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Identification and Function Analysis of Anthocyanin Biosynthesis Related MYB Genes in Mulberry

桑树花青素合成相关MYB类转录因子的鉴定与功能分析



全 文 :书!"#$%&
,2016,36(6):1110-1116
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CDEFGHI
! "1,#$%2,&(2,)*+2,&,-1,./02
(1567%8 5550025;2!\]% S$^_%8,` a400715)
J K:bJKLcS$Md%ef,EVW;Ig6hij8klmnS$4opq/rstIu(MYB)vw
;I

xystgz{|}~€‚PCR^_,ƒ„j…;I†‡ˆg‰|Š},Zc‹6ŒŽ。ƒ„
‘Š’“
,4k MYB;I”•–、—˜™š›lmnpqœ/ MYB;Iž,Ÿ1k MYB;I” \¡、¢
Šlmnpqœ/ MYB;Iž。stgz{’“£z;I†¤l6¥—¦Ž。}~€‚PCR‘
Š§
,2k犕犢犅;I(犕狀犕犢犅犑™犕狀犕犢犅4)†Š},Zc‹6¨©ªp,1k犕犢犅;I(犕狀犕犢犅330)†Š}
,Zc‹6’«¬p

ƒ­”lmn†V®6Œ¯°o±œ/™²œ//³



VW MYB;Iµ¶·lmn
Œ¯°¸H¹†²pq”±pqº[»¼


VW

lmn

stIu

pq

Žƒ„
NOH=$
:Q786 !PQRS:A
犐犱犲狀狋犻犳犻犮犪狋犻狅狀犪狀犱犉狌狀犮狋犻狅狀犃狀犪犾狔狊犻狊狅犳犃狀狋犺狅犮狔犪狀犻狀犅犻狅狊狔狀狋犺犲狊犻狊
犚犲犾犪狋犲犱犕犢犅犌犲狀犲狊犻狀犕狌犾犫犲狉狉狔
LIJun1,ZHAOAichun2,LIUChangying2,LRuihua2,LIUXiaoqing1,YUMaode2
(1.GuiyangcolegeofTraditionalChineseMedicine,Guiyang550025,China;2.ColageofBiotechnologyofSouthwestUniversi
ty,Chongqing400715,China)
犃犫狊狋狉犪犮狋:Inourstudy,8anthocyaninsbiosynthesisrelatedMYBcandidategeneswereidentifiedbyusing
bioinformaticstechnologiesandtheexpressionprofilesindifferenttissuesandstagesoffruitsduringthe
developmentanddeterminedbytranscriptomedataandrealtimePCR.Phylogeneticanalysisindicatedthat
4MYBgeneswereclosedtoMYBgenesfrom犞犻狋犻狊狏犻狀犻犳犲狉犪L.,犗狉狔狕犪狊犪狋犻狏犪and犣犲犪犿犪狔狊L.,while
only1MYBgenewasgroupedintooneclusterwiththosefrom犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪and犕犪犾狌狊犱狅犿犲狊狋犻犮犪
L..Highleveloftranscriptsofmajorgenesinmaleflowerwasdeterminedbytranscriptomedata.Contin
uousdownregulationexpressionsof犕狀犕犢犅犑and犕狀犕犢犅4duringthedevelopmentofmulberryfruits
wereconfirmed,while犕狀犕犢犅330hadacontinuousupregulationexpression.Therefore,MYBgenes
havepositiveregulationandnegativeregulationduringtheaccumutionofanthocyanins.
犓犲狔狑狅狉犱狊:mulberry;anthocyanins;transcriptionfactors;regulation;expressionanalysis
  lmn½ž¾¿¹†À#$6Œ—ÁÂà n,ÄÀŐÆ4$6ŒÇÈ[1]。lmnŒ‘É
ÊËÌ2kÍΙ1kÏÐÑÒÎÉo,2Í;Í
ÓÔÕ5Öuά…×،OH ÙOCH3 ÚÛ;
Œz‚™ØÜ݁jlmnŒÞ
[2]。
lmnς
ߥŒ—ŠCDàáâ



•–ã

ä6V®6
lmnςå`5.768mg/g[3],敖åŠ
(0.059mg/g)[4]、áâåŠ(0.25~4.38mg/g)[5]
¥çè£
。Hassimottoã[6]”Kangã[7]éêë§
jV®lmnìÚ$·ÀíîïðñŒòÂóôõ
ö÷àßøŒù¼Ny

V®lmnHúù¼ûü
ýþÿŒ!"#Æ|$%GH&
[8]。
V®lmn
ìÚ$¸ù¼()þÿ‡*™+,-.TŒ+,4
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/0126+,ÐƜ/;IŒŽÓ/3+
,45H6

E7ù¼+,†126Œ¯°
[910]。
lmn†V®6Œ¯°89ÀäS$4o:;
|pq»¼

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ÄÀ½ÇÈ
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DÍEF-ñGH$

Ilmn4oJ
(ANS)ã/rJŒNyªSol
ÃK

>êIcû;ÆLMNolmn
[1]。
1JKþgPIQoVWlmnS$4oŒ/rJ;
IŒR™GHƒ„
[11],
·pq»¼ŒJKSTÀ
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S_,Zã

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€

abD|ýcdeã

ŒfˆNy

ä6€™a
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[12]。
JK§MYBstI
u

gä½R2R3MYB)½£[MhYTŒijk
ölm

nop”pqlmnS$4o/rJŒ
Ž—¦
。犕犢犅10™ 犕犢犅犃 ½qr1#$¢Š、s
|tâlmn4oŒpqIu
[13]。
š›6犕犢犅;
I犆1pqlmnŒ4o,½tO#$JKlmn
S$4oŒIuvÞ
[14]。
OJKLcS$Md%
efEVW;Ig6Rlmn4oœ/Œ犕犢犅
;Iµ¶ow

Lcstgz{|}~€‚
PCR^_JK犕犢犅;IŒŽvx,ƒ„yz;
I”V®lmn¯°/³

{|VWlmnS$
4oŒpq»}~;

1 €™ef
1.1 T U
2014‚3~5ƒ„À!\]%VW…†‡ˆÚ
V‰[
(‘
Š‹30h’)6k~Œ(lê0、1、2、3、4
™5Ž)ŒŠ},ƒ­“~ŒS0~S5,y‘’
“ꔹÀ-80℃•–6—y。˜™š›pMD19
T、TaKaRaEx犜犪狇J、õstJ MMLV、RNAiso
Plusœž、DNaseⅠ、DL2000DNAMarkerãŸ
 ¡S$¢‹



थ¦

]§¨©
(犈狊犮犺犲
狉犻犮犺犻犪犮狅犾犻)©ªDH5០«¬SÆ3^("­)à
¤¥¦

ð$Ì\­<®¯¥¦4o

1.2 V W
1.2.1 45 犕犢犅,@XY:ZBCD ENCBI
ªšlmn4oœ/ MYB;I(¢Š MdMYB10、
MdMYB110a™ MdMYBA,•– VvMYBA, ¡PAP1、PAP2,š›ZmC1)ŒF;-°±,Lc
BlastP‹°²|VW;Ig6Œ³´lm°±,Œ
µ¶e10。·hiŒŒµ¶ß¸ŒF;-°±N
query°±,²|¢Š、•–、tâŒ;Ig|³
´;I°±

\2@‘Šž¹,º»n³ˆ†
;I

1.2.2 45 犕犢犅,@XYB[\]^_HI`
a,bcdef lm˜‘ɼ³´†SMART
(http://smart.Emblheidelberg.De/smart/set_
mode.cgiNORMA)¬½¾,lm˜;O˜ƒ„
¿y ExPasy (http://web.Expasy.Org/)。y
ClustalW ½¾£°±æ·,>êyGeneDoc·æ·
‘Š½¾ÀÁ

¿y MEGA4.0ÂÃ6ŒÄof
(NeighborJoining,NJ)ÉųƽÆW。
1.2.3 45678gh 犕犢犅,@XYB>?i
jkHI stŽz{džÀµÈɵ;Ig`
Ê}ËÌQoŒVWstg´°‘Š

÷à¬

Í

ÎÏ

¤l™Ð5kg‰,¬{stg;IŽÑb
RPKM¶[Reads/(Fragments)PerKilobaseofex
onmodelperMilionmappedreads]҂ 犕犢犅 ;
I†…g‰6ŒŽ—¦

1.2.4 678lmBno V®lmnìڔ´
‚pÓRabinoã[15]Œef。·-80℃”¹ŒV®
†‘6JÔoÕ

ÖÚ0.1gÕiY2mLÖ×Ø
6

3Ù600μL³ÚŒ-ÂÛÜ(Ï1% HCl),4℃
ªìÚ12hê,3Ù400μLddH2O™400μLÝ
Þ

߃àáê12000r/minÖ×10min,Ú¬4
ƒ­²´›†530™570nmâ_ªŒã€¶,l
mnœ·Ï‚=犃530-0.25犃570。
1.2.5 45l 犚犖犃 pq`犮犇犖犃 9: …Ú
0.1gäåå‰À³ÚŒJæ63‘ç’JÔo
Õiè

péRNAisoê윝ž(TaKaRa)ëNì
§ìÚíRNA,IDNAJîÆêy50μLDEPC
—Áï

¿y1%ð+ûñïòó²´Óxyôõ
ƒ€€bö´íRNA÷b。DíRNAvø,
DOligo(dT)15ð$,xy MMLV õstJ
11116Œ           = >,ã:VWlmn4oœ/ MYBstIuŒR”GHƒ„
(TaKaRa)4ocDNAù1ú,Ü-80℃”¹—y。
1.2.6 r\stEuvwxDm犘犆犚no ÌÀ
lmnœ/犕犢犅;IŒŽ”lmn¯°÷àû
0Œœ/Â

=†Š},ZêŒàüýþŒÿÆ

I
´wÚj

Š‹30h’4k~Œ(S0、S3、S4™S5)Œ
Š}ÇJK犕犢犅;Iµ¶owŽ—¦,!w”
lmn4oœ/Œ犕犢犅;I。¬{ 犕犢犅;IË
K-"#°±¿yPrimer5.0ƒ­$ö8kvw
;IŒ}~€‚PCRð$(1),‘Š¿y
2-ΔCt “。} ~  €  ‚ PCR % y SYBR
PremixExTaqTM(TliRNaseHPlus)Ÿ TaKaRa
¥¦

%&|YŒäPÆ%œÉ(ƒ„)

2 ‘Š”ƒ„
2.1 45=yz[\9:;<犕犢犅,@BCD
ªšPI&*ŒpqlmnS$4oŒ犕犢犅
;IF;-°±

LcBlastP‹°²|ˆ† 犕犢犅
;I

EVW;Ig6Rç8k”ÇÈS$4
oœ/Œ 犕犢犅 vw;I(2),ƒ+À‡ˆŒ
scaffold¬,2k;IŸ1kZÏu,5k;I¹†2
kZÏu
,犕狀犕犢犅犅 à11kZÏu,¸ H¹†ß
,-Œ./ex

yz;IÀ0261~478kF;
-

œ·ƒu‚]þE29.2~54.0kD。3k MYB
lm
(MnMYB86、MnMYB11™ MnMYB21)ÄÀ
1-Âlm˜
,MnMYB330ã5klmÄÀ2Âl


£°±æ·’“ 6 k MYB lm (Mn
MYB308、MnMYB330、MnMYB86、MnMYB4、Mn
MYB11、MnMYB21)ŒF;-°±6Ïà”3Œ
R2R3‘ɼ(41),MnMYBB” MnMYBJ”ä
5;I6#ß]
。MYBlm6Ïà5k”3ŒÃ
F-
(W)7;,MnMYBJ;IŒù3kÃF-89F
-
(Glu,E),MnMYBJ™ MnMYB4Œù4kÃF-
ƒ­8ÍEF-
(Phe,F)™:F-(Ser,S)ÚÛ,Mn
MYBB” MnMYBJŒù5kÃF-ƒ­8;F-
(Tyr,Y)™Û=ÿ¸H” MYBlmŒGHàžŒ>³。
2.2 45 犕犢犅,@XYB{=HI
j?´8kVW MYBlmŒGH,ÉÅj
MYBlmF;-°±Œ³Æ½ÆW(42)。VW
5k MYBlm¸ƒ­@Ù2kA,ÇÈ
4oœ/ MYB;I。MnMYB11、MnMYB330、
MnMYB308、MnMYB86”BuDkMyb4(犇犻狅狊狆狔狉狅狊
j1 |}~€rTable1 Primersusedinthisstudy
;ICÖGenename ¬Dð$Forwardprimer(5′→3′) ªDð$Reverseprimer(5′→3′)
犕狀犕犢犅308 GGTGGATTTGTGAGCAAAGA GCTGTTGTGGCTGATAAGGA
犕狀犕犢犅330 AAAGCCCACACCAATAAAGG TATCCATCTCAGCCTGCAAC
犕狀犕犢犅86 GCGCGAGCTATTTGTAATGA TGCTGTCAAGGCTTCCATAG
犕狀犕犢犅犅 TCATGTCCCATACAGCAGGT ATCCCTTTATCGCATTCACC
犕狀犕犢犅4 GTTTCGCGAGATCAAAGTCA TCTTAGTGCGACATGCTTCC
犕狀犕犢犅犑 CAAGCATCCATGACATCACA ATTGTCGCAGAGTGATCTGG
犕狀犕犢犅11 AAAGCCACCCGTACTACCAC CATCAACCGCAGGTTTAGAA
犕狀犕犢犅21 AATAATCCCAAGTGCCCAAG GTTTGGCAGAATTGACGATG
j2 45678[\9:;<犕犢犅,@XY:Z
Table2 Identificationofflavonoidsbiosynthesisrelated犕犢犅genesinmulberry
;ICÖ
Genenane
;IEth
GeneaccessionNO.
;IgØ
Genomelocation
ZÏukz
Intronnumber
À0F;-
Encodedaminoacids
ƒu‚
Molecularweight/kD
}FãòÊ
Isoelectricpoint
犕狀犕犢犅330 XP_010104477.1 scaffold485 2 305 34.1 9.22
犕狀犕犢犅86 XP_010089049.1 scaffold297 2 298 33.8 6.56
犕狀犕犢犅犅 XP_010096371.1 scaffold155 11 478 54.0 8.79
犕狀犕犢犅4 XP_010107438.1 scaffold759 2 304 34.8 8.79
犕狀犕犢犅犑 XP_010109645.1 scaffold751 1 319 36.3 8.81
犕狀犕犢犅11 XP_010107990.1 scaffold959 2 364 41.0 5.85
犕狀犕犢犅21 XP_010099251.1 scaffold20 2 327 37.1 6.06
犕狀犕犢犅308 XP_010090332.1 scaffold137 1 261 29.2 8.97
2111 ! " # $ % &                   36G
HIJR2、R3lm‘ɼ;KÃeLJ”3ŒÃF-(W)7;;MnMYBJ.MV(XP_010109645.1);MnMYBB.MV(XP_010096371.1);
MnMYB21.MV(XP_010099251.1);MnMYB86.MV(XP_010089049.1);MnMYB4.MV(XP_010107438.1);MdMYB110a.
¢Š
(BAJ24837);MdMYB1a.¢Š(ACQ45201);MnMYB330.MV(XP_010104477.1);MnMYB308.MV(XP_010090332.1);
MnMYB11.MV(XP_010107990.1);DkMyb4.Bu(AB503701);PAP1. \¡(NP_176057);C1.—˜(CAA75509);
P.š›(AAK81903)
41 VW MYBlm£°±æ·
TheblacklinesindicatedconservedR2andR3domain;conservedTryresidueswasshownbysolidbox;MnMYBJ.犕狅狉狌狊狀狅狋犪犫犻犾犻狊
(XP_010109645.1);MnMYBB.犕狅狉狌狊狀狅狋犪犫犻犾犻狊(XP_010096371.1);MnMYB21.犕狅狉狌狊狀狅狋犪犫犻犾犻狊(XP_010099251.1);MnMYB86.
犕狅狉狌狊狀狅狋犪犫犻犾犻狊(XP_010089049.1);MnMYB4.犕狅狉狌狊狀狅狋犪犫犻犾犻狊(XP_010107438.1);MdMYB110a.犕犪犾狌狊犱狅犿犲狊狋犻犮犪L.(BAJ24837);
MdMYB1a.犕犪犾狌狊犱狅犿犲狊狋犻犮犪L.(ACQ45201);MnMYB330.犕狅狉狌狊狀狅狋犪犫犻犾犻狊(XP_010104477.1);MnMYB308.犕狅狉狌狊狀狅狋犪犫犻犾犻狊
(XP_010090332.1);MnMYB11.犕狅狉狌狊狀狅狋犪犫犻犾犻狊(XP_010107990.1);DkMyb4.犇犻狅狊狆狔狉狅狊犽犪犽犻Thunb.(AB503701);PAP1.
犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪(NP_176057);C1.犗狉狔狕犪狊犪狋犻狏犪(CAA75509);P.犣犲犪犿犪狔狊L.(AAK81903)
Fig.1 ProteinmulialigmentofMYBgenesinmulberry
犽犪犽犻Thunb.,AB503701)、•– VvMybPA1(犞犻狋犻狊
狏犻狀犻犳犲狉犪,NM_001281231)、—˜C1protein(犗狉狔狕犪
狊犪狋犻狏犪,CAA75509)| š › P (犣犲犪 犿犪狔狊,
AAK81903)@Ù GroupⅠ。MnMYB21” ¡
(犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪)ŒPAP1(NP_176057)™
PAP2(NP_176813)、¢Š(犕犪犾狌狊犱狅犿犲狊狋犻犮犪)ŒMd
MYB10(ACQ45201)™ MdMYB110a(BAJ24837)@Ù
GroupⅡ。MnMYB4、MnMYBB™ MnMYBJNà
@Ù2kA,””3ÂØÊÃF-7;=ÿàß
¥Œž¹Â
,¸
Hp”ÇÈS$4oDõŒä5
ÛAO;Œpq

2.3 45犕犢犅,@B>?iHI
Lc·VWstgz{PŒ²||ƒ„

,Q
8k 犕犢犅 ;I†Rl6ूŒŽ(43)。
ä6犕狀犕犢犅86Ÿ†Rl6]‚Ž,†ä5g‰
6N಴YstO
;犕狀犕犢犅犅 †Rl|Ð6
Ž‚ߥ

†¬

Í|ÎÏ6Ž‚߸
;犕狀
犕犢犅4†Rl6H²´YŽ,†ä5g‰6Ž
‚߸
;犕狀犕犢犅犑†Í™Rl6²´YŽ,†
¬

ÎÏ|Ð6N಴Y
;犕狀犕犢犅11†Rl|Ð
6S÷àߥŒŽ

†¬

Í™ÎÏ6Žß
T
;犕狀犕犢犅21†Rl6]‚Ž,†Í|Ð6à
31116Œ           = >,ã:VWlmn4oœ/ MYBstIuŒR”GHƒ„
46ƒCʌzU“;À1000@`,bVʌ -C¨W;XۍXYYÖ
42 MYB;Iµ¶½Æ/³
Thenodesinthefigureshowthebootstrapvalueonreplications,andthescalerepresentthegeneticdistance
Fig.2 ThephylogenetictreeofMYBgenefamilyinmulberry
43 VWlmnœ/犕犢犅;IŽ
Fig.3 Transcriptionanalysisofanthocyaninrelated犕犢犅genesinmulberry
T‚ŒŽ

¬|ÎÏN಴YŽ
;犕狀
犕犢犅330†Í|Rl6Ž‚ߥ;犕狀犕犢犅308
†Rl|Ð6¥‚Ž

Í6àT‚Ž

¬|Î
Ï6Ž‚߸

2.4 4:‚ƒ„N678…mBoD
V®½uތlŠ

EBÕYŠ}QZo[
\DI]]^35~40d,¬{Š}"_”Š}õ`
·akŠ},Z~ŒƒS0~S5 ã6k~Œ,ä6
S0 bBÕQc,S5 Š}QZo[,dk~Œ]
efg7d。Lc´‡ˆ~Œlmnς,,QE
S0~S4 Œ5k~Œ6lmnŒÏ‚߸,7†S4~
S5 S_c‹6,lmnςÿÆj·h8þ,ì§
yk~Œ½lmnŒi’4o~Œ

44)。
2.5 犕犢犅,@XY†4‡ˆƒ„NBjkHI
j½žU!w”VWlmn4oœ/Œ
犕犢犅;I,Lc}~€‚PCRƒ„;I†l
mn4oßiŒük~Œ
(S0、S3、S4 ™S5)ŒV®
6ŒŽ—¦

‘Š’“8k;IjQ‡ˆŒŽ
vx

45),犕狀犕犢犅308†Š},ZŒ4k~ŒS
÷àߥŒŽ
,犕狀犕犢犅犑™犕狀犕犢犅4klŠ}
Œ,ZjQªpŽŒmn
;犕狀犕犢犅6†Š},Z
6jQoŒŽ‚¥™pŒŽ‚߸
;犕狀犕犢犅犅
jQéªpŽq¬pŽŒmn
;犕狀犕犢犅330
†Š}Œ,Zc‹6jQ¬pŽ

=†Š},Z
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