RRKL．RGLIAEKN〔APIWRLAWH5AGTFD〔QSRTGGPFGTM
1：一打KNYPA〉SEEYqKA工EK〔
1：NrTKSYPTVTEDYQNAIEK〔 ∃＝
1：叶「ドNYPTYSEDYKKA〉EK〔
β0−APX」
β0−Aアズ2 A．ぬaムa皿aAPg 月0−APXJ β0−Aアズ2 A．地aムa刀aAPX
β0−Aアズl β0−AP方2
61：RFDDELAHGANNGLHIALRLLEPIREQFPTIS仙DFHQしAGVV」叩EVTGGPEIPFtlPGRE 61：RFDAEQGHGANSGIHIALRしLEPIREQFPTISFADFHqLAGV叩VEyTGGPEIPFHRGRE
61：RFDAEQAHG叫SGIHIALRLLDPIREQFPTISFADFHQしAGVVAVEVTGGPDIPF＝PGRE
1Zl：8 1Zl：
A．地a五a8aAP方1Zl：DK
：
：
：
β0，APXl 181:
月0−APX2 181:
A．地a左丘皿aAPX181 β0＿APXI Z41：
β0，AP方2 Z41：
A．地aムadaAP方Z41：
Fig・10 ComparisonofaminoacidsequencesofBO−Aml（accessionno．
ABO78599），BO−APX2（accession no．ABO78600）and A．thaliana APX
（accessionnumberX59600）■Theidenticalresiduesbetweenthreealignmentare
representedbytheshadedarea．
β0−A夕方
DIG−1abeledBOrAPX1 8
DIG−1abeledBOrAP方2
舞Fig．11Dot−blotanalysISShowlngthespecificityofthegene−SPeCincprobes．
RNAs for BO−AEXl（1）and BO−APX2（2）wereimmobilized onto a
membraneandhybridizedtoDIG−1abeledBO−APXlandBO−AfX2probes．
80耶血加 01ヱZ■36■さ8012Zヰ3石嶋6001Z Zヰ泌4さ占00122ヰ3￠j鳩60 1叙h押r 21d如r
姐叩tわn Ⅲon匁0【⊂Ⅶr血
M2 rRNA
Ho耶血血■r咄 O IZ2■詭■8石0122ヰ3る4膿6￠01ヱ：か136■さ6001Z 王43も48麒）
1或如r 加d如 鮎al匹I爪ねn
馳tb dr⊂u血抗g．12 Cl岬hmRNÅ血匹1eYelsd■bmh血hⅣ鰐t．
Nb血udanalys塔職匹血m妃d血g群肝づ匹癒cp血負汀
β0〟ⅩJむdβ（）」LP貫2．（A）stemtissues
襲御 ㈱抄‥r 竃芸二 三＝：ご二二鳥 二＝ニ →脚詳 0 12 12 0 12
Hoursafterharvest
rBO−APXl lstlayer 2ndlayer Basalportion orcurds
（B）norets
∵二＝完←ニニニこ＝＝ニ ニ触 が一柳 丁二言慧禦
Hours after harvest O 12 24 36
r王】0−APXl Rorets
Fig．13 Changesinproteinlevelsofbroccoliafterharvest．Fivemicrograms
Ofproteinextractedfromvariousparts ofbroccoliwereloadedineachlane
exceptthelaneloadingrecombinantBO−APXl・Immunoblotswereperfbrmed
uslngPOlyclonalantibodiesforrecombinantBO−APXl・
伊鵬 劇画趣睡
軸測温．宣
抗g．14 SDS−PAGE戚cr■血鉱山騨虚血叫愕d轡
1（B）血相2（印W鮮叩頭dba12・5％如鵬○騨脚絆l・
Tkp血iI蛤Ⅴ醒庵血吋C師勝i￠血溺眈・
︵lJlb∈−1・已召こ○∈羊こ︻ゝ 1 2 3 4
〇． 0 0 0 0 0
10
Ascorbate（mM−1）
20
︵lJ一也∈・l・已召こ○己ヱ︶l・＞
︶ B ︵ 0
0．5
Hydrogenperoxide（FLM−1）
Fig．15Lineweaver−BurkdoublereciprocalplotsofBO−APXl（○）andBO−APX 2（△）．TheAPXactivitywasmeasuredwithvaryingAAconcentrationsat4（刃根M
H202（A）orvaryingttO2COnCentrationsat500LLMAA（B）・BarsrepresentS・D・S
WhenlargerthansymboIs・
CIIAPTER Ⅳ
AscorbateMetabolisminHarvestedBroccoli
l．Introduction
Inthepreviouschapter，IobservedarapiddegradationofAAoccumnginbroccoli floretsafterharveStbutnotinthestemtissue，inwhichIstudiedthechangeSinboth
activitiesandgeneexpressionsofAPXafterharveSt・ConsideringthechangesinAA
POOIsize andtheimportance ofscavenglngAOS，itis necessary tounderstandthe
regulationofotherenzymesinadditiontoAPX・However，Verylittleworkhasbeen doneonsimultaneousanalysisofAA−relatedenzymesindistinctcellularcompartments．
Inthisch叩ter，IwilldescribethechangeSingeneexpressionofenzymeSinvoIvedin AAsynthesisandbreakdowninharveStedbroccoli（BTuSSicaoleraceaL・Var・italica）・
Inaddition，IwilldescribetheeffectsofplanthormOneS（ethylene，6−benzylaminopurin
（BA），methyljasmonate（MJ）andabscisicacid（ABA））orethyleneactioninhibitor，2，5−
norbornadiene（NBD），OnthesenescenceinharveStedbroccoliflorets．
2．MaterialsandMethods
2．J．P由〝J∽αJerねね
Whole，intact broccoli（Brassica oleracea L．var．italica）plants with roots were
harveStedinafarmandtransportedtothelaboratorywherethestemendswerecut・
HarveSted broccoliheads wereincubated at20℃ under a humidified condition
（RH＞100）．AtO，2，4，6，12，24，48and72hafterharveSt，SamPlesofstemtissue（2mm thick）forthefirstandsecondlayers，andbasalportion（ca・2mmthick）ofcurds，and
floretswereexcisedfrombroccoli（Fig．4）．Theouterportion（greenpart）ofthestem tissue was excised and used for RNA extraction．The excised stem tissue andflorets
wereimmediatelyfrozeninliquidnitrogenandstoredat−80℃untilreadyforuse．
2．2．升eα加e旭W油〆α乃Jゐor〝10乃e∫
Broccoliobtained from alocalmarket was used for treatments with chemical
COmPOunds・ForNBDorethylenetreatments，broccoliheadswereexposedto4，000LL l・l−1NBDor50LLl・1−1ethylenefor12handthenfloretswereremovedandfrozenin liquidN2．ForBAandMJtreatments，SamPles were dissectedintocurds（segments averaging17．1g）whichwereirrmersedwithgentlestimingforlhinasolutionofO・22
mMBAorlmMMJcontainlngl％ethanolandO・1％Tween20・Controlbroccoli Curdsweretreatedinthesamewaywithl％ethanolandO・1％Tween20・Thesecurds
wereremovedfromthesolutionandheldat20℃underhighhumidity．After23h，
florets were separated from the curds and used forthe subsequent analyses・For
treatmentwithABA，floretsexcisedfromaharveStedbroccoliheadwerefloatedinl
mMABAorwaterwithgentleagltationfor24h・Afterthetreatment，thefloretswere frozeninliquidN2andstoredat−80℃untilreadyforuse・
2．j．A∫∫叩げe卸ね乃ピタrO血cJわ乃α乃加∫COr∂αreCO乃加r
Ethyleneproductionwasmeasuredasdescribedinsection2．2．ofCHAPTER Ⅱ．
AAcontentinreducedandoxidizedformwasassayedasdescribedinsection2．3．of CHAFrER Ⅱ．
2・4・R〃Aα打αCfわ乃，属rPC凡cわ花王乃g，α乃d∫e冒以e柁C王乃g
TotalRNA wasisolated according to the method as describedin section2．5．of
CHAFrERⅡ．TheprimersusedfbrRT−PCRweredesignedbythecommonsequences
inthegenesofvariousplantS・ForBO−SAPXandBO−tbAPXthepnmersweredesigned
bythecommonsequencesbasedonArabidqpsisthalianasAPX（accessionno．X98925）
andtbAPX（accessionno．X98926），SpinaciaoleraceachloroplasticAPX（accessionno．
ABOO2467），NicotiafW tabacum sAPX（accession no．ABO22274）and Cucurbita cv．
KurokawaAmakurisAPX（accession no．D88420）．BO−GL，DHwasisolated withthe
Primersdesignedonthebasisofthecommonseq11enCeSinsweetpotato（accessionno・
ABO17357）andcauliflower（accessionno・Z97060）GLDH（Table4）・Theprimersfor
BO−MDARIweredesignedbythecorrmonsequencesinA・thaliana（accessionno・
D84417），Brassicajuncea（accession no．AFlO9695）and S・Oleracea（accession no・
ABO63289）MDAR，andforBO−MDAR2weredesignedonthebasisofA・thaliana
（accession no．AF360197）and OT7Za Sativus（accession no．D85764）and tomato
（accessionno．L41345）MDARnucleotidesequences．DHARcDNAwasisolatedusing the primers designed by the common sequencesin A・thaliana（accession no・
A円01597），S．oleracea（accession no．AF195783）and O．sativus（accession no．
ABO37970）DHAR・BO−AOwasisolatedwiththeprimersdesignedby thecommon
SequenCeSin A・thaliana（accession no・ABOO4798），B．juncea（accession no．
AF206721）andCucumismelo（accessionno・AF233593）AO．TheprimersforGRwere designed on the basis ofthe common sequencesin A．thaliana（accession no．
AYO40029），Brassica rqpa（accession no・AFOO8441），0・Sativus（accession no．
D85751），Pisum sativum（accession no．X98274）and S．oIwacea（accession no．
D37870）GRandusedforRT−PCR．ThePCRprocedurestartedwithlOminat95℃
andwascarriedoutfor35cyclesof30sat95℃，30sat52℃and30sat72℃，and endedwith10minat72℃withGeneAmpPCRSystem9600（PerkinElmer）．The
amplified cDNAwas clonedwithTACloningKit（Invitrogen）・The sequences were
deteminedusingTaqDyePrimerCycleSequenclngkitandTaqDyeTeminatorCycle
Sequencingkit（PerkinElmer）with373SDNASequencingSystem（PerkinElmer）．
2．5．A叩PJ研cαJわ乃翻払Je乃gJんc∂ルA妙見ACg−PC尺
Amplificationoffu1ト1engthcDNAbyRACE−PCRwasperformedasdescribedin
section23．ofCHAFrERⅢ．EachprimerusedforRACE−PCRwasdesignedbythe nucleotidesequencesofthecDNAfragmentsforBO−SAPX，BO−tbAm，BO−MDARl，
BO−MM2，BO−DLuRandBO−GRobtainedfromRT−PCRwiththepnmersdesigned
bythecommonsequencesinthegenesreportedinvariousplantS・
2．6．ⅣorJんer柁あわgαJ∽桓∫f∫
Northernblotanalysiswasperformedasdescribedinsection2．7．ofCHAFrERII・
TotalRNA（10LLgPerlane）wasseparatedinformaldehyde−agarOSegelsandRNAwas
eachlane．ProbeswerepreparedusingDIGRNAlabelingkit（Roche）andthedetection
afterblottingwasperformedbychemi1uminescencewithCDP−Star（Roche）exposingto Hyper員1mECL（AmershamPharmaciaBiotech）・
3．Results
j．ノ．C厄乃ge∫加α∫C（フrねgeco乃Je乃J
AAcontentwasmeasuredinfourpartsofbroccoli：thenrstlayerofthecutstem（0−2 mm，CutSurfacelayer），thesecondlayer（2−4mm，adjacenttothefirstlayer），thebasal portionofcurdsandflorets（Fig・4）・
TheamountoftotalAAinfloretswasthehighestofallportionsmeasuredatOh（Fig・
16）．Itstayedatrelativelythehighleveluntilabout12h，fo1lowedbyarapiddeclineto alowlevelduringsenescence・Inthestemtissue，thelevelsofAAremainedalmost unchangedorincreasedslightlyastimeprogressed・DHAcontentexhibitedlessthan
10％oftotalAAinallportionsthroughoutthepostharveStPeriod（datanotshown）・
TheseresultsconsistedwiththoseobtainedinCHAFrER Ⅱ．
j．2．ム0ぬfわ乃α乃dねね乃古漬Cαfわ和げ血c∂仙i乃VOgVed加α∫COrあα′e椚eぬあ0地肌
TodeterminethechangesinthelevelsoftranSCnptSCOrreSPOndingtoAPX，AO，
GLDH，MDAR，DKARandGR，eaChcDNAwasisolatedfrombroccolibyRT−PCR
withthepnmersshowninTable4andusedasatemplateforprdbesinnorthernblot
analysis・Additionally，nuCleotidesequencesincludingcompletecodingreglOnforBO−
SAPX（accessionno．AB125635），BO−tbAPX（accessionno．AB125634），BO一山旺旭丘
（accession no．AB125636），BO−MZW2（accession no．AB125637），BO−
（accessionno．AB125638）andBO−GR（accessionno．AB125639）weredeterminedand theaminoacidsequenceswerepredicted，reSPeCtively・IisolatedtwochloroplasticAPX CDNAs by RT−PCR uslng the pnmers designed by the common sequencesin
ChloroplasticAPXsreportedinvariousplantS・Thenucleotidesequences，BO−SALTand BO−tbAPX，Showedahighsimi1aritytothoseofstromalandthylakoidboundAPXfrom Arabidopsis（accessionno・X98925，X98926），reSPeCtively・BO−SAPXandBO−tbjm
CDNAssharedahighnucleotideidentityinthecodingreglOnSSOthatprobesweremade
using3 −untranSlatedregionsinwhichtheysharedalowernucleotideide皿tityofless
than70％（Fig．17）．ThepredictedaminoacidsequencesofBO−tbAtWandBO−SAPXare ShowninFig．18．ThenucleotidesequenceofBO−GLDHobtainedwasidenticalwith thatofcauliflower（accessionno．Z97060）．Twodistinctcloneshavingahighnucleotide
identitytothesequencesencodingMDARwereisolatedfrombroccoli（Fig・19）・The
PartialcDNAusedforprobesofBO−MMlandBO−MM2innorthemblotanalysis were328and411bplongrespectivelyandsharedlessthan60％nucleotideidentity・
ThenucleotidesequenceofBO一朗DARIshowedmorethan90％nucleotideidentity
withthose of A．thaliana（accession no．D84417）and B．juncea（accession no．
AFlO9695）MDAR．BO−MDAR2cDNAgavevaluesof89・8％and87・8％identityto thoseofA．thaliana（accessionno．AF360197）andB・rqPa（accessionno・AYO39786）
MDARrespectively・PredictedaminoacidsequencesforBO−MDARsareshowninFig・
20．BO−DEL4RcDNAobtainedshowedmorethan80％identitytothoseofA．thaliana
（accessionno．AYO85616）andB．juncea（accessionno・AF536329）（Fig・21）・BO−AO
cDNAshowedmorethan80％nucleotideidentitywiththecorrespondingreglOnSOfA・
thaliana（accessionno．ABOO4798）andB・juncea（accessionno・AF206721）AO（Fig・
22）．BO−GR obtained showed more than80％nucleotideidentitywith A．thaliana
（accession no，U37697）and B．rqpa（AFOO8441）GR（Fig・23）・According tothe simi1aritytothesequencesfromotherplants，putativelocalizationoftheirencoding
protein was estimated・BO−SAL％BO−tbAPX，BO−MDARland BO−DEL4R were
SuPPOSedtoencodetheproteinsinchloroplastsandBO−MlhR2andBO−GRshoweda highsimi1aritytothenucleotidesequencesoftheproteinsinthecytosol・Thepredicted
amino acid sequences for BO−5月PX，BO−tbAPX，BO−MDARland BO−DEL4R were confirmedtoincludeatranSltPePtideinananalysiswithChloroPVer・1・1・
j．j．Cゐα乃ge∫f乃∽尺八仏ねveね
ChangesinthetranscriptlevelsforBO−ALWl（accessionno・ABO78599）andBO−
AET2（accessionno．ABO78600）weredetectedusingprobesdescribedinCHAFrER
Ⅲ．ExpressionsofbothgeneswereinducedmarkedlyjustafterharveStinallportions examined（Fig．24）．The mRNAlevels ofboth BO−SAPXand BO−tbAPXinflorets remainedhighunti16hafterharvestandthendiminishedrapidly（Fig・24）・Transcripts
ofbothchloroplasticAPXsincreasedgraduallyinthestemtissueexceptthatBO−SMX
inthebasalportionofcurdsshowedthesimi1arpatt占mtothatinflorets・TranSCript
abundanceofBO−GLDHinfloretsandthebasalportionofcurdsstayedatahighlevel
unti16hafterharvestandthendecreasedrapidly・Inthelstand2ndlayerofthestem，
mRNAlevelsofBO−GLDHwereinducedandmaintainedatacomparativelyhigher
levelovertheexperimentalperiod・ThemRNAlevelsofbothBO−MDARlandBO−
MDAR2inthelstand2ndlayerwereinducedgraduallyafterharveStWithapeakat12
hand24h，reSPeCtively・Innorets，BO−MDARIwashighlyexpressedunti16handthen declinedrapidlyat12h，WhileBO−MM2tranSCrlPtSdetectedatalowleveloverthe first24hincreasedmarkedlyat48and72h．BO−DEL4RmRNAlevelinnoretsshowed arapidincrease until12hafterharveStbutitbecame almostundetectable at24h．
TranSCnptSOfBO−DEL4Rinthethreeportionsofthestemtissueexhibitedalmostthe
SamePatternShowingagradualincreaseduringtheexperimentalperiod・
Accordingtotheputativelocalizationoftheencodingproteins，thegenesexamined
WereClassifiedintothreegroupsofcytosol，mitochondrionandchloroplast．BO−APX and BO−APX2were described as cytosolic APXin CHAFrERⅢ．BO−AO was Classifiedinthecytosolicgroupbecause ofAOlocalizationproposedinthecytosol
besides apoplast・Whenthe mRNAexpression patternSinflorets after harveStWere
dividedintotwo groupsofcytosolandothers（chloroplastandmitochondrion），their PatternSWerequitesimilarineachgroup（Fig・25A，B）・Geneexpressionsofcytosolic
enzymes（BO−AO，BO−APXl，BO−jm2，BO−MDAR2andBO−GR）werestimulated
activelywithin4hafterharveStinflorets・Afteratransientdecreasetoalmosttheinitial
level，theyincreased significantly after12h（Fig・25A）・Changesin mRNA of Chloroplastic genes（BO−SjU％BO−tbAPX，BO−MDARl，andBO−DLL4R）and thatof mitochondrialgene（BO−GLDH）inflorets after harveSt tended to exhibit a simi1ar
PatternOfthemRNAlevelincreaslngWithin6h，andthendecreaslngraPidlyat6to12
hafterharveSt（Fig．25B）・Inthelaterperiodofthestorage，thisgroupofgenesshowed thelowerexpressioninthetranSCriptlevels・
j．4．聯CJげ打ぞα加e乃J∫W助〃βか，eJ如才e乃e，βA，九打α乃dAβAo乃α∫COrあαJe椚efαあoJね肌
ToinvestigatethefactorsthatmayhaveplayedaroleinaltenngtheAApooIsizeand thetranscnptlevelsofthegenesrelatedtoAAmetabolismafterharvest，broccolinorets
WeretreatedwithNBD，ethylene，BA，MJandABA・TheNBDtreatmentenhancedthe levels ofmost mRNAsdetectedwith the except10n forBO−GR，While the ethylene treatmenthadnoeffectonAAcontentandgeneexpressioninvoIvedinAAmetabolism
（Fig．26）．AAdegradationwassuppressedslightlybytheBAtreatmentandevidently acceleratedbybothMJandABA（Fig．26）・ThemRNAlevelsofBO−GエD耳BO−SAPX，
BO−tbAPX，andBO−MLuRlincreasedbytheapplicationofBA，WhereasthoseofBO−
GLD〃，BO−SAEtX，BO−tbAPX，BO一朗かA尺1andBO−DEL4Rdecreasedbythetreatments
WithMJandABAincorrelationwiththechangeSinAAconcentrationinnorets．
4．Discussion
TounderstandtheeffectofharveStlngOnAAmetabolisminbroccoliplants，SeVeral
isoenzymesinvoIvedinAAmetabolism（APX，MDAR，DHAR，GR，AOandGLDH）
Wereinvestigated．Inthischapter，Iconcentratedonthegeneexpressionbecauseitwas difficulttoassaytheactivityofeachisoenzymeseparately，eXCePtforAPXisoenzymes・
Thepresentresultshelptoexpectthechangesintheactivityofeachisoenzyme，Which
maybetheoppositepattemincytosolicandchloroplasticonesunderstressconditions・
ThenndingledbygrouplngOfthegenesindicatesthatthesegenesinchloroplastsand
thecytosolactinaco−regulatedbutdistinctmamerunderstressconditions（Fig・25and 26）．ItseemsthatthesechangeSCOuldeffecttheAOSandAAlevelsanditsredoxstatus
in each compartment.
4・J・Cゐα乃ge∫f乃りわ∫OJ〜cge乃ee坪re∫∫わ乃∫reJαJedわα∫COrあαJe肌erαあ0揖肌〟托滋r∫打e∫∫
co〝dgJわ旧
IncreasesafterharveStlngandthetreatmentwithMJorABAanddecreasesafterthe BA treatment were observedin the expressions of cytosolic genes related to AA
metabolismin broccoliflorets・ItseemslikelythatAAmetabolisminthe cytosolis
regulatedbythesamemannerinthetranSCriptionallevelandthattheseincreasesafter harvestingandthetreatmentwithMJorABAmaybearesponsetooxidativedamages
CauSed by treated stresses・General1y，AOS−SCaVenglng SyStemin the cytosolis COnSideredto play arole astheprotection ofimportantcellularcompartmentsffom
OXidativestresscausedbyenvironmentalchanges・Itiswe11knownthattheformationof
AOSisacceleratedunderstressconditionssuchaschilling，hightemperature，intensive lightingandwounding，Whichareassociatedwiththeprocessofsenescence（Okudaet al．1991，Foyer et al．1994，1997，Dat et al・1998，Watanabe and Sakai1998）・
HarveStingcouldalsocausetheincreaseofAOSinthecell，relatingtotheactivationof
AOS−SCaVenglng SyStemin the cytosol・Many have reportedthat cytosolicAPXis
induced by environmentalchanges（Mittler andZilinskas1992，Morita etal・1999，
Yoshimuraetal．2000）．Understressconditions，theplantCellmaytendtoscavenge AOSandprotectcellularcompartmentsbyactivatingAA−regeneratingsystemsbesides
APXinthecytosol・
4．2．C如乃ge∫加c肋rqpね∫庇ge乃ge甲re∫∫わ乃∫reJαJedわα∫COrあαJe椚eJαわ揖刑〟乃鹿r
∫打e∫∫CO乃d〜rわ乃∫
Down regulation of chloroplastic genes observed after harveSt and MJor ABA
treatment couldlead to a breakdown of AOS−SCaVenglng SyStemin chloroplasts，
resultingintheinhibitionofCO2fixationinCalvincycle（Kaiser1976）・Theseresults indicatethatAPX，MDARandDHARinchloroplastscanberegulatedinmRNAlevel
andthatnotonlyAPXsbutalsoAA−regeneratingsystemincludingMDARandDHAR
COuldbeinactivatedinchloroplastsunderthestressconditions・Asaconsequenceof
these down regulation，Chloroplasts may startto disintegrate at earlier stage before OCCumngOfchlorophylldegradationduringsenescencebecausedegreenlngOfsepals
Observedvisual1y at48h afterharveStinthis experiment・Ithasbeenreportedthat
Chloroplastic APXisoenzymes areinactivated by excess generation of H202and
depletionofAA（Shikanaietal・1998，Manoetal・2001）・APXmaybeinactivatedin chloroplastsbeforethesuppressionoftheirtranscnpt10ninharveStedbroccoliflorets
becauseoftheexcessAOScausedbyharveStlngOrAAredistributionfromchloroplasts tothecytosoIwhichmayhaveahighdemandofAAtoprotectcellsfromoxidative
damageduringsenescence・IthasbeenreportedthattbAPXactivitydecreasedrapidly
underphoto−OXidativestress（ManOetal・2001）・Yabutaetal・（2002）havedemonstrated thattbAPXisalimitingfactorofantioxidativesystemsunderphoto−OXidativestress
uslngtranSgenictabaccoplantsthatexpressedtbAPXcDNA・Thereforeitisthoughtthat
theloss oftbAPX activityis moreimportantfactorin the postharveSt SeneSCenCe・
However，itseems thatthe disruPtionofstromalAOS−SCaVenglng SyStemincluding sAPX，MDARandDHARisalsooneoftheimportantfactorstoalterAOS andAA
levelsunderthestressconditionsandthatthedownregulationofMDARandDHARin ChloroplastsleadstoAAdegradationdirectly・
4・j・Cゐα乃ge∫f乃α∫COrあαJeあわ叩乃伽∫∫∫〟耽ねr∫打e∫∫CO乃劫わ乃∫
BO−GLDH exhibited a similar expression pattem to the chloroplastic genesin harveSted broccoliflorets，Which showedthe rapid decrease at the early stage of
postharveSt，indicatlngadecreaseoftherateofAAbiosynthesis．AdecreaseinAA biosynthesismaybeexpectedbecauseofsugardenciencycausedbyaninactivationof
Photosynthesisinfloretsand／orthestopofitssupplytofloretsfromleavesafterharveSt．
Therapiddeclineofsucrosehasbeenobservedinbroccoliflorets（seeCHAFrER V）
andasparagusspears（Daviesetal・1996）justafterharveSt・Thesuppressedexpression
OfBO−GLDHafterharveStmayberelatedtothelossofsugars．
4・4・Cゐα乃ge∫〜乃ge乃eeスpre∫∫わ乃∫reJαJedわα∫COrあαね椚eねあ0鮎mf乃血∫ねmめ∫〃eげ ゐαrVe∫fedあroccoJg
Inthebasalportionofcurdswhichisapartofthestemtissuefarfromthecutsurface，
AA levels remained unchanged over the experimental period despite the expression
PattemSOfmanygenesshowedahighsimilaritytothoseofflorets・Incomparisonof
thestemparts withflorets，a greatdifferencemaybeintheamountOfchloroplasts，
Sincefloretsarerichinchlorophyllintheirsepals・IthasbeenobservedthatpostharveSt
ChangeSinbroccolifloretsshowmanysimilaritiestotheprocessesofdevelopmenta11eafsenescence（Pageetal．2001）．AssumingthatamqjorsourceofAAdegradationin
noretsispnmari1ycausedinchloroplasts，itcouldbesupposedthatthelossofAAafter harveStisinfluencedbytheamountofchloroplastsandtheregulationofchloroplastic
geneexpressionsinvoIvedwithAOS−SCaVenglngSyStem・TheconstitutivelevelsofAA
Of chloroplasts or different response to floretsin BO−tbAPX and BO−DfL4R gene expressions・
4．5．7為e∈辟crげゐor〝10乃e∫埋Pgね∫0乃α∫C（フ「あαJe椚eねあoJf∫椚α乃dα∫COrあα〜eJeveJ
HarvestlngdisruptS Water，energy，nutrientandhormOneSuPPlies andalsocauses
WOundingstress・HeadsofbroccoliareharveStedattheimmaturestageandshowthe rapidyellowlngduringstorage・Ithasbeenobservedthattherateofethyleneproduction
infloretsincreasedtoamaximumthendeclininglnaPatternalmostparal1elingthatof
ACCoxidaseactivity（Katoetal・2002）・Ethyleneproductionregulatestheyellowingof Sepals after harveSt based on chlorophy11degradation・The activation of APX by ethylene has also been documented（Mehlhorn1990，Ievinsh et al・1995）・In this experiment，therateofethyleneproductionbegantOincreaseat12hafterharvestand
degreeming of sepals became obvious after48hinflorets（data not shown）・To
understand the relation betweenAA catabolismand ethylene production，NBD，the inhibitorofethyleneaction，andethyleneweretreatedinharvestedbroccoli．However，I COuldnotfindconsiderableefftctsonAAmetabolismbyethylenetreatment，indicating
that AA degradationin harveSted broccoliflorets was not regulated by ethylene・
Increasesin the expression of some genesinvoIvedin AA metabolism by NBD
treatment suggest that these genes could be usually suppressed to some extent by
ethylene・ItwasreportedthattheMJandABAwereinducedbywoundingandwater
StreSS，reSpeCtively（Le6netal・2001，Zh112002）・ThechangeSintranscriptlevelsof mRNArelatedtoAAmetabolismafterharvestseemedrelatedtothoseobservedinthe
SamPlestreatedwithMJandABA，SuggeStlngthatMJand／orABAmayberelatedto
Table 4
PrimersdesignedforgenesinvoIvedinascorbatemetabolisminbroccoli．
Oligonucleotide pnmers showed below were usedforamPlification of CDNAs byRT−PCRandobtainedcDNAs wereusedas atempletefor
PrObesinnorthernblotanalysis．
Genename Sensepnmer Antisensepnmer
5▼−〔A（TGAm〔G（TGAGG〔−3■
5−−⊂AA（AGAA〔TG〔GA（AAA〔T〔A−3▼
5■−G〔AGATGATGAAGAÅG〔GT−3▼
5 イAA〔〔AG〔A仙AG〔G〔AA（ÅG−3▼
5▼−T〔CAGAGTGGITGAAGTTTG−3−
5T−TTA⊂GACAATT〔AA（〔ATGA〔−3，
5一−GA〔AGTGAAATGG〔T〔AAGT−3−
5 −AA（〔AGAGTÅAT⊂AGAGGAG−3−
5 イ′（（TA）／GA／（AG）′G〔′（〔A）′GA（GG〔AA〔TA−3
5 −（〔T）′TG／（AG〔T）／GTGTTGAG／（AG）／AG／（AG）／A／（AT）／（G⊂）／A−3 5 −AAGAAG′（A（）／A／（〔仏）／A／（GÅ）／GAT（〔GA〔⊂−3
β0−AP方J β0−AP方2 β0一頭しPg β0−めAPX β0−AO β0−GムD打
5 −〔／（AG）／AG／（GT）′（〔A〔AT〔GAG〔′（AT）／AGA−3
BO−んのAR15．−A〔TAT／（AG）′TT⊂G／（GA）／GA／（GT）′GT／（TG）／G⊂−3 5 −GTGT〔／（TA）／G（／（〔T）′T（／（AT）／AT／（TG）／GTAGq3
BO−んのAR25 −T／（GTA）／AG／（AG）／GA／（GA）／（AG）′T／（TA〔）／GATGATG（／（〔T）−3 5T−〔′（GAT）／GT〔TT／（AG）／AT／（TG）／〔〔′（AT）／〔（／（TA（）T−3
BO−DEL4R 5 −GTG／（T〔）′T／（AG）′（〔T）／TGA〔／（AT）／（AT⊂）／T／（GA）／GA／（GA）／G−3 5T−T⊂′（T〔A）／G〔／（TA）′G〔／（〔AT）／GA／（GA）／AT／（〔G）′「TCT−3．
BO−GR 5 −ATATGGG〔T／（GA）′T′（AGT）′GG／（AT）／GATG／（〔T）一3T
5 −T〔／（AT）′GG／（AG〔T）′⊂〔／（GA）′〔A〔AT／（GTA）′GAT〔一3
．■／（CrA）／t−meansthattherecouldba■ C ，a−1T一 oran A． betweentheslashes．
︵l≧急こ○∈立︶望蜃七︒︺Sヱd︼︒ト 0
12 24 36 48 60 72
HoursafterharveSt
0
Fig．16ChangesinTotalAAcontentinthefirstlayer（○），SeCOndlayer￠），
basalportion of curds（□），andflorets払）of broccoliafter harvest・
Means±SD ofthree replicates are shown，but S・D・S are nOt Shown where
SmallerthansymboIs．
β0一路APg β0・エ1P．Y
1：Q⊂gCg9gggd gtg8gtqd⊂t tく888亡8 t⊂⊂q8g七g9⊂□8q9Cqt⊂Ct⊂t8d⊂ttt⊂⊂g8 ⊂8tCt⊂⊂ロロ⊂t（q亡tt亡t⊂t⊂⊂t⊂⊂⊂8⊂⊂gtttくgCCロ⊂CgtローCgt9d⊂ddt8ATG
●●●●●●●● ●●●●●●◆■暮●●●●●●■●◆●●●●●●●◆●●●●◆●●◆●●
1：q⊂g⊂999g−一一一＝一−−−−−−一口t□tt⊂−t⊂⊂ttgくg9⊂g tg匂いCg七□t⊂ロdt⊂g⊂⊂g tttdqt⊂gく一一dくt⊂tt□dd tdロロC⊂tq−一口ttq□OdC9C⊂dQ8七CqC七t⊂ロー一也TG
120：G⊂T⊂TAT亡T（T〔一T〔⊂G（CG（ATCT（A−−C T（A（T（T・G〔T〔⊂丁（（T〔〔▲C−C▲C（CG▲G T〔丁（・T（T〔〔⊂⊂亡（〔G（〔G（（GTIT（AT⊂⊂T⊂TT（（TCCT CC（（ATCTTC T⊂〔（丁〔T〔T（ ■●●●●●■●●●●●■●●●●■●●●●●●●●●●●●●●■●●●●●●●●■●●●●●■●●●●●●●■●●■●●●●
98： G（一AGAG⊂G〔G⊂GT〔TCTCG CA−CT（AACA⊂CA（TATGG〔TT∝T〔TCTT（GCACT（AAG T〔T〔AGCTIT T（GTく：TCCTC⊂Gm〔一−−− −−−TCCTCCT〔TGGAT（ ⊂AAA（T（T（T−
Z）4： m〔〔TTTT⊂TT（T〔TC（G AAGC⊂T〔G〔（T〔⊂T⊂−〔T〔〔TC（T（〔T（〔T（CTC（：T⊂T〔T｛TT〔〔〔（（AT T〔（（CAT（〔（T〔GTT⊂AGAG AWCAT（〔G A▲CAGA（；G〔T亡TAGTAA（AC
●●●●●●■●●■●■●●●●●■●●●●●■●●●●●■■●●●■●●●■●●●■●■●●●
206： TTT〔⊂⊂T⊂GT CAT〔lTT−−− AT⊂GTTCA（〔AGAT（T〔TCG T−くT〔AT−〔G C亡Gm亡T〔T（TCAGA＾GA（；ATGT亡AAG（T GくGTT−GGTG A−＿−＿−＿＿ AT⊂GGAGTTT〔＿− AGCT（
353： GG−TGGl．TTC TC〔⊂A⊂A⊂GT G（TG⊂r（；〔（T〔T（；ATく；（AGC T（A（；CTGAAA AG⊂G〔TA＾AG ▲AGA亡AT〔AA AGT⊂〔lT〔T⊂ ⊂G（ACAAAGT T⊂TG⊂仏T⊂〔CATTぐT（GTT AGGTTGGGAT ●● ●● ● ●◆… ＝ ◆● ●● ● ■●● ● ● ●●● ●●●● ●◆ …… ………●●● ●● ●●■ ●●● ◆… …… ●● ●●◆●■ ●●● ● ●●●●●◆●
307； GGCTGCAA〔C A（〔〔A⊂一T（；⊂A（AG（AGCTA（GGAT⊂（T（；A A（AGTTGAAG AGTG〔TAGAG AAGACATCAA AGAG（：TC亡TC AA⊂A（（AAGT mG（CA⊂⊂⊂AATT⊂Tく；GTT CGmGGGAT
◆72； GG（A⊂GATG（TGGAA（GTAC AA〔AAGAACA TA（；AGGAGTG G（CACAG＾GA GGTGGA（；r「A A（GGAAGT（T TAGGTTCGAG〔〔TGAGmA AACACGCTG（AAA（G⊂TGGT CT（GTTAATG
●●●● ●l■●●● ●●● ●● ●●● ■●●■■■●●●● ● ●●■●● ●●● ●■●●●● ■●■●●●●●●● ● ■● ■●■●● ●● ● ●●●● ■●●●■●■■ ● ●●●■●●■● ■● ●■■●■■ ●● ●● ●●●●
126： GGCATGATG⊂TGG（A⊂⊂TA〔AACAAGAACA T〔TCTGAGTG G⊂⊂T〔AGAGA GGTGGAGCTA ATG（；CAGTCT CAGATA⊂GAG ATl GAGmA AGCACG（TG〔TAAT（；（TGGT〔TTGTAAATG
59Z：（GTTAAAG⊂T ⊂ATTGAA〔CT GTAAAAGAAA AGTA⊂T〔TA＾ CATCT（TTA（G（⊂GACTTGT TC⊂AmAG（TAGTG⊂⊂A⊂T GCTGTAGAGG AGG⊂丁（；GTGG TC（TGAAATA 亡（CATGAAGT ● ……＝■◆ ● ■ ● ●●●●● ● ◆●●●◆●● ◆＝＝◆◆ ●● ■●● ● ◆ ……＝●◆……… … ◆……●◆◆●●● ●
S4名：くTTTAAACCT AATTAAGCA⊂AT（：AAAGACA TGTA〔T（TG（；GAT（：T（CTAC GCTGA（⊂TAT T（CAA（TGG（（AGTGCTA〔T GCTATAGAGG AAG（TGGAGG A（CAAAATA C（⊂ATGAAAT 712： ATGGGAGAGT TGATく．T〔T（G G（GC〔TGAGC AGTGT〔（AGA AGAAGGAAG▲（T（（（T（；ATG（TGGA（（A（C TT（A（〔AG（G GAT〔ATTTGA GAGAAGT口T（TA（AGAATG GGACTAATG …● ■●■■■ ◆… … ＝◆● ● ■ ■●●●● ●■ ●●◆●●◆●●● ■● ●●●■■ ● ●●●■ ●● ●● ●●●●●● ■● ●●●●■ ● ◆ ………● ◆●●●◆●●●●● ●● ●● ●●●
666： AT（；GAAG▲GT TGATA（く丁（T GGT⊂（T⊂AT（；AATGTCCTGA AGAAGGAAGG（TTC（T（；ACG CTGGT〔⊂T（（TTCA（：C（；GCT AAT（ATmA（；AGAAGTCTT CTA（AGAAl−G Gく；TTTAG＾TG
832： ATAAGGAGAT AGTTGCmA TCTGGT（；⊂Gく ACA（CCTAGG C（GATCCAGA（〔AGACC TA G（GG（TGGGG ⊂AAA〔⊂TGAG ACAAAGTACA⊂GAAAG（TGG A（CTGGAGAA ⊂⊂AGGAGGAC
■●●●●●● ■● ●●●●●● ●●t ●●●■●●●● ● ●●●●● ●●●● ●●●● ●● ●●●●● ●●■● ● ■■ ●●●●● ●● ■■ ■■ ●● ●●●●●●● ●●●●■● ■● ●● ■●●● ● ●●●●■●●●■●
786： ATAAGGA（AT AmG（ATTA TCTGGTGCA⊂A（AC（TTAGG AAGAT〔TAGG CCAGAACGTA GT（；GTTGGGG GAAG（CAGAA A（TAAGTA〔A ⊂GAAAGAGGG G⊂CGG（；AGCA ⊂CAGGAGGAく■
95Z：AGT（ATG蓬ヨ
9舶： AGT⊂▲TGG▲〔
u7Z： ＝＝＝
119Z：
11ヰ；：
1さ1Z：
1Zさ5：
143Z：t＝tgtg（】0亡at tgtttttL］Ot ttt（】亡Ot（】td tdC8tOOCtC［tgtCdt⊂ttC dOOtdtt⊂tt tgt（】OQCdOg⊂QCqt⊂attg t￠Otddt（】tg gtgCgOgaC
●●●●■■■■■■●●●●■■●■●●●●■●●●
1∋S5：野間叱tgttt−−一−t ttgロ8qt8一‥8⊂ttgd□8d tgggq…−−C⊂忙tttt00gt tgtCddttdg…−OCdgtg g⊂⊂亡tgtttg g−…−−−−
Fig・17 ComparisonofnucleotidesequencesofBO−tbAPX（upperline，
accession no．AB125634）and BO−SAPX（lowerline，aCCeSS10n nO．
AB125635）．Identicalresidues are marked with asterisks（＊）．Gaps
introducedfbroptimalalignmentareindicated（−）・CapitalandsTlall
lettersindicatecodinganduntranslatedreg10nS，reSPeCtively・Thereg10nS
usedfbrprobesareshownbytheshadedarea・
β0イあAタⅩ
β0−∫AタⅩ ≡：三：三：三三三≡ニミ：三三三≡こ三豊荘：三三二三三悪三三三三：亘三三璃二≡避…音臣三三悪三
三三≦三二審；；≡妻妾蔓：三三三：三三…室；主賓二三三三三三溝二；主…彊；監羽：謳
55 43
1◎8 1◎◎
168
16◎
ZZ8
22◎
Z88
28（）
348
34◎
488 351 437 351
169 K 161 M
2Z9 ZZ1
2る9
281 349 341
DNGPAQGEKFVAAKYSTQKKELSDSMKKKIRAEYEAIGGSPDKPLPTNY 4◎9 FLNIIIA工SVLVLLFTFLGNNNSSDYSGF
351 −−−…＝…−…一一一一一一一−−−一一一−−一一
Fig．18 Comparisonofthepredictedaminoacid sequencesofBO−tbAPX（upper line，aCCeSSion no．AB125634）and BO−SAPX（lowerline，aCCeSSion no・
AB125635）．Theidenticalresiduesbetweentwoalignmentsarerepresentedby
theshadedarea．BO−AのARl l：qtd⊂ tCtgO t⊂tC）Ot ⊂t CtgttgttgC d−−gttCgaa gdg⊂CATGGC GTTAG⊂AT（〔A⊂CA（GTTGC（▲G〔GAAGA⊂（GGATTGTCT（mGGTGTC C〔ACTTCTCC CT⊂⊂⊂T（G〔T β0−んのA月2 1：8⊂gCgggggC dC・8dd亡用8亡tt⊂8gCg8C qq⊂⊂t⊂tg8⊂g8tC−−−−−−−・−…一一・−g gdtdddATGG⊂GG▲GAAGAG（−−−・−−−−−−−−−−−−一丁T（AA一丁Å（▲T（AT⊂一−・−一口
119： ⊂G（（GTCTT⊂ ⊂CGCTCGGTT TT（丁（CGATT GGTT⊂⊂AGAA T（：G⊂CT（CAG AGG⊂GT〔GT⊂A〔T（；⊂TT⊂CT TCG〔⊂AA（GA GAATCGくGAG TTTGTGATAA TTGGGGGAGGJuAT（；⊂GG（G
＆3：（GG（G＿一一 一一G（−−−GGA GT⊂T〔T−−T− GG C〔GGAT A（GC・− −− −−一一一一一一一一 一−＝−一−−＝ −AGCTAAGGA G−…−−・−−− −−TTTG（TAA T−＝−−−−・−（AA一…−一一G
●●● ◆ ◆◆ ● ◆●◆● ● ●● ●● ●●●●●●●●● ● ●● ■● ◆…＝ ◆…＝
Z39： GGGTAT（；CAG〔TA（；GACCTT TGTAく；AAAAC GGAATく；G⊂CG ATGGT⊂G（⊂T TT（；TATTGT〔A⊂CAAAGAGG mA⊂G⊂G⊂CITATGAGAGA⊂CTG⊂CTTGA CAAAAGCTTA CTT（；TT〔CCT ◆ ●■ ● ●● ●●◆● ●●●
1まう：GGG TTAAG CCAGGA一…・一一一・一−−− −GAAT−− − −TGG−−−− −−CT（；TTAT（T（CAAAGAGG⊂⊂GTGG⊂T⊂〔ATATGAA⊂G⊂CCTGCA⊂TTA GCAAGG（；GTA TCT（；TTT（⊂T
）59：〔〔AGAGAAGA AG〔（TG⊂T（G TCTT⊂（AGGG mCATACTT GTmGGAGG TGGTGGAGA（；AGG〔仙⊂A⊂（AGATTGGTA（AAGGAGAAA GGGATTGAGA TGATA−TATG A−−AGAT〔〔A
■●●● …… …■●●●●● ■◆ ●●● ◆ ……… ＝…● ●●◆ …●● ◆●●●●●●●● ■●… ● ◆ ●■● ● 222： 一一−GAAG− GGG（TGCTAG ACTT⊂CAGGTIT〔⊂ATTG〔T GTGTTG（；CAG CGGTGGAGAA AAACTG⊂TT⊂〔l GAAT⊂CTA CAAACAGAAA GGGATT（；AGT TGATACT（；AG〔A⊂AGAAATA
◆76： GT（；ACTGGAG（TGATmGA AAAG（A▲lCく．⊂TCACGACA− AACATCAG（；G AAGCAGTTAA AATATGG＾T⊂CCT（；ATTATC G〔TACAGGGT GTA（AG・CCT⊂TAGGTT⊂（⊂AGAT一−−…−
…＝ …… …＝…＝▲ ……＝… ◆ ● ■● ●… …… ＝…＝… ＝●●
ま36： GTGA−・−AAG CAGAT⊂T〔G− −⊂TG⊂⊂AAGA（T（：TTGTCAG TGCAく；CTGG（；GATGTm⊂A AATA⊂GAGAC TCTCATAATT（；CAACTGG⊂T CTA⊂TGTT⊂T⊂−AG＾TTGA⊂ TGATTTGGA
S87： ん11ATTG（；TG GAAAm（；〔（TGGG（；［（遜■i正
●●■■●●■■●t●●●●●‖■●●●●●●●■●●■●■●●●●●●●●●●●
45◎： GTAAAJLGGTG（AGA⊂TCTA〔GAA（AT⊂（T〔TAT⊂
alS： ● ●■● ●● ●●● ● ● ● ●● ●● ●● ● ● ● ●●● ●● ● ● ● ● ●● ●●● ● ● ●■●●
68ヰ： ■□ 一 由 ・由血 団 山 囲 厨 山口一 如 円薗山一一叫闘鵬噌由l山村巾 叫中村用l叫
934：画GTTGTGATCG GAATTGGAG⊂TAAG⊂CTG（T ATTGGCC〔CTTTGAAACTTT GT〔〔A−−TGA A（AAAT（TAT TGGAGGCATT（AGGTTGATG G⊂TTGTT⊂AG AA（AAGTA⊂⊂ ●● ●●●● ●● ● ●●● ●● ●●●● ●● ■● ●● ●●◆● ● … … ……● ● ●● ◆● ●●● ●● ●●●● ●●● ●●●● ●●●●●●● ●
d●】： Tl（m亡▲lんMて1て
1（I5之：〔〔TGGA＾TT T⊂G〔TATTGG AGATGTGGCA G（（TT〔〔m TAAAGATATA TGATCGGATG A（⊂⊂GAGTTG AA〔ATGTTGA TCACGC（CG（（GIT⊂TGCA〔AACA⊂TG⊂GT GAA−−−−ATC ●●■■ ◆＝… ●◆●● ●● ●● ●● ● ●●●●■ ● ■ ●● ●● ●● ●● ●●● ● ● ●● ■ ● ●●●●●●●● ●● ● ●●● ●● ●●● ● ●● ●● ●● ●●◆
91烏：〔⊂TGATGTTT ACGCTGTTGG TGA⊂mG⊂T A⊂TTT〔〔CCT TGAAAATGTA TGGAGA（ATG A（；AA（；GGT⊂G AG⊂ATGTTGA（（ATT⊂T（G⊂AAAT⊂（GCAG A（；CAAG⊂TGT TAAGGCGAT（
●●●●■●●●●●●●●●●●■●■■●■
116＆：−−−TCTGCTC ACTG一一一−CA〔A⊂ACT−GAC AmAT（；ATT ATCT⊂CCGTA〔TT〔TACTCA AGAGTAITCG AGTATGAACG〔AG（T〔AAGA AAAGTCT（；GT GG（AGTmA TG（；GGATAAT ●■●●●●●●■●●●●■●●●●●■●●■●●●●
10∋＆：AAAG（GG（TG AGGGAGG⊂GG AA⊂GGTG（；AG GAATACGA〔T AT（T⊂（⊂AIT〔TT⊂TA（T（一 −−一− −−−・−−・ − −G〔−G（T〔m GAT（T〔TCAT GGCAm⊂TA TGG▲GACAA⊂
1280：GTGGGAGAAA⊂AGTTGAAGT TGG（AAmC GAT（C−−（AA GAT⊂G⊂TA⊂⊂＝−TT〔TGGA TTGATT⊂TGG −⊂AGGITG AAAGGTGT T−−−・…（TT GTAGAAAGTG GTT〔T⊂⊂AGA ●● ●●◆●● ● ●● ● ● ●●● ●● ● ●●●● ●●● ● ■＝■＝… …… … ＝… ●● ● ■ ● ●●● ● ● ●●●1 ■●
1137：GT▲GGAGACT CTGT⊂TTATT TGGAGA⊂AGC AAT（CATCAA AC〔（AAAA⊂⊂AA（；ATTTGGA GCG−TACTGG AT⊂（AAGATG GTAAAGTGGT TGGAGCATT（：ATGGAAG（；AG mGTGGTGA
13糾：G（；AG−TTT〔A G⊂TTCTC（CA AAGCTAG（AA（；AAGT⊂AA（（AATTGT〔GAT AAGG⊂TA．AA⊂T〔G〔⊂AGCGC AT（TT⊂AGTく；GAAGAAG（CA T（GAGATTG〔T（AAGCCG⊂丁−〔TA（AGAGT
■●●●●●●■■●●■■■●●●●●■●■■■■■■■■●●●●●
1256；（GA（；AAqu▲GC⊂TTG（；CTA AAG−TCGCCA A▲G〔T（GA（〔TGTTG⊂AGA−GAG（CITGAC GAG〔TGG〔・一一−−＝CA −−AA〔AA（；GCA T（，・−−T⊂（T− 一丁（GCT GCTA−AGAIT 15￠Z：イ仙gdg8gt tg8tg卯ttt td−−一一−−tt8tqg⊂tttCg gtttCdgdgd d8g⊂88g⊂qロgC8呵（dd t亡ttt8tt⊂t⊂ggtg8ロdtt□ttCdtCttt Ct旺ロー・一夕d g8g Ctくt一
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u53：tgqggdgO−d dgdtggdOgt gOgOgt⊂七tt tttttttt（t gt9t⊂ローtgt ttgtd⊂9ta⊂dtttg⊂−tgO dOtOqgtt9t gtgtOttgtg dtgCet⊂ttt tC喝qt⊂⊂gt taCZgd9t⊂t□
1611：tgt亡ddtddt⊂tg⊂8tg88t qdgtdt⊂gC⊂t⊂tt一一−tt七⊂叫qOtt亡8t dttttgくtt⊂ロtqqg叩09亡ttg亡t⊂C
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1◆70：t！俳一喝8gg□ddく8t卯￠⊂⊂gC8tt用印ttttdgd仕t8tg88tt−−g gtttttC句亡dddddd8qQq8888d伽
Fig・19 ComparisonofnucleotidesequencesofBO−MLuRl（upperline，
accession no．AB125636）and BO−A4DA尺 2（lowerline，aCCeSSlOn nO．
AB125637）．Identicalresiduesaremarkedwithasterisks（＊）・Gapsintroduced foroptimalalignmeサtareindicated（−）・Capitalandsmalllettersindicatecoding
anduntranslatedreglOnS，reSpeCtively．Thereg10nSuSedforprobesareshown
bytheshadedarea・