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Cloning and Expression Analysis of Sulforaphane Synthesis related Gene BoCYP83A1 in Broccoli

青花菜萝卜硫素合成相关基因BoCYP83A1的克隆与表达分析



全 文 :书!"#$%&
,2016,36(7):1302-1307
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
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RTPCR…†,‡ˆ犅狅犆犢犘83犃1,mv‰Š‹Œ。y,m‰Š1509bp,Ž502,‘,’“”•vP450–
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Q™š›œ4žPCRŸ ¡犅狅犆犢犘83犃1,m¢£¤K]、£¤¥¦|§£¤¨r©ª«v¬­V®。
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¢}~JK]CDBY10v¾、¿、À3¥¦º¬­V®Á¼ÂÃ,¢K]CDBY12u¬e¿>À>¾,¢
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Ë
,犅狅犆犢犘83犃1,m¬­žÌ?ÍÎÏv1.9Ð,ÑËiҞ«Ó;ÊSA©ªË,犅狅犆犢犘83犃1,m¬­V®ÔÕ
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Ú¬­ÆÇÛ MeJA„SAÜÝÞ。}~J犅狅犆犢犘83犃1vßà§á4â_?opq
r“žv}~JãK]ä4¡ªå,æ

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opqr
;犅狅犆犢犘83犃1;çè‘éê;Vë‘
LMFN$
:Q785;Q786 !OPQR:      !"#R:
犆犾狅狀犻狀犵犪狀犱犈狓狆狉犲狊狊犻狅狀犃狀犪犾狔狊犻狊狅犳犛狌犾犳狅狉犪狆犺犪狀犲犛狔狀狋犺犲狊犻狊狉犲犾犪狋犲犱
犌犲狀犲犅狅犆犢犘83犃1犻狀犅狉狅犮犮狅犾犻
GAOCanhong1,DONGLili1,GUANXiaowan1,ZHAOLiangxia1,LINJuncheng2,
XUFule2,ZHANGShuiming1
(1AnhuiAgriculturalUniversity,Hefei230036,China;2FuzhouChaodaModernAgricultureDevelopmentCo.Ltd.,Fuzhou
350003,China)
犃犫狊狋狉犪犮狋:犆犢犘83犃1isakeygeneinsulforaphanebiosynthesisandmetabolism.Thefullengthof犅狅犆
犢犘83犃1wasobtainedfrombroccoliCDBY10bythemethodsofRACEandRTPCR.犅狅犆犢犘83犃1was1
509bpinlength,encodedadeducedproteinof502aminoacids,andcontainedtheconservedP450domain.
Theexpressionlevelof犅狅犆犢犘83犃1indifferentcultivars,tissues,orseedlingstreatedwithdifferenthor
moneswasanalyzedbyrealtimefluorescencequantitativePCR.Phylogenetictreeanalysisshowedthat
BoCYP83A1hadtheclosestrelationshipwith犅狉犪狊狊犻犮犪狅犾犲狉犪犮犲犪var.狅犾犲狉犪犮犲犪BoCYP83A1.Tissuespecific
expressionof犅狅犆犢犘83犃1incultivarswasdifferent:expressiondifferenceinCDBY10wassmal.It
showedstem>leaf>rootinCDBY12,whileroot>stem >leafinCDBY14.ThetreatmentsofMeJA
andSAledtothechangesof犅狅犆犢犘83犃1expression:aftertheMeJAtreatment,theexpressionof犅狅犆
犢犘83犃1wasraisedto1.9timesofthecontrol,andthendecreasedalittle;AftertheSAtreatment,theex
pressionof犅狅犆犢犘83犃1reducedquickly,andupto0.1timeofthecontrolat6h,thenrestoredgradualy
totheexpressionlevelofcontrol.Theresultsindicatedthattheexpressioncharacteristicsof犅狅犆犢犘83犃1
inbroccolicultivarsweredifferent,and犅狅犆犢犘83犃1expressioncanberegulatedbyMeJAandSA.Clo
ningandcharacterizationof犅狅犆犢犘83犃1geneprovidethetheoreticalbasisforbreedingnewbroccoliculti
varswithhighsulforaphanecontent.
犓犲狔狑狅狉犱狊:broccoli;sulforaphane;犅狅犆犢犘83犃1;MeJA;SA
    } ~ J (犅狉犪狊狊犻犮犪狅犾犲狉犪犮犲犪.var.犻狋犪犾犻犮犪
Planch)ìíîïðñi”òóô,ng­(7õ
ž¸2vIJö÷

Ûø

IJùú
”[1]。
}~J
¸ñ»ûvnï“üÆsŸ
———
opqr
(Sul
foraphane,SF),ysŸñiýþvÿ7!"$#
$

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¢")*+

u+

,-
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n/01¢IJugev2"
3½¸þv4*3½sŸ
[23]。
56Ÿ7M$%v
gh

‚ƒ,m89:;}~J_]vã…=

opqr>1992?¢}~JuÛge[4],n@
ABCqD
(Aliphaticglucosinolates)4é,EF
GH,qD
(Glucoraphanin,RAA)Ó#Msv÷
]¼qI‘J

:P¬W

opqrv`sftK
LÂMN

EFŸ5O;Ps。QR`s*S
T$éq‘

5Ë|éq‘ÈUV$

ʙW
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qD[\–—v]s„WXv^_`a
b3O;]sRAA。¸Ë,RAA¢cd7eS
ƒ«Ÿ#sopqr
[57]。
fM$™9g§犎犕犜、
犕犃犕、犆犢犘79/犆犢犘83、犃犗犘 bh`s,m(
C
[6]。
i犅狅犎犕犜 ©>opqr`sftKLv
¸*S

Îéq‘v“žV®ÈwxÝÞSƒ
[5];
犕犃犕1n犕犃犕 ,m(CuÞ&V$éq‘W
XYZ

]sjXqDkT$v,m
[5];犆犢犘79/
犆犢犘83,m(CnqD[\–—]svwx,m,
Úu犆犢犘79犉1l±`sŠXqD[6],犆犢犘83犃1Ä
ÝÞqD=ABmqD`s

qDWX^_`a™
9uEFn 犃犗犘 ,m(CÝÞ,Úu 犃犗犘2„
犃犗犘3Ÿol±]spq,qD„rs,qD[7]。
犆犢犘83犃1,mn(ûrt±eP450,m(C
ö÷
[8],
>*¢uvd
[911]、
wJ
[12]、
ÃxJ
[13]、
!
y~
[14]
b h ] # $ u Û á 4

z u v d u
犆犢犘83犃1,m¢xJu™ž¬­,xJuABC
d7wD„{d7wD“žŸo|}4.5„2
Ð
[15]。
!y~犆犢犘83犃1,m¢uvdu™ž¬
­

~,m#uABCd7wD 4MSOB „
4MTBÅiW€Ì,ðABCd7wD{žÌ
?
[14]。
z‚Juv犅犼狌犆犢犘83犃11¢uvdu™
¬­6È¡ABCd7wDvƒ„

{À…d7w
D|}1.41~1.78Ð,~,m#uABCd7
wD|} 1.53~1.96 Ð[16]。†`‡:P,
犆犢犘83犃1,mv™ž¬­ÆÇ6ÈABCd7w
Dˆ{d7wD“žv|}

Ñopqrv]s|
ABCd7wDvŸ#*<

mfABCd7w
D“žv
Œ:P‚ƒ RACE …†G}~Jußà¡
犅狅犆犢犘83犃1,mv‰Š‹Œ,Ÿ ¡ 犅狅犆
犢犘83犃1¢£¤K]、£¤¥¦º§ MeJA „SA
¨r©ªŽ«v¬­»½

y:P£7÷‘
:P犅狅犆犢犘83犃1vŸ7M$%üÆä4,æ,’
?“žopqrvãK]â_<“¡,m”‚

1 €5…†
1.1 STUV
•3opqr“ž£¤v}~JK]CD
BY10、CDBY12„CDBY14z{€,–h}
~J£¤K]

£¤¥¦º,m¬­Á¼v:P

z3K]—>˜™128š›œu,K]10

,3_žM。Ÿ _¡¢ªÍ3À1\›£¤。
Ÿoz¤÷žMv10Ÿ¾、¿、À3]¥¦£¤,
ÔՃ§¨Õ©Ë~ª-80℃«¬­®¯ƒ。
|CDBY12z{€,–h°^¨rÝÞ«
,mv¬­Á¼:P

±ƒ²â†zCDBY12—
]>g³´
(10cm×10cm)u,ƒ5mLµ¶Vz
·²¸¹

*Ëi‹—]100º|”»¼½÷!,
©ª—]3žM。¾g³5d›,Ÿoƒ
1mmol/LMeJA„3mmol/LSA¿À©ª,|¿
Àµ¶Vv©ªÎÏ

¿ÀžÞ&÷!

¿À©
ªË3、6„9hÁ£¤1_,ÔÕª§¨Õ©,Ë
~ª-80℃¯ƒ。
1.2 W X
1.2.1 ,?YZ[\@AB ±ƒTrizol†Ã<
30317+          ?@A,b:}~Jopqr`sÄw,m犅狅犆犢犘83犃1vßà5¬­Ÿ 
}~JÀ…v{ RNA,ÅƒSMARTTM RACE
cDNAAmplicationKit(Clontech)7¼Æ~LJˆ
cDNA。¾ÈNCBIÉÊÇvuvd、wJ、op
b$]v犆犢犘83犃1‹ŒËÌ6$83PF„83
PR,7¼,||‡ˆuº…;。¾È‡ˆv‹ŒË
Ì3’Ÿ=6$F3′1、F3′2„Æ=6$R5′1、R5′2,
5Ë5Í46$ UPM、NUP7¼5′„3′ÎSv,
|

zÏП7¼Ñ‹ˆÒ,mv‰Š‹Œ

ËÌ
‰Š6$83HSF„83HSR,‚ƒ?”ÓePrim
erSTARHS7¼PCR,|ÆÔ,|Îыv‰Š
‹Œ7¼ÕŸ

1.2.2 ,?]^_`aFGbcdefg@hi
 ‚ƒDNAMAN2.67¼£¤$]ºÖx‹Œv
×Î

±ƒØ‹†¢ MEGAu—Ù¯°g_²,Ë
ÚBootstrapžMŸ 500_。‚ƒSignalP7¼
RÛÜÝÞ

‚ƒPDB7¼Pfamdomain„ Motif
ÝÞ

1.2.3 ,?DEFG ±ƒTRzol(Invitrogen)
†Ã<¤Kv{RNA,ÅƒDNaseⅠ(TaKaRa)
zßàvDNAᱩª,5Ë£2μgRNAŃ
PrimeScript RTreagentkit(Takara)7¼Æ~
Ç

¾Èɇˆv犅狅犆犢犘83犃1,m‰ŠËÌ6$
83RTF „ 83RTR,| 18S  â ã , m,ä Ï
SYBRPremixExTaqTMⅡ (Takara)zå´v
ñæçS‘è¢ABI7500œ4žé7¼,|,
Äά­žvÌêŃ2-ΔΔCT…†。¥¦»¼½¬
D1 jST=>@k^[Table1 Relatedprimersequencesof犅狅犆犢犘83犃1
intheexperiment
6$ëì
Primername
6$‹Œ
Primersequence(5′→3′)
83PF ATGGAAGATATCATCATCGG
83PR GAACGGTATAAACTCGTAGT
UPM
CTAATACGACTCACTATAGGGCAAG
CAGTGGTATCAACGCAGAGT
CTAATACGACTCACTATAGGGC
NUP AAGCAGTGGTATCAACGCAGAGT
F3′1 ACAGGTCTCACGGCTTATAT
F3′2 TAGAGAATGTCAAAGGCGTG
R5′1 CATTCCCATCTTCCTCATCT
R5′2 CCACCATTGTTTGACTTCCT
83HSF ATGGAAGATATCATCATCGG
83HFR TTATTTGCTCACTTTCTCCG
83RTF CTTCAAAGGCACGGACTA
83RTR AACGGAACCTCAAGCATCG
­Ÿ |À…uv¬­žÎÏ

£¤¨r©ªu
|CKv¬­žSÎÏ。
íȱƒExcel2003、DPS7.05、Agilent1100
offlinestudioî7¼°ÌŸ 。
2 –ô5Ÿ 
2.1 456犅狅犆犢犘83犃1,?犮犇犖犃YZ@AB
|83PF/83PR6$7¼PCR,|,‡ˆ
犅狅犆犢犘83犃1vuº…; (ï1,A)。Ń F3′1/
UPM、R5′1/UPM6$7¼ð÷ñvPCR,|Æ
Ô

|ÆÔT$Sòó

ŸoƒF3′2/NUP、R5′2/
NUP6$7¼ð^ñvPCR,|ÆÔ,‡ˆ750
bpôõv3′…; (ï1,C)„260bpôõv5′…;

ï
,1,B)。zÏП7¼Ñ‹ˆÒ1630bp‰Š
‹Œ

5Ë|83HSF/83HSR6$,Ń?”
Óe7¼,|

|Îыv‰Š‹Œ7¼ÕŸ

|f
ö4}~Ju犆犢犘83犃1v‰ŠcDNA‹Œ(ï1,
D)。‚ƒ NCBIv ORFFinder7¼Ÿ ,}~J
犆犢犘83犃1,m‰Š‹Œ1509bp,Ž502,
‘

ï2),÷ë犅狅犆犢犘83犃1。
2.2 犅狅犆犢犘83犃1,?@[\befgFG
z犅狅犆犢犘83犃1,mŽv,‘‹Œ5Úø
$]v,‘‹Œ7¼×Î

geBoCYP83A15
wJBnCYP83A1vÄù½¸?,­Ò97.01%。
5uvdAtCYP83A1、opRsCYP83A1Äù½Ÿ
o87.67%„96.02%。
‚ƒESyPred3D¢úÝÞBoCYP83A1Öx
LÏû–—

Ú?ü–—ñi H x306,ýþí
20,~ÿí44,ÖxLŸ7ž57.52kD,b
M.DL2000;A.uº…;T$;B.5’RACET$;
C.3’RACET$;D.‰ŠcDNA
ï1 犅狅犆犢犘83犃1,mv,|–ô
M.DL2000;A.Internalfragment;B.5’RACEfragment;
C.3’RACEfragment,D.ThefullengthcDNA
Fig.1 PCRamplificationresultsof犅狅犆犢犘83犃1
4031 ! " # $ % &                   36!
"#7.96。Q™SignalPNNÝÞyÖx¢ð25~
26,‘v$Úñi1RÛܖ—。Q™
PfamíÈ%×Ο ,BoCYP83A1Öxöš
P450(C,m,–—˜&ð31~494,‘,
ðð4~21,‘‹Œ1&(Äw–—(ï2)。
¡ö4BoCYP83A15Úø$]CYP83A1
v¶·w¯

‚ƒ MEGA5.05 î,z BoC
YP83A15Éßàvuvd、wJ、op、´ µ、dJ
„E)‚b6$]vCYP83A1s7¼hž‹Œ
×΄¯°7±Ÿ 

–ô€*
,BoCYP83A1„
´µBoCYP83A1(2)v¶·w¯¸¹,+÷,,
Ñ5uvdAtCYP83A1„E)‚CsCYP83A1¶
·w¯Â-

ï3)。
2.3 犅狅犆犢犘83犃1,?lmnopmnqrs@D
Etu
  Ÿ }~J犅狅犆犢犘83犃1,m¢£¤K]v
¾

¿

À¥¦uv¬­V®

–ôiï4Ü*。Ú
u
,犅狅犆犢犘83犃1,m¢CDBY10v3¥¦º¬
­V®Á¼ÂÃ

¢CDBY12u¬e¿>À>
¾

Ñ¢CDBY14uĬe¾>¿>À。¢¾¯
u
,CDBY14犅狅犆犢犘83犃1,mv¬­ž€.?>
Ú/K]

¢¿u
,CDBY14犅狅犆犢犘83犃1,mv
¬­V®’ÄÎÚøK]Â?

ÄÁÂÃ

Ѣ…u
,CDBY10犅狅犆犢犘83犃1,mv¬­ž?>
CDBY14„CDBY12。
2.4 v:wxy犅狅犆犢犘83犃1,?@DEtu
Σ¤¨r£¤›º©ªË
,犅狅犆犢犘83犃1,
m¬­ž7¼0Þge

Ê MeJA©ª3h›,犅狅
犆犢犘83犃1,m¬­žÌ?ÍÎÏv1.9Ð;56›
ºv12
,犅狅犆犢犘83犃1,m¬­V®¢6„9h›
iҞ«Ó

3?>ÎÏ

ÊSA ©ª3h›,
犅狅犆犢犘83犃1,m¬­V®ÓÖ,ð¢6h›­Ò¸
Ö

ÎÏv0.1Ð,Ñ9h›iÜÙÌ,ð4?>
CKv¬­V®(ï5)。
AtCYP83A1.uvd (NP_193113);BnCYP83A1.wJ (NP_001303017);RsCYP83A1.op(AHB11194.1);
ïucû

Aû§µûŸof¬,‘P‰”•

П”•§Äù

ï2 犅狅犆犢犘83犃1,m14v,‘5Úø$]CYP83A1sv×Î
AtCYP83A1.犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪(NP_193113);BnCYP83A1.犅狉犪狊狊犻犮犪狀犪狆狌狊(NP_001303017);RsCYP83A1.犚犪狆犺犪狀狌狊狊犪狋犻狏狌狊
(AHB11194.1);Aminoacidshighlightedinblack,redandblue,respectivelyrepresentresiduescompletelyconsewed,partialyconservedand
similartoconsensus.
Fig.2 Alignmentofthepredictedaminoacidsequencesof犅狅犆犢犘83犃1withotherCYP83A1s
50317+          ?@A,b:}~Jopqr`sÄw,m犅狅犆犢犘83犃1vßà5¬­Ÿ 
£¤$]vCYP83A1ÖxÊÇÛi«:BoCYP83A1(2).´µ
(XP_013636864);BnCYP83A1.wJ (NP_001303017);RsCYP83A1.
op
(AHB11194.1);BjCYP83A1.‚J (AGO37757);AtCYP83A1.
uvd
(NP_193113)CsCYP83A1.E)‚ (XP_010495830)
ï3 £¤$]CYP83A1sv¯°7±Ÿ 
CYP83A1proteinaccessionnumbersfordifferentspeciesareas
folows:BoCYP83A1(2).犅狉犪狊狊犻犮犪狅犾犲狉犪犮犲犪var.狅犾犲狉犪犮犲犪
(XP_013636864);BnCYP83A1.犅狉犪狊狊犻犮犪狀犪狆狌狊(NP_001303017);
RsCYP83A1.犚犪狆犺犪狀狌狊狊犪狋犻狏狌狊(AHB11194.1);BjCYP83A1.
犅狉犪狊狊犻犮犪犼狌狀犮犲犪(AGO37757);AtCYP83A1.犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪
(NP_193113)CsCYP83A1.犆犪犿犲犾犻狀犪狊犪狋犻狏犪(XP_010495830)
Fig.3 PhylogeneticanalysisamongdifferentCYP83A1s
£¤Ã567¬*£¤K]}~J8¡¥¦º,m
¬­ž¢0.05V®Á¼€.½
ï4 £¤K]}~J8¡¥¦º犅狅犆犢犘83犃1
,m¬­Á¼
Normallettersindicatesignificantdifferenceof犅狅犆犢犘83犃1
expressionamongdifferentcultivarsortissuesofbroccoli
at0.05level
Fig.4 Expressiondifferencesof犅狅犆犢犘83犃1gene
amongdifferenttissuesorcultivarsofbroccoli
3 9 å
Œ:PG}~Jußà¡犆犢犘83犃1v¤^,
m

‹Œu’“”•vP450üƘ–—,mf÷ë
犅狅犆犢犘83犃1(ÊÇÛKU573063)。¯°7±²
Ÿ €*
:BoCYP83A15Úø$]vCYP83A1È
^Ĥ

öšnCYP83A1(Cv¤^,m,¢Ë
:7±v£¤›+

5Úø$]Ÿ;–:
。BoC
YP83A15–³´µBoCYP83A1(2)v¶·w¯¸
£¤Ã567¬*£¤¨r£¤©ª›º
¢0.05V®vÁ¼€.½
ï5 £¤¨r£¤›º©ª«犅狅犆犢犘83犃1
,m¬­Á¼
Normallettersindicatesignificantdifferenceanomgtreatments
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[2] STU,VWX,?@A,b.}~Jq、YftZ[§ÚΔ
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