Brassica rapaにおけるMSAP法による器官特異的なDNAのメチル化の解析

誌名誌名新潟大学農学部研究報告 = Bulletin of the Faculty of Agriculture, NiigataUniversity

ISSNISSN 03858634

著者著者佐々木, 卓川辺, 隆大藤本, 龍

巻/号巻/号 64巻1号

掲載ページ掲載ページ p. 7-16

発行年月発行年月 2011年9月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Analysis of Organ-specific Regulation of DNA Methylation in Brassica r~ραby MSAP.

Taku SASAI(II， Takahiro KAWANABE2 and Ryo FUJIMOT03*

(Received J uly 4， 2011)

Summarv

DNA 111巴tlwJarioniぉan1口lpOn<lnt巴plg巴口巴ticll1odification regllJming g巴neexpr巴ssion.ln lhis study. w巴 invesligateddiIferellces of DN f¥ ll1ethyJζ1110日 status belW巴enJeaves. stamens. <lnd pistiJs by 111巴lhylations巴日日itiveamplification polymorphisll1 UvrSf¥ P) analysis in Bras~、 ica mta. The differ巴nceof DNf¥ m巴thylationstatlls was the largest b巴lweenJ巴avesand slal11ens/piSliJs. Abollt haJf of th巴 differentiallyl11ClhyJ日t巴ds巴qllcnccsw巴regenic regions. but thcir expression Jevels did not differ betwecn organs. Th巴 percentageof genic regions delect巴dby 'vISAP anaJysis was higher than thal of lh巴 totaJJenglh of th巴 g巴nl仁 r巴gionsin lhe g巴nOll1e.Sllgg巴stinglh日lg巴nicrcgions <!re diffεr巴mially111巴thyJatcdb巴tweenorgans IIIβ-rαta.

β ulμU~α ClII.A‘'¥g♂riC.Nへ;¥/i引lμtぽ'g(αltωピ白IUI川I口11札J人.，64ρ): 7 -16，ι~ 2011 !(ey WOl'ds : DN A mClhylation. ivlSf¥P. Brassica rata， organ-sp巴じifiじIty

Gen巴 expressionis regulaled not only g巴neticallyby the

nucl巴otidesequences of genes but also epigenetically by

DNA 111巴thylationand hislon巴 lllodifications. 1n m日日llllalian

C【~lI s. cytosines at CG siles are m巴thylat巴d.while those al

CNG (N is eithe1' A. T¥C. 01' G) sites and CHH (H is巴itherA.

T. 01' C) sites as well as CG sites are 111巴thylatedin plants

DNJ.¥ l11ethyll1'ansferases illlpo1'tant fo1' r巴gulationof the

DNA III巴lhylalionare widely conserved in living o1'ganisl11s.

bllt not det巴ctedin yeasl ancl n巴matoclc(Chan el al.， 2005). 1n

.'lrabidopsis l!w/ulI1a. DDMl (decrease in DN A lllethylation 1)

which is an SWI2/SNF2 sllbfamily ch1'omatin remocleling

factor. is (1lso reqllired for DNA methylalion as are fouJ' DNA

mcthyltransferases. i.巴.JlfETl (m巴thyltransfe1'ase1). CMT3

(Chl一omom巴thylas巴3).DRi1l1 (domains rcarranged methylase 1).

and DRJlf2 (Finnegan ancl Kovac. 2000). Some hypomethylatecl

l1l1llants show mo1'phological abno1'malities. inclicating that g巴n巴

rcgulation by DNλmethylation is required [01' normal

d巴1モlopment(Ronemus el a/.. 1996. Kakutani el ([1.. 1996). Ruiz-

Garcia el al. (2005) h日V巴1'evealedthat the DNA methylalIon

lev巴1in A. thalianCi ch日ngesthroughollt its life cycle and have

sugg巴stecldev巴lopm巴ntal1'egulation by DNA melhylation. A

genome-wide c1ecrease in DNA methylalion has been obs巴rved

in th巴 endospermcompared with lh巴巴mbryo(Gehring el al、

2009. Hsicll et a/.， 2009)

R巴centst.lIdies have r巴veal巴dthal ag1'iculturally impo1'tant

trmts ¥、'erer巴gulatecl by som巴 epigeneticmocliIication

(lvlanning et a/.. 2006. I-Iaub巴netα/.. 2009. lvlartin el a/.. 2009). ln

Brassica ra戸a.th1'ee D;-JA melhyltransferas巴 genes.i.e

1 GI巴gorMendel lnstilute

" Walanab巳Se巴clCo.. Ltd.

・IGraduale 5choo¥ of Scic日ceand Technology. Niigal<l University

BrMETla， BrMETlb， and BI'CMT. and lwo genes for

chromatin remodeling factors. i.e.. BrDDMla and BrDDMlb，

hav巴 beenisolat巴d.and hYPoll1巴thylat巴CIplants with an ['(NAi

construct of BrDDMl (ddmJ-RNAi planls) have b巴巴11oblained

Wujimoto et al.. 2006. 2008. Sasaki et al.. 2011). Comprch巴nSlv巴

in[0l'I11ation on lhe DNf¥ methylalion statlls has be巴nobtained

in some organiSll1s. Howev巴rthc1'巴 islittle inforll1ation on lh巴

DNA m巴lhylationstalus of each lissue in B. ra戸a.which

includes variolls veg巴lables.although expression of rccessiv巴

alleles of SP 11メSCRin B. raρa have b巴巴nrevealecl to be

reglllatecl by tiSSllCωspecific DNA methylalion (Shiba et a/" 2006. Tarutani et al.. 2011)

1n lhis study. we per[orm巴div!SAP (methylation-sensitive

ι1m)コlificalionpolYl11orphism) analy日isto inv巴sligalcth巴

巴pigeneticregulaUon in B. rata. MSAP is a 1110difi巴CImethod

of AFLP (al11plification fragm巴ntlength polymorphisl11)

analysis llsing Hゆ Il/Mst1 instead o[ ilIse I. in which th巴

c1ifference of DNA l11elhylation at 5'-CCGG-3' sites can be

d巴tecl巴d日spolymo1'phiSI11 (Xiong el a/.. 1999). Hat II ancl M.\~ρ

1 al巴 isoschizom巴1'swhich recognize CCGG sil巴S.but tbei1'

resp巴じ【IV巴 5巴nsitivitieslo DN f¥ methylaLion cliffel‘ Hαρ II

cannot perform digestion when either cytosin巴 isflllly

methylaled. while i1fsρ1 can digesl C"CGG but nOl川 CCGG

We detectecl sequ巴nceswhose DNA methylation is

clifferen tially l'巴gulateclbeい、'eendiff巴r巴nto1'gans

7 ←

新潟大学炭~~ì~IS研究報告第 64 巻 l 号 (2011 )

MA TERIALS AND METHODS Plant matel"ials

An F， cultivar of BrassIca raρα. 'Osome' (Takii Seed Co

Kyoto. ]apan). was used. Genomic DNAs wer巴 isolatedfrom

leaves. stamens.日ndpistils by the DNeasy Plant Mini Kit

(Qiag巴n.USA). R~As were isolated from leav巴s.stamens. and

pistils by the SV τotal R~ A Isolation Kit (Prom巴ga.USA).

and used for RTPCR. Slamens and pistils w巴retaken from

flowcr buds 3-5 111m in 1巴ngth

Table 1. Adaptors and primers used for lvISAP analysis

Adaptors

Eco RI-ad-F

Eco RI-ad-R

Pre-selective promers

E仁0+0

S巴lectiveprimers

Eco+CAA

Eco+CAC

Eco+CAG

Eco+CAT

Eco+ACG

Eco+TAC

Eco+GTA

Eco+CGT

Eco+ATG

Eco+'1'GC

Eco+GCA

Eco+AGC

5'-CTCGTAGACTGCGTACC

~AATTGGTACGCAGTCTAC

5'.GACTGCGTACCAATTC

5'-GACTGCGT ACCAATTCC!lA

5'-GACTGCGTACCAATTCCAC

5'.Gf-¥CTGCGTACCAA TTCCA C

5'-GACTGCGTACCAA TTCCA T

5'-GACTGCGT ACCAATTCACC

5'-GAC'1'GCG'1' ACCAATTC T.4 C

5'.GAC'1'GCG'1'ACCAATTCGTA

5'-GAC'1'GCGTACCAA'1'TCCCア

;:;'-GAC'1'GCG'1' ACCAA '1''1'CA TC

5'.GACTGCG'1' ACCAATTC TCC

5'-GAC'1'GCGTACCAA'1'TCCC4

5'-GAC'1'GCG'1'ACCAA '1'TCACC

MSAPαnαlysis

lvJSAP analysis ¥Vas performed following Xiong et al.

(1999). DNAs (100 ng) were digesLed with Htゅ II/EcoRI or

MsρI1Eco m (TaKaRa Bio. Shiga. japan). Oligonucleotides used in ;¥lISAP analysis are shown in Tabl巴1.PCR products

wer巴electrophor巴sedin 5% polyacrylamide g巴1containing 8.5

M urea. After Lhe 巴l巴じtrophor巴sis.DNA fragments w巴re

stained by lhe silver staining m巴thod.)vISAP analysis was

repeaLed twice using the same primer pair¥and reproduじibl巴

HaρII/Mst I-ad日F

HaρII/J¥lsρI-ad-R

HM+O

HM+CAA

HM+ CAC

H1vl+ACG

IIlvl+TAG

HM十TGC

H:VI+GAT

;:;'-GATCATGAGTCCTGCT

5'-CGAGCAGGACTCATGf¥

5'-ATCATGAGTCCTGCTCGG

5'-ATCA '1'GAGTCCTGC'1'CGGCAメ4ιlしAぷl

5'-ATCA'1'GAG'1'CCTGCT、'CGGCAメAC

5'-ATCATGAG'1'CCTGC'1'CGGACC

5'-A TCA '1'G AG'1'CCTGC'1'CGG T.4 C ?

5

5'-AT、CA'1'GAGTCCTGC'1'CGGC.4T

1. U nderlincd s巴quencesindicate complem巴ntarys巴quencesbetween for可I'ard(F) and reverse (R) sequences o[ adaptors.

2. Italiじtrinucl巴otidesindicate added sequences to pre-seleclive primers for sel巴ctivcamplification

Table 2. Primer pairs us巴din RT.PCR and bisulfile s巴quenじinganalysis

target sequences primer pairs for R'1'-PCIt

MSAPt-4(4) F: 5¥CAGCCAGAAAGCGTCTAT AG-3'

MSAPt・12

MSAPt-13

iVISAPt-19

lvISAPt-54(1)

R: 5'-TGTGCAG'1'GGAT ATCATGAT AG-3'

F: 5'.GAATGC'1、'1''1'CACAGACCATCGTG-3'

R:5'-AAGCTCGG'1'CATTGAACCCAG-3'

F: 5'-GACA'1'CTGAACAAAGCGTTGGC-3'

It:5'-GAGA '1' AGTGAGAAC'1'GGGCCTCC3'

F: 5'-CCACGTGG'1'GCCA'1'TTGGTC-3'

R5'-GCAC'1'C'1'GAGTGCGTG'1'GCC-3'

F: 5'-ACCTCGGCTTTTG'1'AAACAGC-3'

R: 5'-AAT'1'TCGGAAGAAGGGAATGG-3'

- 8

Primer pairs for bisulfite scqu巴nじII1g

F: 5'-AAAAGGGAAGTT'1'GAGAAGYAAAAT-3'

R: 5'-AR'1''1''1' ACRATAAAATTAAAACCAAC-3'

F: 5'-AAT'1''1'GATTAAA YAGGYA'1'YGA'1'GG-3'

R 5'-CCARCACA T ARCACACCH.CCA'1'CA '1'C-3'

F: 5'-AGAYAGAGAGA'1'AGTGAGAA YTGGG-3'

R: 5'-CTRACACCf¥Cf¥RAf¥ ARCCCAAAACC-3'

F: 5'-A'1'GGY f¥GGTYTTGAGTTGGGGAGA'1'-3'

R: 5'-CTCCA'1' f¥CCRTT'1'TCCACATCCCC-3'

F: 5'-TG'1''1'YGAGAGGAGATf¥ YTAAGGGGG-3'

R: 5'-AAACTAAARTCCCRACCA'1''1'C'1'CTC'1'C-3'

Organ-specific regulation o[ DNA methylation in Brassicαrαta

polymorphic bands wcre counted

Characterization of polymorphic bands

Bands showing polymorphism were cut from

polyacrylamide gels and heated with 50μ1 of PCR buffer at

65.C for 2 hours. DN As were r巴amplifiedby PCR using

HiVI+O/Eco+O primers. The PCR condition was 94"(; for 1

minute. followed by 45 cycles of 94"(; for 30 seconds. 58t for

30 seconds句 and72"C for 1 minute句 andfinal extension at 72t

for 3 minutes. Reamplified PCR products were cloned using

pGElVI-'1' Easy System 1 (Promega) and tbe nucleotide

sequences were determined with a CEQ 2000XL DNA

Analyzcr (B巴ckmanCoulter. USA). Thc sequence data were

analyzcd using Sequencher (Gen巴 CodesCorporation. USA).

and BLAS'1' search of DDB] was performed (http://blast.ddlコJnig.ac.jp/top-j.html)

'1'0 characterize the flanking sequences. suppression PCR

was performed following Siebert et al. (1995). Amplified PCR

products were cloned and sequenced

RT-PCR

cDNAs were synthesized using thc First-Strand cDN A

Synth巴sisKit (GE healthcare. UK). Expression of genes

diffel巴ntiallymethylated between organs was analyzed by

RT-PCR using total RNA as a template with primer pairs

listed in Table 2. The actin genc was used as a conLrol. The

R'1'-PCR condition was 940

C for 1 minute followed by 28-35

cycles of 940

C for 30 seconds. 550

C for 30 seconds. and 72"(;

for 1 minut巴.and final cxtension at 720

C for 3 minutes

lミco +ACG HM-ιACG +TAC ム GTA

L P L P L P

pnv

hμhu

nυAu

nυAU

63

400bp

300bp

200bp

--・・・・・・・・・・・・6

..， ・e・

lOObp

Figure 1. Polymorphic bands in I¥IISAP analysis bet wcen

leaves. stamens. and pistils. HG+ ACG and Eco+ NNN (N: A. '1'.

C. or G) inclicate primer name. H. Hat II/ Eco RI cligestion: i'v1.

Msρ1/ Eco RI cligestion. Arrowheacl shows polymorphic

bancls between organs. L. leaves: S. st日mens:P. pistils

Table 3. The numb巴rsof polymorphic bancls b巴twe巴nleaves. stamens. ancl pistils in MSAP

leaf stamen pistil No. of sites

H lvl H JVI 1-1 M

+ + + 十 9

十 + + + 8

十 + 3

+ 4 reprocluctive tissue specific

+ 十 + + 4

+ + 2

+ 十 + + + l

十 + + 十 3 stamen specific

+ 十 + + +

+ + + + + 8

+ + + + 2 pistil speci白c

÷ +

+

+ + + others

÷ + +

51

H. Hat II cligestion: 1¥'1. 111sρ1 cligestion: +. bancl present:ー.bandabsent

9

新潟大字決学古1¥研究宇lttlr 第64巻 l与 (2011)

Bisulfite sequencing

Genomic bisulfit巴 sequencinganalysis was performed as

described by Paulin et al. (1998) using DNAs indepcndently

sampled for I¥IISAP analysis. Genomic DNAs (1μg) extracted

from leaves. stamens.正lI1dpistils were digested with Eco RI

and Sal 1 in 200μ1 of the reaction mixtur巴.After ethanol

precipitation. DNAs were dissolved in 20μ1 of water. Aftcr

b巴ingheat巴dat 94"C for 10 minutes and then cooled on ice.

DNAs were denaturcd by the addition of 2.2μ1 of 3 N i¥1aOB

and incubated at 37'C for 30 minutes. '1、h巴 denaturedDNAs

were dissolved in 208μ1 of ur巴a/bisulfitesolution (7.5 g of

urea (Wako. Osaka. J apan)日nd7.6 g of sodium metabisulfite

(MERCFζGermany) dissolv巴din 20 ml of water， adjusted to

pB 5.0) and 12μ1 of 10 mlVI hydroquinone (SIGlvIA守 USA)and

overlaid with mineral oil. The samples werc subjccted to 30

cycles of 95'C for 30 seconds and 55"C for 15 minutes.

followed by 550

C for 15 hours in a PCR instrument. After the

r巴action.DNAs wcre purified using the Gene Clean Kit

(Q-BIOgene. USA) and eluted with 20μ1 of water. For th巴

desu][onation. 3 N Na01-1 was added and thc DNAs wcre

incubated at 370

C [or 15 minutes. After 巴thanol pr 巴c口:コIp仰川)11川t凶~aζa山I

DNAs weαre 巴elut巴d with 20 μ 1 of wat 巴I¥ PCR was p 巴引rfor‘ll1η1 巴d

l 川n50 μ 1 of reaction 1口Il1lxtur喝e cont 日山111山11川ng5 μ 1 o[ DNA as a

1ほ巴Il1pl凶at匂巴.The PCR condition was 94"C for 2 ll1inutes followed

by 40 cycles of 94"C for 20 seconds. 50.C for 30 seconds.日nd

720C for 1 minute句日nda final extension at 72't for 10

mll1ut巴s.The primers employed are Iisted in Tabl巴 2.Th巴

PCR products were g巴I-purifiedand c1oned. Tcn to twelv巴

ind巴pendentc10nes w巴res巴quenced

RESULTS

lVISAP analysis was performed to detect sequences

differ巴ntiallymethylated betwe巴norgans using DNAs

extracted from leaves. stamens. and pistils. 1n fvISAP analysis

using 72 primer pairs. 51 of l86l bands show巴d

polymorphism b巴tweenorgans (Fig. 1. Tabl巴 3).Thirty-three

polymorphisms (64.7%) were observed betw巴巴nthe

vegetative organ (1巴aDand reproductive organs (stam巴nand

pistil). There were 12 polymorphisms specific to pistils (23.5%)

日ndfour specific to stamens (7.8%). O[ these polymorphic

bands. 29 bands were sequenced.日ndBLAST search o[

DDB] revealed 23 of these sequences to bc similar to thc

known sequences守 i.e..13 putative geni じ regions.fiv巴

transposon-lik巴 sequ巴nces.on巴 microsatellitesequence. and

four sequences with no annotation (Tabl巴 4)

Bisulfite-sequencing analysis was performed to detect

DNA m巴thylationof these sequences. Flanking s巴quencesof

:VISAPt-4(4). -12. -13. -19. and -54(1). wbicb are parts of th巴

putζItive genic regions showing polymorphism between

different organs in MSAP. were characterized by suppression

PCR. Bisullite-sequencing analyses rcveal巴ddifferentially

mcthylated cytosincs bctw巴enorgans at CCGG sites of

S巴quencesof MSAPt-4(4). -12. and -19 (Fig. 2 and Table 5). A

197 bp sequence of tvISAPt-4(4). which corr巴spondsto the 10il•

to 11，h intron of the putative serine carboxypcptidase 1 gen巴

of A. tlzaliana. contained 42 cytosin巴s.i.e.. tb r巴巴 in a CG

cont巴xtand 39 in日 non-CGcont巴xt.Only two cytosines in

the CG context were m巴thylated.and one of them was th巴

recognition site of 1ヲdρIIand J11sρ1 used 口lVISAPanalysis.

CCGG. These m巴thylationstatuses diffcred between organs.

and methylation levels of cytosines in leaves were higher

than those in other organs (Fig. 2A) consistent with lVISAP

analysis. indicating more m巴thylationof the CCGG site in

leaves than in stamens and pistils. The methylation status of

a 303 bp sequence of lVISAPt-12 containing 58 cytosines

which corresponds to the 10，h to 12，h exon of a putativ巴

protein gene (homologous to A t3g45045 o[ A. tlzaliana)守

differed in two CG contexts between organs (Fig. 2B). A 287

bp flanking sequence of lVISAPt-19. which corresponds to th巴

3，d exon to the 3'【1in tron of a hypothetical protein gene

(homologous to At4g14850 o[ A. t1zaliana). contain巴d51

cytosines. 13 of which arc of lhe CG contcxt and 38 of which

ar巴 ofthe non-CG cont巴xt.Ther巴wcreninc methylated sit巴s.

all of which were of the CG context. Of thes巴 Slt巴s.five

dif[ered in DNA methylation status between ol-gans (Fig. 2C).

Consist巴nt九NithMSAP analysis守 th巴 targ巴tsile was highly

methylatcd in stamens. 1-lowcver. th巴 melhylationstalus in

pistils was th巴 sameas that in leav巴s.1n sIISAPt-54(l).

methylation levels did not differ b巴tweenorgans at the CCGG

sitc recognized by ivISAP analysis守 butsome cytosines were

di旺crentiallymcthylatcd bet¥¥'e巴norgans (Fig. 2D. Table 5). A

270 bp flanking sequence of MSAPι54(1). which corrcsponds

to putative trehalose-6-phosphate synthase. contained 23

m巴thylatedcytosines including 14 in the CG context and 9 in

the non-CG contexl. 1n this seqllence. all the cytosin巴S

di[fcrentially methylat巴db巴twecnorgans were in the non-CG

じontexts.mainly in the CHH contextsλ 278 bp []anking

sequence o[ MSAPt-13 conlained 103 cytosines. but there was

no methylated cylosin巴 inany of the samples. Expression

levels of th巴sefour genes analyzed by RT-PCE did not show

the negative correlation betw巴cnDNA m巴thylationand

expression lev巴Is(Fig. 3).

DISCUSSION

Since MSAP analysis守 whichcan c1etecl differences of

DNA methylation in the whole g巴nomeusing mcthylation

sensitive and -insensitive restriction endonucleases. c10es not

r巴qlllr巴 informationof genome seqllences. it is suitabl巴 for

日nalyzingth巴 diffcrcnccof Dl¥A methylation in Brassica

whose g巴nomeseq u巴ncinghas not been compl巴led.M日ny

stlldi巴shave revealed differ巴ncesof DNA m巴thylationb，・

MS_..¥P analysis in various plants (Xiong et al.. 1999. Xu et al ..

2004. J aligot etαl句 2004町 Labraet al.. 2004. Salmon et al.. 2005.

2008守 Smykalet al句 2007).but few reports have shown

q lIa n tl ta tl v巴 differencesof methylation levels at each locus

detected by lvISAP analysis.

1n thc present study. we applied MSAP analysis to

investigate the s巴qucnceshaving c1iffcrent methylation levels

between vegetative organs and reproductiv巴 organsin B

rata. The bisulfite sequence method revealed differences of

methylation levcls in thrcc organs. but lhese differences did

not complet巴lycorrespond to the results of MSAP an日Iysis.

- 10-

Organ-specific r巴gulationo[ DNA methyl日tionin Bra.川 lcaraρσ

Table 4. .'¥nnotation of seCjllenc巴sshowing organ-specificity of DNA I11cthylation in fllISAP

band paltern

band name leaf 日tam巴n pistil

(MSAPt) 1I ?v1 H :¥'1 11 :vl length(bp) g巴n巴 SOllthern blot'

4(2) + + 司ト + 320 日11CrOSal巴lIitcs巴qll巴日じ巴

4(4) + 田舎 + 十 232 Serinc carboxypcptidasc 1 +

6 + + + + ÷ 209 plltatlve 1・巴tro巴lem巴ntpol polyprotein

8 + + 400 A thalian日chloroplastgcnol11ic DNA. photosystcm IT G +

prot巴1ll

10 + + + + 29'1 no hit

11 + + 141 B. rapa subsp. pckine日sisclone KBrH070IIO.coi11pleIC 4十

S巴qll巴nce.B. 01巴!日ccavar. alboglabra ES1、

12 ÷ + 4・ 十 2~0 pllt日uv巴ρrolcinsimilal一ity10 KIAf¥ ¥094 protcin. HOll1o -ト

saplcns

13 + 246 '¥l<lbidopsis t ha li an日 cD~A c1onc:Rf¥FL07-17-L 13 +

17(1) + + + + + 284 A inlaCl r巴trolransposon.Centrol11巴ricRelrotransposon +

of BrassiじaI(CRBl)

17(2) ÷ + + + 238 no hit +

18 + + + + + 269 Brassica oleraじeaComig C. cOl11pl巴tescqllcnc巴

19 十 + 179 A. thaliana Il1RNA for hYPolhetical prot巴in.cOl11pleteじ【1$. +

c1one:RAFLI'I-50.J09

27 + + + 十 209 Arabidopsis th日lianachloroplast g巴nomicDNA.ATPase +

alplla sllbllnit

28(2) + + + マF + 285 Brassica rapa sllbsp. p日ki日巴nsisclOll巴 K8r8080J15 十

complc1e seqll巴日ιE

28(3) + + + + + 221 Brassiω l<lpa subsp. pekinensisじlone](13rBOlO.¥'J 1 9. +

COl11plCIC sequenじ巴

29(1) + + ÷ 4・ 4:19 Brassiじζ¥napus pllta1ivc diacylglyccl'Ol日じyltlε¥llsfcrasc +

111!(.N f¥. compl巴ほじds

30 + 247 clhyl巴neI・cspon日Ive巴l巴me11lbincling factor-relatccl +

34 + + + + '190 Arabidopsis thali日日日 chloroplas1genomic DN A

35 + + + + I11 8. napu日1111ωchondorialDNA. ribosol1l日1prot巴inS12 +

37 + 173 A. thaliana pllrative clisea日cl'eSlstancc prolcm g巴日巴 +

38 + 十 + + 479 no hit +

39(4) 十 + + + J29 no hit ート

<¥0 + 十d‘- + 290 no hil +

43 + + + 十 607 B. napllS pa rti日1RT g巴ne[or rcv巴rse1ranscriptase frol11 +

Ty3-gypsy、liker巴1ro巴!巴ll1巴nt21G42-04

4(j + + + + + 234 t¥IV¥ TH Copia-like retroelement pol polyprotein

54(1) + + + ÷ + 435 A. thalian<l Il1RN A for Plllative trchalosc-6-phosphat巴 ÷ S}・nthase.col1lplate cds

54(2) + 416 no hit +

54(3) 十 + + ÷ + 269 I3r臼ssicaoleracea Contig C. coll1plere scqllcnce

72 + + + + + 408 ARATH Plllali、cretroelel1lenl pol polyprotcin

H.H，ゆndigestion: M. JlJ.ゅ 1digestion; +. band present:ヘbandabsent

へSoulhern blot analysis was perform巴d(+) or not p巴rformed(ー).Tissue speci!ic band pattern consistent with ¥，lSAP was 日ppearedonly in No. 38

← 11-

新潟大学l史学部研究報告 第 64巻 1号 (2011)

Table 5. Percentages of methylated cytosines in CCGG sites

rates of methylsted cytosine (%)

band nam巴 locus事 organ ollter cytosll1e ll1ner cytosll1e

leaf O 54.5

4(4) 41 stamen O 40

pistil 10 40

leaf 9.l 36.4

12 39 stam巴n O 72.2

pistil O 45.5

leaf O 。13 151 stam巴n 。 O

pistil O O

leaf O 58.3

19*' 230 stamen O 91.7

pistil O 50

leaf O O

19 235 stam巴n O O

pistil 8.3 8.3

leaf 80 100

54(1) 113 stam巴n 90 100

pistil 70 100

leaf 20 100

54(1)帥 119 stamcn 30 100

pistil 10 100

leaf 10 100

54(1) 131 stamen O 100

pistil O 100

• indicates position of inner C

•• indicates CCGG sites recognized in our MSAP analysis

One possible explanation for the difference betw巴巴nthe

reslllts of lvlSAP and bislllfite seqllencing is that MSAP

analysis is not quantitative and m日yd巴tecta sma11 differ巴nce

of methylation 1巴V巴lsas D:¥' A polymorphism

The seqllences showing polymorphism between differ巴nt

organs detected by MSAP were mostly g巴n巴s.44.8% of thc

determined s巴qllences.This percentage is mllch higher than

th巴 percentageof genic regions in the Brassica genome

(Rabinowicz et al.. 2005). suggesting that the regions of genes

are subjected to organ-specific methylation more freqllenUy

than other regions. The ChIP-on-chip or bislll五teseq uenCll1g

analyses have revealed that about one-third of巴xpressed

genes are methylated in their ORFs in A. thalial1a (Zhang et

al.. 2006. Zilberman etα!.. 2007. Cokus et al.. 2008. Lister el al ..

2008). The present stlldy showed that expression levels of genes whose ORFs w巴r巴 m巴thylatedorgan-specifica11y did

not differ between organs. suggesting low involvement of

ORF methylation in the control of gene expression. Zhang el

al. (2006) have reported that about 5% of genes methylat巴din

promoter regions showed organ-specific expr巴ssion

iVlethylation in the promoter region may be more important

than that in the coding region for tissu巴-specificg巴ne

regulation

Th巴 majorityof differenc巴sof DNA methylation statllses

between differen t organs in B. rata detected by j¥ASAP

analysis were the differences betwe巴nleaves and stamens/

pistils. Differences in the methylation status among different

organs or between diff巴rentdevelopmental stages have been

fOllnd in several plants. HPLC analysis has revealcd less

methylation in immatllre tomato tissues. st巴ms.leaves. and

roots than in seeds守 matllreleaves. and fruits (Mess巴glleret

a!.. 1991). Immunohistochemical analysis has detected change

of methylation statuses dllring plant development in Silene

!atifolia (Zlllvova el a!.. 2001). Di妊erentmethylation patterns

during development of A. thalianαhave been reported守 and

differenc巴sin methylation b巴tweenseedlings and adult plants

つ山

methylation in BrassIcαratα Organ-specific reglllation of DN，

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Figure 2. Differen仁esof methylation levcls between organs analyzed by bislIlfite seqllencing

Analyz巴dgene strllctllres ar巴shownat the left. Large arrows indicatc homologolls g巴nesin A. Ihalimza守 andmiddle bars indicate

lhe strllctllre of th巴g巴ncin A. tha!icma. Lower bars rcprcsent sequ巴nじesobtained by tllISAP analysis (gray) and suppression

PCH (black). .Arrowhcads show primers lIsed in bisulfit巴S巴quencinganalysis. Rates of rnethylat巴dじytosinesare shown at th巴

right. Small circles and numbers indicat巴methylatedcytosines and lheir positions. r巴sp巴ctlv巴Iy.and numbers with・indicatethe

recognition site in MSAP an日Iysis.Black. gray. and while circles represent cytosines in CG. C:¥fG. and CHH contexls. respectively.

For cytosines differ巴ntiallymethylated between organs. proportions of methylated cytosine are sho¥¥'n as black parts of th巴 ple

chan. (a). (b). (c) and (d) indicate putative s巴nn巴carboxypeputidaseI (tvISAPt-4(4)). putative protein (MSAPt-12). hypothetical

protein (MSAPt-19). and plltative trehalose-6-phosphate synthase (MS.APl-54(1)) respectively. Cytosines methylated in more than

three clones in each organ were r巴gardedas methylated cytosines

-13ー

第 64巻 1万 (2011)新潟大学農学i'fliNf究報告

Cao. X.. S. E. ]acobsen. 2002. Role of the Arabidotsis DRJ1f

m巴thyltransferasesin de 11口vom巴thylationand g巴n巴

silencing. CZIIγ Biol.， 12: 1138句1144

Chan. S. W.. I. R. H巴nd巴rson.S. E. .l acobsen. 2005. Gard巴nll1g

the genom巴 DNA methylation in Arabidotsis thαliana

へiat.Rev. G'ellel.， 6: 351-360

Cokus. S. ].. S. Feng. X. Zhang. Z. Chen. s. Merriman、C.D

llalldenschild. S. Pradhan. S. F. Nelson. ~k Pcllegrini. S. E

]acobsen. 2008. Sbotogun bisulfite sequencing o[ the

:-l rabidotsis genome reveals DN A m巴lhylation

patterI1lng.入1αture. 452: 215-219

Finnegan. E. ].. K. A. Kovac. 2000. Plant DNA

methyltransferases. Plallt Mol. Biol.， 4:3: 189-20l.

Fujimoto. R.. T. Sasaki. 1¥Nishio. 2006. Charaじterizationof

DNA m巴lhyltransferascsg巴nesin srassica rata. (;enes

Genet. Syst.， 81: 235-242

Fujimoto. R.. T. Sasaki. 11. Inoue. T. Nishio. 2008

I-Iypom巴thylationand lransじriptionalreactivation of

r巴trotrans poson-li k巴 sequ巴ncesin ddml transg巴nlc

planls of Brasseicαrata. Plant 11101. siol.， 66: 463-473

Gehl・ing.11'I.. K. L. Bubb. S. Henikoff. 2009. Extensive

demethylation of repetitiv巴 elementsdllring se巴d

dcvelopm巴nlllnderlies genc imprinting. Sciellce， 324:

1447-1451

I七lL1b巴n.¥11.. B. Haesendonckx. E. St.andaert. K. Van Der

Kel巴n.A AZl1li. 11. Akpo. F. Van Brells巴gem.Y. Guis巴z.ivl. Bots. B. Lambert. B. Laga， t-.'i. De Block. 2009. Energy

lISC efficiency is characterized by an epig巴n巴tic

じompon巴ntthat can be direct巴dthrough artificial

selection to increase yield. Proc. Natl. Acad. S('i.μSA守

106: 20109←20114.

Hsieh. C. L. 2000. Dynal1lics of DN.A methylaLion pattern.

Curr. 0.ρil7. Gelle/. Dev.， 10: 22'1-228

Hsieh. T.F.. C. J¥. Ibarra， P. Silva. A. Z巴ma仁h.L. Eshed.

'vVilliams. R. L. Fisch巴1'.D. Zillコ巴1・man.2009. Genome-wiclc

demethylation of Arabidopsis endosp巴rm.S('ience， 324

1451.1454

]aligot. E.. T. B巴ule.F. C. Balll巴ns.N. Billotte. A. Rival. 2004

Search fo1' m巴thylation-sensiliveamplification

polymo1'phisms associated with the mantled variant

ph巴notyp巴 inoil palm (Elaeis guilleellsis ] acq.). (;ellome，

'17: 224-228.

Kaklltani T.、 J.A.]巴ddeloh，S. K. Flowers. K. Munakata. E. ]

Richards. 1996. Developmental abnormalities and

epimutations associated with DNA hypomethylation

mutations. Proc， Natl. Acad. Sci. USA， 9:3: 12406-12411

Labra. 1VJ.. F. G1'assi. S. Imazio， T. D. Fabio. S. Citterio， S

Sgorbati， E

References

E翠=盟

MSAPt・4(4)

-rー司-"l1・L _ ]

- 】・・-=-:"_II

MSAPt-12

MSAPt・19

』』轟国

MSAPt -54(1 )

。ctm-H

∞-t

Figure :3. Wf-PCR of genes showing polymorphic bands in

MSAP. MS.APt-4(4). -12. -19. and -54(1)

cogsm

』

cuu

have also been identifi巴din rice (RlIiz-Garcia et ([1.. 2005. Sha

et al.. 2005). Differences of DNA methylation patterns

b巴tweendiff巴r巴nlorgans or tlSSU巴日 rev巴aledin these

p1'evious stlldies and in lhe pr巴senLstudy suggest the

presence of some mecl1anisms controlli口glissue-specific

methyl日tion.Since l11elhylation pa此ernsare Lhe results of de

IIOVO 111巴thylation.demethylalion. and lhe maintenance of

existing methylation (Hsieh. 2000). these m巴chanismsmay

function during plant c1evelopment. A. tlzalialla has DRJ1イ'2as

de 11000 methylation enzyme and DME and its paralogs as

DNf¥ d巴methylationenzymes (Cao and ]acobsen 2002.

Morilles-RlIiz et al.. 2006. Penlerman et al.. 2007). 1n B. )"[~ρ[1，

orthologs of these g巴neshave nor been idcnti自己d.bllt gen巴5

involv巴din maint巴nanceof DNf¥ methylation hav巴 b巴巴n

charactel一izccl(Fujimoto et al.. 2006). Interestingly， expr巴ssion

levels of BrCMT and BrMETla have been shown to change

drastically throllghollt slamen development (FlIjimoto et al ..

2006). and may be involvecl in tisslIe speじificityof DN A

methylation. Further inv巴stlgatlOnsar巴 r巴CJ1I1r巴clfor

elllcidation of organ-and tisslIe-sp巴cificcontrol of DNA

methylation. Since th巴 organsin B. rata ar巴 mllchlarger

than those in A. t!za!1ω1(/. IL m日ybe advantageous to use B.

raρa ralher than A. thα!im/{/.

- 14 -

ACI(NOWLEDGEMENT

This work was slIpported in part by NIG Cooperalive

Research Program (2006.;¥-76). and v，;e are grat巴flllto Dl¥T

Nishio for c1'ilical じommentson this manusc1'ipt. Dr. T

Kakutani for his aclvices to ou1' experiments. and Dr. K.

Shirasawa for his technical advic巴

Organ-sp巴じificr巴gulationof DN A methylation in Brassica rata

Manning. K.. lvI. 1'01¥ivI. Pool巴目Y.F!ong. A. ]. Thompson. G. J

I(ing. ]. ]. Giovannoni. G. s. S巳ymour.2006. A naturally

ocじurringepigen巴tlCmutaliol1s 111 a g巴neencoding an

SBP-box transcriplion factor inhibits tomato fruit

ripening. Nal. Genel.， :38: 948-952

Martin. A.. C. Troadec. A soualem. iv1. Rajab. It. F巴rnancl巴Z

H. ~vrorin . M. Pitrat. C. Dogimont. C. A. Bendahmane.

2009. A transposon-il1c1l1ced epigen巴ticchange leacls to

sex detcl・l1linationin I1lclon. !Va!lIre， 461: ll35-1138

M巴ss巴gllCr.R.. iv1. VV. Ganal. ]. C. Steffcns. S. O. Tanksley

J 991. Charactcrization of the level. targ巴( sites ancl

inheritance of cYlosinc m巴thylatiollil1 tomato nllcl巴日r

O¥' A. Plalll Mol. Biol.. 16: 753-770

ivloralcs-Ruiz. T川 A.P. Ortcga-Galisteo. iVI. r. Ponferracla-

iVlarin. M. 1. Martinez-:vlacias. R. R. Ariza. T. Rolclan-

Arjona. 2006. DEMETER ancl REPRESSOR OF

5ILENC1!VG 1巴ncocle5・mcthylcytosincDNA

glycosylases. Proc.人iatl.Acad. Sci. USA， 10:3: 6853-6858 Paulin. R、G.W. Grigg. NI. W. Da¥巴y.A. .'¥. Piper. 1998. U rea

il1lproves efficiency of bislllphitc司 m巴c1ial巴ds巴qll巴ncingof

ST_m巴thylcytosinein genol11ic ONA. NlIcleic Acid Res.， 26

5009-5010. Penterman. ].. D. Zilb巴rl11an.]. H. Huh. T. Ballinger. S

Henikoff. R L. Fisじh巴r.2007. DNA CI巴melhylaLionin lhe

Arabiclopsis genome. Proじ.Nall. Acad. 5ci. USA， 1114:

6752-6757.

Rabinowicz. P. 0.. R. Citek. ivl. A. Bucliman. A. NlInberg. ]. A

Becl巴11.N. Lakey. A. L. O'Shaughnessy. L. U. Nascimento切

羽'.R ¥11ιCombi巴.R. A. lVlartienssen. 2005. Oifferential

m巴thylationof genes ancl r巴pealsin lancl plants. Genome

Res.. 15: 1431-1440

Roncl11l1s. M. J.. lVl. Galbiati. C. TicknoJ¥J. Chen. S. L

o巴lIaporta.1996. Demethylation-inclllc巴dc1ev巴lopmental

pleiotropy in Arabidotsis. Scieilce， 27:3: 654-657.

IulIz-Garcia. L.. M. T¥Cerv巴ra.]. M. lvlartincz-Zapat巴1'.2005

DNA m巴thylalionincr巴asesthroughollt Arabiclopsis

c1evclopment. Plailla， 222: 301-306 Salmon. A.. 1111. L. Ainollche. J r. Wenclel. 2005. G巴nelicancl

epigenelic conseqllences of rec巴nlhybridization ancl

polyploicly in Starlil1a (Poaじ叩巴).Mol. Eω1.， 14: 1163-

1175

Salmon. A.. J. Cloault. E. J巴nczewski.V. Chable. 111. ].

lvlanzanares-Dalllellx. 2008. Brassica oleracea c1isplays a

high level of DNA melhylalion polymorphism. Plallt Sci.，

174: 61-70 Sasaki. T.. R. FlIjimoto. S. Kishitani. 1'. Nishio. 2011 Analysis

of targel sequences of DDtlt[ls in Brassica rata by

:VISAP. Plcml Cell Ret.， :3

Si巴bert.P. D.. A. Chenchik. D. E. Kellogg. K. A. LlIkyanov. S

A. LlIkyanov. 1995. An improvecl PCR mClhod 1'01

walking in lInclonecl genomic DNA. NZlcleic Acid Res.. 2:{

1087-1088 Smykal. P.. L. Valleclor. R. RonclrigllezφM. Griga. 2007.

t¥ssessm巴ntof genetiじ ancl巴plgenetlじ stabililyin long-

term in vitro shoot cllltllre of pea (Pisum sativlI m L.)

P/a711 Cell Ret.， 26: 1985-1998

Tarutani. Y.. H. Shiba. }.I!. Iwano. T. Kakizaki守 G.SlIzuki. M

Watanab巴.A. Tsogai. S. Takayama. 2010. Trans-acting

small RN A c1etermines c10minance relationships in

Brassica self-incol11patibility.八ialure，466: 983-986

Xiong. L. Z.. C. G. XlI. ivI. A. S. Maroof. Q. Zhang. 1999.

Patt巴rnsof cytosine methylation in an eli(e rice hybrid

ancl its parental lines. c1et巴じteclby a methylation-s巴nsitive

amplificalIon polymorphiSI11 techniqlle. J1fol. GeJ/. Genet.，

261: 439-446 XlI. ivI.. X. Li. S. S. Korban. 2004. DNA-l11elhvlalion alteraLions

ancl exじhangesdllring in vitro celllllar cliff巴renliationIII

rose (Rosa hybrida L.). Theor. Atμ Genet回目 109: 899-910

Zhang. X.. J Yazaki. A. Sunclaresan. S. Coklls. S. W. L. Chan

H. Chen. 1. R. I-Ienclcrson. P. Shinn. tvI. Pellegrini. S. E.

Jacobs日n.J. R. Eck巴r.2006. Genome-wicle high-resollltion

mapping ancl functional analysis of DN f¥ m巴thylationin

Arαbidotsis. Cell， 126: 1189-1201

Zilb巴rman.D ‘ M. Gehring. R. K. Tran. T. Ballinger、S

Henikoff. 2007. Genomc-wicle analysis of Arabidotsi五

thaLialla DNA methylation lInじovcrsan II1t巴rcl巴penclenじ巴

b巴t、¥'eenm巴thylationancl transcription. Nat. Genet.， :{9:

61-69

Zluvova. J.. B. Janousek. B. Vyskot. 2001. Il11munohistoch巴micalstucly o[ DNA melhylalion

c1ynamics c1l1ring plant CI巴velopmenl.].Ex戸 801.，52

2265-2273

ζd

:i!JiiP}大学:;立学m;研究ヰ11;;ii U~ 64巻 I号 (2011)

Brassicθrapaにおける MSAP法による器官特異的な DNAのメチル化の解析

佐々木卓1・}11辺隆大2・藤本 龍引

(':ド成23年 7月4日受付)

要約

Dl\A のメチル化は泣伝子の I I!~ "匂: ílilJ御 lこm:裂なエピ ジェネティ ッ クな変化である。 本研究では、 Brassica ra戸。の束、却しべ、

雌しべの DNAのメチル化レベルの迎いについて ivlSAP法をJTJいて IV，~べた。 DNA のメチル化はぷE と }jj~ しべ / 雌しべで最も巡っていた。 DN.'\ のメチル化がiもなっていた円êijlj のず分は巡伝子何l域であったが、 j当{工 :r の発JJI批は3つの日佐官で~いが見られなかった。ivlSAPìl;で見出された'ÍÌÍ.[域の内、遺伝子領域が I~i める :， i {;lj {:tは、ゲノム rj.1に占めるii'1u;r飢J或の制合よりも高かったこ

とから、 B.raþa において、日誌'I~\ q;\・ j'Ul0 な DPJA のメチル化の沿いはJ2伝子制域に起こりやすい可能性が示された。

f!/U;;tW/l/i. 6マω.7.16.2011

キーワードー DNAのメチルイヒ.lvISAP. Brassica rata. r.~'i~'ネ，Y' Ntl:

I Gregor ivlendel I ilstitute

ど株式会社渡辺採符[場

:1 新潟大学大学院自然科学科

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