcurlandveinbandingsymptomsinFujianProvince, China.PartialviralDNAfragment, approximately
500bplongwasamplifiedfromalthesixisolatesbyPCRusingthedegenerateprimerpairPA/PB, specific
forbegomoviruses(FamilyGeminiviridae).Thepartialfragmentssharedthehighestnucleotidesequenceiden-
tity(90%-93%)withthepreviouslyreportedMalvastrumleafcurlGuangdongvirus(MLCuGdV).Isolate
FJ3 wasrandomlyselectedforcloningandsequencingthecompleteviralgenomicDNA.FJ3 completeDNA
was2 765 nucleotideslongwithalthecharacteristicfeaturesofabegomovirusgenomicorganizationand
shared93% nucleotidesequenceidentitywiththepreviouslydescribedMLCuGdVisolatefromGuangdong,
andwasthereforeconsideredtobeanisolateofMLCuGdV.Inphylogeneticanalysis, FJ3showedlowafinity
tootherreportedMalvastrum-infectingbegomovirusesexceptMLCuGdV.Instead, itshowedhighafinityand
clusterstogetherwithpapaya-infectingbegomovirusesisolatedfromthesameagro-ecologicalzoneofSouth
China.FurthercomparisonsofaminoacidsequencesofencodedproteinsindicatethatFJ3 isachimericmole-
culethathasarisenbyinterspecificrecombinationbetweenPapayaleafcurlChinavirusorPapayaleafcurl
Guangdongvirusasthemajorparentandotherunidentifiedancestorsasminorparents.
Keywords:MalvastrumleafcurlGuangdongvirus;Malvastrumcoromandelianum;sequence;recombina-
tion;Fujian
从福建分离的广东赛葵曲叶病毒的分子特征　RoyB.Mugira1 , 吴剑丙 1 , 吴祖建 2 , 李桂新 1 ,
周雪平 1　(1浙江大学生物技术研究所 , 杭州 310029;2福建农林大学植物病毒研究所 , 福州 350002)
摘要:从中国福建省表现曲叶和脉突症状的赛葵上分离到病毒分离物 FJ1 ～ FJ6。利用菜豆金色花叶病毒属病毒的特异性
简并引物 PA/PB, 在所有 6个分离物中都分离到了约 500 bp长度的病毒部分 DNA片段。这些 DNA片段与已报道的广东
赛葵曲叶病毒(MalvastrumleafcurlGuangdongvirus, MLCuGdV)的核苷酸序列同源性高达 90%～ 93%。随机挑选 FJ3分离
物进行全基因组 DNA的克隆测序。结果表明 , FJ3 DNA全长 2 765个核苷酸 ,具有典型的双生病毒科病毒的基因组结构特
征 , 与 MLCuGdV的同源性为 92.9%,表明 FJ3是 MLCuGdV的一个分离物。系统进化分析表明 ,除了 MLCuGdV, FJ3与其
它赛葵上分离到的双生病毒的亲缘关系都较远 , 而与分离自中国南方番木瓜上的双生病毒聚成簇 , 有较近的亲缘关系。
　
　3期 RoyB.Mugira, etal.:MolecularCharacterizationofMalvastrumleafcurlGuangdongvirusIsolatedfromFujian, China
进一步比较分析各蛋白编码的氨基酸序列发现 , FJ3可能是一个种间重组分子 ,它可能是由中国番木瓜曲叶病毒或广东番
木瓜曲叶病毒和另外的未知病毒重组产生的。
关键词:广东赛葵曲叶病毒;赛葵;序列;重组;福建
中图分类号:S432.41　　　文献标识码:A　　　文章编号:0412-0914(2008)03-0258-05
　 　 Geminiviruses(FamilyGeminiviridae)are
characterizedbytheircircularsinglestrandedDNA
genomes, encapsidatedintwinnedicosahedraparti-
clesandareemergingasimportantplantpathogens,
whichcausesignificantcroplossesthroughoutthe
world[ 1 -3] .Themosteconomicalyimportantgemi-
nivirusesbelongtothegenusBegomovirus, manyof
whichhavebipartitegenomeorganizationwithtwo
circularDNAcomponents, referredtoasDNA-A
andDNA-B[ 4] .Anumberofbegomoviruseshavea
singlegenomicDNAcomponent(monopartite)re-
semblingtheDNA-Aoftheirbipartitecounterparts,
andanovelsateliteDNAcomponent, designatedas
DNAβ hasbeenfoundassociatedwithsomemono-
partitebegomoviruses[ 5-8] .
Recently, fourdistinctmonopartitebegomo-
viruseshavebeenisolatedfromM.coromandelianum,
aperennialweedplantspeciesthatgrowswidelyin
fieldswithin theagro-ecologicalzoneofSouth
China[ 9-12] .Leafcurlandveinbandingsymptoms
werefoundwidespreadamongM.coromandelianum
plantsinFujianProvince, SouthChina, andbased
onthesymptomsexpression, abegomoviruswas
suspectedtobethecauseagent.Amoleculardiag-
nosiswascariedouttoidentifyandcharacterizethe
specificviralpathogenresponsible.
1　MaterialsandMethods
1.1　Samplecolection
LeafsampleswerecolectedfromM.coroman-
delianumplantsshowingleafcurlandveinbanding
symptomsindifferentlocationswithinFujianPro-
vinceofChinainMarch2005.Thesampleswere
transportedtotheviruslaboratoryoftheBiotechno-
logyInstitute, ZhejiangUniversityandlabeledFJ1,
FJ2, FJ3, FJ4 , FJ5 andFJ6.
1.2　ViralDNAcloningandsequencing
Totalnucleicacidswereextractedfromsymp-
tomaticleavesusingtheCTABmethodasdescribed
byXie, etal.[ 13] andthedegenerateprimerpair
PA/PBwasusedtoamplifypartial, approximately
500bpviralDNAfragmentsasdescribedbyZhou,
etal.[ 14] .SequencesofprimersusedforPCRare
showninTable1.ProductsofPCRamplification
wereclonedintopMD18-Tvector(TaKaRaBiotech-
nology[ Dalian] Co.Ltd.), andsequencedusing
theautomatedmodel3 700 DNAsequencingsystem
(Perkin-ElemerAppliedBiosystemsInc., Foster
City, CA, USA).Basedontheanalysisofthepar-
tialviralDNAsequences, thepreviouslydesigned
primerpairGD6BamHIF/GD6BamHIR[ 12] wasused
toamplifythecompleteviralDNAfromrandomly
selectedFJ3 isolate.ThecompleteviralDNAfrag-
ment, approximately2.7 kblongwasclonedinto
pMD18-T vectorandsequenced.Atemptswere
madetoamplifyeithertheful(1.3 kb)orpartial
(750 bp)fragmentsoftheputativesateliteDNAβ
byPCRfromalthesamplesusingpreviouslyde-
signedprimersβ 01/β 02[ 15] andβ 01/BetaR1[ 16] .
1.3　Sequenceanalysis
Sequencedatawereassembledandanalyzed
withtheaidoftheDNAStarsoftwareversion6.0
(DNAStarInc., Madison, WI, USA)andDNA-
MANversion5.2.2 programs(LynnonBiosoft,
Quebec, Canada).Multiplesequencealignments
wereperformedwithCLUSTALVmultiplesequence
alignmentprogram(MegAlign)inDNAStar, and
similarity searcheswereperformedusing NCBI
BLASTprogram(htp://www.ncbi.nlm.nih.gov/
BLAST).Phylogenetictreesweregeneratedusing
theNeighbor-joiningmethodavailableinDNAMAN.
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植物病理学报 38卷
Table1　NucleotidesequencesofprimersusedforPCR
Primer Sequence5′-3′
PA TAATATTACCKGWKGVCCSC
PB TGGACYTTRCAWGGBCCTTCACA
GD6BamHI/F GGATCCACTTGTGAACGAGTTTCC
GD6BamHI/R GGATCCCACATTTTTGAAGCAAAAC
β 01 GGTACCACTACGCTACGCAGCAGCC
β 02 GGTACCTACCCTCCCAGGGGTACAC
BetaR1 GACGATCARATACAVCAVCA
R=A/G, W=A/T, B=G/C/T, Y=C/T, V=A/C/G.
Thefolowingpreviouslyreportedbegomoviruses
wereusedfornucleotidesequencecomparisonwith
FJ3 andphylogeneticanalysis:Ageratum leafcurl
virus(ALCuV, AJ851005), Ageratumyelowvein
virus(AYVV, X74516), Euphorbialeafcurlvirus
(EuLCV, AJ558121), Malvastrum leafcurlvirus
(MLCV, AJ971263), Malvastrumyelowveinvirus
(MYVV, AJ457824), MalvastrumyelowveinYun-
nanvirus(MYVYNV, AJ786711), Papayaleafcurl
Chinavirus(PaLCuCNV, AY650283), Papayaleaf
curlGuangdongvirus(PaLCuGdV, AJ558122),
Soybeancrinkleleafvirus(SbCLV, AB050781),
TomatoleafcurlGuangdongvirus(ToLCGdV,
AY602166), Tobacco leafcurlYunnan virus
(TbLCYNV, AJ566744), TomatoleafcurlPhilip-
pinesvirus(ToLCPV, AB050597), Malvastrum
leaf curl Guangdong virus (MLCuGdV,
AM236779), Starchytarphetaleafcurlvirus(StaL-
CV, AJ810156)andTomatoleafcurlVietnamvirus
(ToLCVV, AF264063).
2　Results
PartialviralDNAfragments(500 bp)were
amplifiedfrom sixMalvastrum leafsamplesfrom
FujianbyPCRwiththedegenerateprimerpairPA/
PB and then sequenced (Accession numbers
AM503099-104).Comparisonoftheirnucleotide
sequencesandthoseofotherreportedbegomoviruses
showthattheyshare94% -99% sequenceidentity
amongthem and, 90% -93% sequenceidentity
withthepreviouslyreportedisolateGD6ofMLCuG-
dV(AM236779), indicatingthatthesamplesmight
beinfectedbyisolatesofMLCuGdV.
ThecompletegenomicDNA fragmentofthe
randomlyselectedisolateFJ3wasamplifiedbyPCR
usingprimerpairGD6BamHI/FandGD6BamHI/
Randthensequenced.FJ3DNAwas2 765 nucleo-
tides(nt)longandhadatypicalgenomicorganiza-
tionofotherbegomoviruseswithtwoORFs[ AV2
andAV1 (CP)] inthevirion-senseDNAandfour
ORFs[ AC1(Rep)toAC4] inthecomplementary-
senseDNA, separatedbyanintergenicregion(IR)
(AM503104).TheIRcomprised252 ntwithapu-
tativestem-loopstructurecontainingtheconserved
nonanucleotidesequenceTAATATTAConwhich
mapsthenickingsitefortheinitiationofrolingcir-
clereplication;aTATAmotif(at2 713-2 716nt)
anditeratedGGTACCmotifsatposition2 670 -
2 675and2 703-2 708ntonthe5′sideoftheTA-
TAmotif.Together, thesefeaturesdefinetheorigin
ofDNAreplicationforalgeminiviruses.
SequencecomparisonsshowedthatFJ3 DNA
hadthehighestnucleotidesequenceidentity(93%)
withMLCuGdV.Infurtheranalysisofindividual
encodedproteins, FJ3 hadthehighestaminoacidse-
quenceidentity(>93%)withMLCuGdVforAV1,
AV2, AC1, AC3andAC4, andPaLCuGdVforAC2
(92.6%).ExcludingMLCuGdV, theindividual
encodedproteinsofFJ3 sharedthehighestsequence
homologywiththoseofPaLCuGdVandPaLCuCNV
(Table2), previouslyisolatedinSouthChina.The
AC4ORFofFJ3hadthehighesthomology(82.3%)
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　3期 RoyB.Mugira, etal.:MolecularCharacterizationofMalvastrumleafcurlGuangdongvirusIsolatedfromFujian, China
Table2　PercentagesofnucleotideandaminoacidsequenceidentitiesbetweentheFJ3
andothercloselyrelatedbegomoviruses
Virus Nucleotide AminoacidsDNA IR AV1 AV2 AC1 AC2 AC3 AC4
MLCuGdV 92.9 91.7 98.1 98.3 93.4 89.6 94.0 95.8
PaLCGdV 84.4 40.5 94.9 94.8 87.0 92.6 91.0 71.9
PaLCuCNV 82.9 54.8 96.5 94.8 87.3 91.1 90.3 70.8
TbLCYNV 71.3 40.9 79.7 75.9 86.2 70.4 71.6 82.3
ToLCPV 71.6 37.7 82.5 46.6 84.2 69.6 68.4 82.3
ToLCGdV 72.6 38.5 93.8 78.4 78.9 69.6 72.4 42.3
StaLCV 73.9 33.7 90.3 75.9 84.7 65.7 74.6 79.2
ToLCVV 70.7 38.5 94.6 77.6 78.7 65.2 60.4 37.1
SbCLV 73.0 38.9 89.9 79.3 80.1 74.4 72.4 76.0
AYVV 74.3 35.7 89.1 77.6 86.9 65.9 70.1 75.3
ALCuV 74.3 37.3 94.9 77.6 82.8 67.2 75.4 70.1
EuLCV 68.0 36.9 80.5 67.5 82.2 60.4 66.4 71.9
MLCV 69.1 38.6 67.5 74.1 79.3 72.6 73.4 71.9
MYVV 66.8 38.9 80.1 65.2 77.6 61.5 62.7 43.3
MYVYNV 65.3 42.9 79.7 64.3 77.4 60.0 61.9 44.3
withthoseofTbLCYNVandToLCPV.Phylogene-
ticanalysisshowedthatisolateFJ3 hadlowafinity
tootherMalvastrum-infectingbegomovirusesexcept
MLCuGdV, buthadhighaffinitytoandclustered
togetherwithpreviouslyreportedpapaya-infecting
begomovirusesisolatedinSouthChina(Fig.1).
ThebegomovirussateliteDNAβ moleculewas
notdetectedinanyofthesamplesbyPCRusing
primersspecificforDNAβ molecules.
3　Discussion
ThegenomicDNAofvirusisolateFJ3 shares
93% nucleotidesequenceidentitywiththatofML-
CuGdV.Accordingtotherevisedcriteriaforspecies
demarcationandnamingofgeminiviruses[ 4] , virus
isolatessharingmorethat89% nucleotidesequence
identityareconsideredtobeisolatesofthesame
virusspecies.ThusvirusisolateFJ3 isanisolateof
MLCuGdV.
MLCuGdV-[ FJ3 ] sharesthehighestamino
acidsequenceidentitywithPaLCuGdV andPaL-
CuCNVfortheAV1, AV2, AC1, AC2 andAC3
ORFs.TheAC4 ofMLCuGdV-[ FJ3 ] sharesthe
highestaminoacidsequenceidentitywiththoseof
ToLCPVandTbLCYNV(Table2).Althoughthe
percentagesofidentitybetweentheAC4 ORFof
thesethreevirusesisnotsufficientlyhigh(82.3%)
toconstitutearecombinationevent, itindicatesthat
theymayshareacommonancestry.MLCuGdV-
[ FJ3] genomeisthereforeachimericmoleculethat
mayhavearisenbyinterspecificrecombinationbe-
tweenPaLCuGdVorPaLCuCNVasmajorparents
andotherunidentifiedancestorsasminorparents.
PhylogeneticanalysisshowthatMLCuGdViso-
lateshavehighaffinitytoandclusterstogetherwith
papaya-infectingbegomovirusesisolatedinthesame
regionofSouthChina, whereastheyhavelowafi-
nitytootherMalvastrum-infectingbegomoviruses
(Fig.1).Thisindicatesthatphylogeneticafinities
amongbegomovirusesdependlargelyonashared
geographicalproximitythanacommonhostrange.
Acknowledgement:Thisworkwassupportedbythe
NationalNaturalScience Foundation ofChina
(GrantNo30471137).
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植物病理学报 38卷
Fig.1　PhylogenetictreebetweenisolateFJ3
andotherpreviouslyreportedbego-
moviruses
Thetreewasgeneratedusingtheneighbor-joiningmethod
availableinDNAMAN, bootstrapped1 000times.Figuresat
thenodesshowthebootstrappingvalue(>90%)supporting
thebranchatthatparticularnode.
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