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Molecular Cloning and Expression of ScABP4 Gene in Sugarcane

甘蔗生长素结合蛋白ScABP4基因的克隆与表达



全 文 :书!"#$%&
,2016,36(5):0865-0873
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‡

ˆ,-‰‡RTPCRYŠKMN‹Œ犅犪犱犻犾犪Ž‘’1“犃犅犘7i,”•U–—˜ˆ7i[™šE›
œ
(ORF)zž615bp,Ÿ›204“ 7¡。¢”•£¤¥?¦§¨©U–ª«¬Q)­(犛狅狉犵犺狌犿犫犻犮狅犾狅狉)¥®
¯
(犣犲犪犿犪狔狊)[ABP4~[°±²?³´,iµ¶ˆ7i·¸ž犛犮犃犅犘4。¶¹ºl‘P»ª¼½>pGEX6P
1,¾gª¼¿À“UVÁž22.5kD[~。ÃÄÅÆÇ|ȗ˜ScABP4IJÆÇÉÄÅÊËÌ|º¥
ÄÅÍ

H$]Î%U–—˜ScABP4ÏÀ“ÐÑ]€Ò[UÓ~,Ô«ScABP4~ÕhÖ×DØÊˍÙD
ÊÍÚÛܹH$%gh

ÝÞÆÁPCRU–|Ȫ«,犛犮犃犅犘47iDMN[ß、à、á、âãѪ¼,¹D
ߍ䤪¼Á³)

Hz{¥åæçèé犛犮犃犅犘47i[ª¼Úƒ,Ô«犛犮犃犅犘47iÕhêQMN¤Hz{
[ëìíQÞ]€\î[ST

µï
,ABA、JA、SA、CuCl2 ðñhòóe犛犮犃犅犘47i[ª¼,CdCl2 ðñÕôX
犛犮犃犅犘4[ª¼。õµö÷,犛犮犃犅犘47iÕhêQMN¤Oø、ùú、ûü、t8ý…ðñ[ìþÿ!。ˆ,-|
Ȟ"#MNHz{|}~7i[gh$%’vw&

FGH

MN

Hz{|}~

()Ž

ª¼U–
IJKL$
:Q785;Q786 !MNOP:A
犕狅犾犲犮狌犾犪狉犆犾狅狀犻狀犵犪狀犱犈狓狆狉犲狊狊犻狅狀狅犳犛犮犃犅犘4犌犲狀犲犻狀犛狌犵犪狉犮犪狀犲
DENGYuqing,ZHAIYushan,PENGLei,DONGMeng,XUQian,
CHENGGuangyuan,LINYanquan,XUJingsheng
(FujianAgricultureandForestryUniversity/KeyLaboratoryofSugarcaneBiologyandGeneticBreeding,MinistryofAgricul
ture,Fuzhou350002,China)
犃犫狊狋狉犪犮狋:Auxinbindingprotein(ABP)playsanimportantroleinauxinsignaltransductionandtheregula
tionofplantgrowthanddevelopment.Tostudythefunctionandexpressionfeaturesofthe犃犅犘genein
sugarcane,weclonedan犃犅犘genefromasugarcanecultivar(犅犪犱犻犾犪)usingRTPCR.Sequenceanalysis
showedthatitcontaineda615bpopenreadingframe(ORF)andencodedadeducedproteinof204amino
acids.Itwasdesignatedas犛犮犃犅犘4becauseofthemultiplesequencealignmentandphylogenetictreeanal
ysisresultsrevealedthatithadcloserrelationshipswith犃犅犘4in犛狅狉犵犺狌犿犫犻犮狅犾狅狉and犣犲犪犿犪狔狊.Itwas
clonedintotheprokaryoticexpressionvectorpGEX6P1andexpresseda22.5kDproteinsuccessfulyin
犈.犮狅犾犻.SubcelularlocalizationassayshowedthatScABP4proteinislocatedinthereticularofcytoplasm
andonthecytomembrane;proteinstructurepredictionshowedthatithasasignalpeptide,speculateit
maybestoredintheendoplasmicreticulum,andexertitsfunctionontheplasmamembrane.qRTPCR
showedthat犛犮犃犅犘4expressedinbud,root,stemandleafinsugarcanewiththehighestexpressionlevel
inbud.犛犮犃犅犘4wasupregulatedbyIAAordarktreatmentsindicatedthat犛犮犃犅犘4mightparticipatein
sugarcaneresponsestoauxin,andinvolvedinlightsignalingpathway(s).Inaddition,theexpressionof
犛犮犃犅犘4insugarcanewasinducedbyABA,JA,SAandCuCl2butwasdownregulatedbyCdCl2.These
resultsindicatedthat犛犮犃犅犘4mightinvolveinthesugarcaneresistancetodisease,osmoticandheavymet
alstress,whichprovidesthebasisforfurtherstudyingthefunctionsof犛犮犃犅犘4.
犓犲狔狑狅狉犱狊:sugarcane;auxinbindingprotein;homologouscloning;expressionanalysis
  Hz{(auxin)Ï#$ƒ„Hz.‚¥*+ë
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2

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E«Hz{DÄÅíUV
FG[]€eWX¤É’H#$Hz.‚[ƒ
„

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Hz{[]€eϹƒ„#$Hz.‚[²
NJO

¹PÀQÏQR>|}

3STUIJ
Ñ3VHz{R>:¹2VÏÇÉÄÅØ[W{
¨X}>
(SCFTIR1/AFB¥SCFSKP2A),1VÏÇÉÊÍ
Ú[ H z { | } ~ 
(auxinbindingprotein1,
ABP1)[1]。Y3VR>Z4[e’1“]€\î,
3S,-\]^[ÏõSCFTIR1/AFB[e[]€_
`

aHz{b\cd

eôXVAux/IAAQ
ARF(ƒ„Hz{7iëì[eiV)ä|}f
g¹|}‘Hz{ëì7ih2VÚ[Hz{iì
jk
(auxinresponseelement,ARE)Ú,ôXHz
{ëì7i[ª¼

aHz{ba)d
,Aux/
IAAlmA‘SCFTIR1/AFBX}>ÚnW{_`o
H
,ARFpš¿qQARE|},h2Hz{érë
ì7i[e

Kstë#$[Hz.‚
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eiV[vÆ>

w§ÄÅ/0efx$[~
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[1,4];ABP1\ÿQHz{|
}

5yQzÍ,{~
(transmembranekinase,
TMK)5¾ÄŪZHz{q|X}>,,1}G
~RhoGTPase(ROP)2B6]€\î,~qI
Hz{]€

Ksƒ„PIN[ÆÇ,ƒ„€eÄ
ÅÊiì?íä²ÿ!

ÙêQnÁ[Hz{0
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§sƒOÄł/¥ÄÅU
6

Hz{[ƒ>„¿Qi…ƒ„…
[510]。
†Ï

õ
ÉABP1D#$>[ª¼Ác‡ˆ‰\Š‹Œ


ŽABP1[e[]€_`,-䤏y。#$
>Ø[‘Hz{]€_`Cـ’“äS”•

sÏ×D–—

³´,-.9
,ABP1ÇÉ TIR1/
AFB[Úr,¬Õ?˜™Aux/IAA~[vÆ>,
¤SCFTIR1/AFB]€_`š‘›ƒ„[T‡,#$>
\ÿYœŒ_`[žGŸKsv9eƒ„
[11]。
‰’ABP1ï,ABPs( ¡Ñ¢“¾c,3®
¯¢£×D5VABPs[1213],‡¤($Œ[ABPs
( ¾c¥3¦¤(

¤(V§[ ABPsÕhRd
¨ƒOÙDghÚ×D©ª
[1415]。ABPs³D®
¯Ú l . 9
[16],
« y D ¬ ­ ®
[17]、¯
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¶·
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…#$
CŽ’ˆV7i٧ܒä²gh,-

†Ï¹
ºžgØﻼ½‘µV7iDMNÚ[,-


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(犛犪犮犮犺犪狉狌犿狅犳犳犻犮犻狀犪狉狌犿L.)ύ³t
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CÏ)H$Á[h)T$

MN[
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H–Hz{
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=KÑtJLM

È,-KMNŽ‘À“
犃犅犘7i,Ù\ÿP»ª¼、ÃÄÅÆÇ、vdÝÞ
ÆÁ*+¤¹§Ü’ª¼U–

žÉQ’HMN
犃犅犘7i[gh$%’vw&。
1 ÊÀ¥YŠ
1.1 QRSTU
Kkl;mn%Ëw̍ÍÎzÏÀŒ[MN
‹Œ犅犪犱犻犾犪ÐÑ#Æ,ÎÒÀâ、á、ÓÔà¥
ß

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tX

ٓH$%tXØÚ3Æ。
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¼

¶5â0[jÜݚÞßàFÜÄ10d,Í
ÎzÏÀŒ[}݇Éçè

àávw|ȞÆ
ÎâdDu

ٓçèØ3“H$%tX,ٓH$
%tXØÚ 3 Æ。çèjçè‘kžFã¡
(5mmol·L-1SA Fäå)、æç¡(100μmol·
L-1JAFäå)、èé¡(100μmol·L
-1ABAFä
å
),
U ê É ç è 3、12 ¥ 24 h Î â;CuCl2
668 ! " # $ % &                   36ë
(100μmol·L
-1
Fäå
)、CdCl2(50μmol·L
-1
F
äå

çèUêÉ12、24¥48hÎâ;Hz{
(200μmol·L
-1IAAFäå)çèUêÉ3、12、24
¥48hÎâ;åæçèUêÉ1、3、6¥12dÎâ。
¼çèìݞ¤í

Îây‡åîï

5yÚ-
80℃ðñò×ó‡。
1.2 VW
1.2.1 犛犮犃犅犘4,=>?@ ?)­¥®¯犃犅犘
7i ” •
(XM _002442193.1、EU975483.1、
JN243771.1、S53630.1、X16308.1、X16309.1 ¥
EU944559.1)Tžêô”•,‰‡PrimerPremier5
õkD7i”•5′、3′öò÷øùØ0Ö×>ú$

ª1)。û‡TrizolŠ$ÎMN‹Œ犅犪犱犻犾犪âü
ýRNA,iecDNAžþÿ§ÜPCR‚˜。i
ì>?ž25μL,! Ex犜犪狇0.125μL,10×PCR
buffer2.5μL,dNTPs2μL,Òi<ú$Z1μL,þ
ÿ1μL,ddH2O17.375μL。iì‘kž94℃á"
>4min;94℃">30s,55℃#$30s,72℃%7
1min,34“&J;72℃%710min。PCR=$n
1.5%(¿)*+,-÷,.Á3[ü/,¶¹m
A‘ pMD19T Vector,5 y  ¨ n 0 1 2
DH5α|R3ÄÅ,nÿ456Í?í2åPCR
w&y

78H9H$9!

Ú:

Ñ;<=÷”

1.2.2 6XYZ[K\ ‰‡DNAMAN¥ORF
Finderõk,¤ŽÂ[犛犮犃犅犘47i[»¡”
•í¹öe[ 7¡”•§ÜU–

‰‡ExPASy
>?@
(http://www.expasy.org/proteomics)[
ProtParam、ComputePI/Mw ¥ ProtScale9Ká
÷ScABP4 ~[è¨>Ê¥°AF>;‰‡
TEPred(http://embnet.vitalit.ch/software/TM
PRED_form.html)9KU– ScABP4~[zÍ
|ºù

‰‡SignalP4.1Server(http://www.cbs.
dtu.dk/Services/SignalP/)á÷ScABP4~[]
€Ò

‰‡GORIV(http://npsapbil.ibcp.fr/cgi
bin/npsa_automat.pl?page=npsa_gor4.html)¤
ScABP4~§ÜBC|ºá÷;‰‡ MEGA6.0
(NeighborJoiningŠ)ºl?¦§¨©。
1.2.3 犛犮犃犅犘4 ]^BC à÷”‘[3[
7iORF,Ø0P»ª¼Ö×>ú$(ª1),?
pMD19T犛犮犃犅犘4ÊDžþÿ,‚˜3[7iü
/

iì>?ž25μL,!Ex犜犪狇0.125μL,10×
PCRbuffer2.5μL,dNTPs2μL,Òi<ú$Z
1μL,þÿ1μL,ddH2O17.375μL。iì‘kž
94℃á">4min;94℃">30s,66℃#$30s,
72℃%71min,34“&J;72 ℃%710min。
PCR=$n犅犪犿HI¥犡犺狅I{E,‰‡T4 mA
{mA‘(â{Eÿ[ª¼½>pGEX6P1,
¨n012DH5α|R3ÄÅ,tjÊDnF{E


ÊD PCR GÆí÷”,H>Ž·¸ž
pGEX6P1犛犮犃犅犘4。¶¨ÊD¥tjÊDUê
ÞII2犈.犮狅犾犻Rosetta(DE3),JÎK2é,D
10mLLBå>ÜÄ7(!50mg·L-1Amp)37
℃ÜÄÿL。Î400μLÜÄ$É30mLLBå>
ÜÄ7

!50mg·L-1Amp)BM1¨¢OD600
ž0.6;NÞObž1mmol·L-1[IPTG,37℃
óe¢10h(Ù2hÎâ1mL),UêÁP2å,
4℃、8000犵QR10minyÁP2>;NÞ30μL
~NâSTå

UFV5min;ŠWé12000犵Q
R5min,XÎ10μLÚ]åuâ,12%SDSPAGE
+,-÷Y}~[ª¼

1.2.4 犛犮犃犅犘4 _`abc   ‰‡DZõk
PSORT(http://psort.hgc.jp/form.html)á ÷
ScABP4[ÃÄÅÆÇÙ§Üvww&。à3[
7iORF,Ø0ÃÄÅÆÇÖ×>ú$(ª1),?
PMD19T犛犮犃犅犘4ÊDžþÿ,‚˜3[7iü
/

[¨y[3[7iü/n犅犪犿HI¥犡犫犪I{
EyQ(â{Eÿ[pCAMBIA2300GFP½>m
A

¨n012DH5α|R3ÄÅ,nÿF{E-
÷

ÊDPCRGÆí÷”,H>Ž·¸ž35S
ScABP4GFP。¶Â[tjÊDH>Ž¥¨½>
B1 犛犮犃犅犘4,=?@ABCdefX
Table1 Primersusedin犛犮犃犅犘4genecloningandexpressionanalysis
ú$¸Primer
Ò<ú$”•
Forwardprimersequence(5′→3′)
i<ú$”•
Reverseprimersequence(5′→3′)
‡_
Strategy
ScABP4 GGAGAACATGGTGCGTGAACTAC GCAGGAAACACTTGTGACCTAGAG 7iŽGenecloning
ScABP4Q CTGCTGCCTCGTTCCTC TTCCCTCCCATAGTTGCT ÝÞÆÁ RealtimePCR
GAPDHQ CACGGCCACTGGAAGCA TCCTCAGGGTTCCTGATGCC ÝÞÆÁ RealtimePCR
ScABP4PE CGCGGATCCATGGTGCGTGAACTAC CCGCTCGAGGAGTTCATCTTTTGGTGC P»ª¼Prokaryoticexpression
ScABP4SL GCTCTAGAATGGTGCGTGAACTACC CGCGGATCCGAGTTCATCTTTTGGTG ÃÄÅÆÇSubcelularlocalization
76850             @AB,…:MNHz{|}~犛犮犃犅犘47i[ŽQª¼
35SGFPUꁨ;12EHA105,DLBå>Ü
Ä7

!50·μgmL
-1Kan¥35·μgmL
-1Rif)
28℃ÜÄÿL,Xó;12]^å,ƒ.2åOD600
ž0.8,_ÞÈ`±âü,2dyabÝÞÆÇ|È。
1.2.5 犛犮犃犅犘4,=BCK\ û‡ TrizolŠ$
ÎMN7z0[ÒÀâ

ß

á

ìàíçèj¥¤
íjâü[ý RNA,ie¾cDNA,TžÆÁ
PCR[þÿ。àŽÂ[犛犮犃犅犘47i[
ORF”•,Ø0vdÝÞÆÁPCRú$(ª1),?
犌犃犘犇犎 TžØê7i[24],cíSYBRGreenPCR
MasterMixKit(Roche)Ô«deÚÆÁiì>?,
D7500§vdÝÞÆÁPCRf(ABI,USA)ڒ
¾ÆÁPCR‚˜,ٓâ‹ØÚ3M*+tX。‚
˜!”ž50 ℃ 2min(gÑaiìåNޒ
UNG{d͇);95℃10min(,1犜犪狇DNAh
}{
);95℃">15s,60℃#$1min,40“&J;
iNäHjZ

iì|:y

û‡2-ΔΔCtkŠU–
¥
[25]。
2 |ÈQU–
2.1 犛犮犃犅犘4,=gh>ijA6XYZ[KŽ‘À“zž640bp[KÀ‘Ð(l
1)。\ÿ÷”¥”•U–—˜,ˆ7i ORFz
ž615bp,Ÿ›204“ 7¡(l2)。‰‡ NCBI
¥ m
(http://www.ncbi.nlm.nih.gov/)[
BlastN9K£¤U,ˆ7iQ)­(犛狅狉犵犺狌犿犫犻
犮狅犾狅狉)、®¯(犣犲犪犿犪狔狊)¥nV(犛犲狋犪狉犻犪犻狋犪犾犻犮犪)…
$Œ犃犅犘47i)(),äo>Uêž95%、
94%¥87%。ª«ˆ7ižMNHz{|}~
7i

iµ·¸ž犛犮犃犅犘4,Ù¶¹pe‘ Gen
Bank,pe€žKT880509。
ProtParam、Compute PI/Mw á ÷ ª «,
ScABP4UVÁž22.5kD,…+u5.87,›+qr
7
(Asp+Glu)22,Ò+qr7(Arg+Lys)17,¤v
Æ?¥42.67,ž¤vÆ~。ProtScaleá÷ª«,
ScABP4P27Ç 7¡Kѳ)Us2.911,P61
Ç 7¡KѳcUs -2.378,GãAF>
(GRAVY)ž-0.119,Žö÷ž°F~。TM
Predá÷ª«,ScABP4~Ñ2“zÍ|ºù,¹
À“ýUž1750,ÑKؑï[zÍht;uÀ
“ýUž1542,ÑKï‘Ø[zÍht。ýGOR
IVá÷ª«,ScABP4vwuëjxy[£z³
)ž55.88%,¹MÏα{|y25.00%,%7}x
M.100bpUVÁ~;1.PCR‚˜=$
l1 MN犛犮犃犅犘47i[PCR‚˜|È
M.100bpladdermarker;1.PCRamplifiedproduct
Fig.1 PCRamplifiedproductsof犛犮犃犅犘4
é€Zª˜‚šƒÇu
;ª˜Og„›V
l2 MN犛犮犃犅犘47i[cDNA”•í¹öe[ 7¡”•
Underlineindicatestranslationinitiationsite;indicatesstopcodon
Fig.2 Thenucleotideacidsequenceof犛犮犃犅犘4anditsdeducedaminoacidsequence
868 ! " # $ % &                   36ë
y£zÏ19.12%,vβ…†|º。]€Òá÷ª
«

P42LJ ¡r7Kѳ)[PƒˆEÇuU
s0.364¥³)[‰}ˆEÇuUs0.546,P28
NJ ¡r7Kѳ)[]€ÒUs0.987。à
³yk 7¡r7[N‹Gãs0.642,ö÷
犛犮犃犅犘47ixŸ›[~×D]€Ò,žUÓ
~

2.2 犛犮犃犅犘4k,lghmnoApqrsKۇNCBI[BlastP!”,¤ScABP4[ 
7¡”•§Üäo>£¤



l3)—˜,¹Q

(犛狅狉犵犺狌犿犫犻犮狅犾狅狉_ref|XP_002442238.1|)、®
¯
(犣犲犪犿犪狔狊_ref|XP_008662629.1|)、nV(犛犲狋犪狉
犻犪犻狋犪犾犻犮犪_ref|XP_004962873.1|)、BŒŽ²
(犅狉犪犮犺狔狆狅犱犻狌犿犱犻狊狋犪犮犺狔狅狀_ref|XP_003575870.1
|)¥Fµ(犗狉狔狕犪狊犪狋犻狏犪犑犪狆狅狀犻犮犪犌狉狅狌狆_ref|NP_
001066918.1|)ABP~[ 7¡”•äo>Uê
ž94%、92%、86%、86%¥85%。¹Ÿ›[~Ê
KÑABP( ~[§Ö(l3),ÅKё!
BoxA、B¥CÏ#$ABP[3“)ò÷|ºù;KDELÏCö[ØÊˏ’]€”•
l3 MNScABP4 7¡”•Q¹“#$äo>£¤
BoxesA,BandCcorrespondtohighlyconserveddomainsamongalplantABP;KDEListheCterminalsequencewhichmay
functionasasignalforretainingtheproteininthelumenoftheER
Fig.3 SimilaritycomparisonofScABP4aminoacidsequencesamongsugarcaneandotherplantspecies
U”OuÚ[¥sž4•s

~–:ªpq—Q

˜€ØŸ€
ž 7¡pe€
l4 ¤($Œ7É犃犅犘7i 7¡”•[?¦§¨©
Numbersabovebranchesontheleftindicatebootstrapvalues
of1000replicates;Thescalebarrepresentsgeneticdistance;
TheaccessionNo.isgiveninbrackets
Fig.4 Phylogenetictreeofaminoacidsequences
encodedby犃犅犘geneamongdifferentplantspecies
M1.15000+2000bpUVÁ~;M2.100bpUVÁ~;
1.tjÊDF{Eü/
l5 pGEX6P1ScABP4tjÊDF{EGÆ
M1.15000+2000bpmarker;M2.100bpmarker;
1.EnzymedigestionfractionsofpGEX6P1ScABP4
Fig.5 PCRidentificationofpGEX6P1ScABP4
with犅犪犿HI/犡犺狅I
96850             @AB,…:MNHz{|}~犛犮犃犅犘47i[ŽQª¼
13~20“ 7¡r7[BoxA、BoxB、BoxC3“)
ò÷[Hz{|}|ºù?íCöØÊË|}
]€ø
(KDEL)。
ScABP4~[?¦§¨U–(l4)|ȗ˜,
§¨©n>Užœ“U”

¹(ýKVâ#$[


®¯



nV

BŒŽ²

™¸
(犃狏犲狀犪
狊犪狋犻狏犪_dbj|BAA25433.1|)、n¸(犎狅狉犱犲狌犿狏狌犾
犵犪狉犲_dbj|BAK02611.1|)¥š›œl}¸(犜狉犻狋犻犮狌犿
狌狉犪狉狋狌_gb|EMS50686.1|)ÇÉÀ“U”,FVâ#
$ž
(犈犾犪犲犻狊犵狌犻狀犲犲狀狊犻狊_ref|XP_010931928.1|)、

(犘犺狅犲狀犻狓犱犪犮狋狔犾犻犳犲狉犪_ref|XP_008793651.1|)、
 ¡
(犇犻犿狅犮犪狉狆狌狊犾狅狀犵犪狀_gb|ACX54195.3|)žu
ÀU”
,ScABP4Q)­、®¯…$Œ ABP§¨—
Q\´

2.3 ]^BCtu>vbAwx;<>犛犇犛
犘犃犌犈K  tjÊD pGEX6P1犛犮犃犅犘4n 犅犪犿HI、
犡犺狅I{Ey=$¢9]£[2‘,Ð,UêD640
bp(3[7iü/)¥5000bp(½>ü/)¤¥(l
5)。¤tjÊD÷”—˜,”• ORFQPŽ”
•ÀŒ

ª«P»ª¼½>ºl¾g

tjÊD
SDSPAGE+,|ȗ˜,DUVÁ48kD¤¥ç
Õ½1‘Ö×>‘Ð,ABP4~[n}è¦Âž
22kD,NÚpGEX6P1½>Ú[GST~§~n
}ž26kD,iµQȨá÷[~n}7Èä
©

Õ?žÆˆY}~¾gª¼

l6)。
M.~ÊUVÊÁ~ª;1.¼óe¨ÊD;2.IPTGóe8h
y[¨ÊD
;3.¼óetjÊD;4~7.IPTGUêóe
2、4、6、8hy[tjÊD
l6 óeª¼=$[SDSPAGEU–
M.Proteinmolecularweightmarker;1.pGEX6P1without
induction;2.pGEX6P1afterinducingfor8h;3.pGEX6P
1ScABP4withoutinduction;4-7.pGEX6P1ScABP4after
inducingfor2,4,6and8h,respectively
Fig.6 AnalysisofexpressedproteinsbySDSPAGE
A.GFP¤í;B.GFP¨½[««;C.GFP¨½[â¬>;D.GFP¨½[†N«;E.ScABP4GFP;F.ScABP4GFP[««;
G.ScABP4GFP[â¬>;H.ScABP4GFP[†N«
l7 ScABP4[ÃÄÅÆÇU–
A.GFPcontrol;B.GFPvisionundernaturallight;C.Chlorophylof35SGFP;D.GFPoverlappedvisionofA,B,andC;
E.ScABP4GFP;F.ScABP4GFPvisionundernaturallight;G.Chlorophylof35SScABP4GFP;
H.ScABP4GFPoverlappedvisionofE,FandG
Fig.7 SubcelularlocalizationofScABP4fusedwithGFPintheepidermalcelsof犖.犫犲狀狋犺犪犿犻犪狀犪(bars=50μm)
078 ! " # $ % &                   36ë
2.4 犛犮犃犅犘4>_`abcKàPsortõká÷,ScABP4~ÆÇÉØÊ
Ë

­
)、
ÊÍ

®>

ÿ¯${>
)、
)œ7>[Õh
>Uêž80.0%、79.0%、63.1%¥30%,¹ÆÇ
ÉØÊË

­

¥ÊÍ[°±³)



ö÷¹³Ñ
ÕhÆÇÉØÊË

­

¥ÊÍÚ

È`±âüD,Þ²h³—®´éab¬ÓÝ
Þ



l7)—˜,¤íj35SGFPÕK”D±
²ªµÄōnÁª¼

¬ÓÝÞ]€DÄÅÊ

Ê
Í¥ÄÅ»ãÑU¶

Q¹ä£
,35SScABP4
GFPY}~¬ÓÝÞ]€IJU¶ÉÄÅËÌ
|º?íÄÅÍ|ºÚ

ˆÆÇ|ÈQPsortõ
ká÷|È7ÈÀŒ

¤(·¸ª˜¹×—º>
(犘<0.05)
l8 犛犮犃犅犘47i[jÕÖ×>ª¼U–
Thedifferentsubscriptionswithdifferentlettersindicate
significantdifference(犘 <0.05)
Fig.8 Tissuespecificexpressionanalysisof犛犮犃犅犘4
fromdifferenttissuesinsugarcane
2.5 犛犮犃犅犘4,=>BCK犛犮犃犅犘4D7z0MN[¤(jՍãѪ
¼

s‡KÑjÕÖ×>

¹Dߍª¼Á³)

¹MÏDâ

D፪¼Á³c

l8)。
犛犮犃犅犘47ihòlï)IAA(200μmol·L
-1)、
åæ
、SA(5mmol·L-1)、JA(100μmol·L
-1)、
ABA(100μmol·L
-1)
¥CuCl2(100μmol·L
-1)
™
»óeڃª¼

sDCdCl2(50μmol·L
-1)
ðñ
é7i[ª¼Á—ºéƒ

l9),Ô«CdCl2 ôX
犛犮犃犅犘47i[ª¼。¹,ABA、IAA、CuCl2 ç
èé犛犮犃犅犘47i[ª¼ÁãD24h¼‘³),U
Ꞥí[1.3、2.3、1.5¼;JA çè3hy,
犛犮犃犅犘47iª¼Á—ºÚƒ,ž¤í[2.8¼,
«yª¼Á½¾oc

†¿«—)ɤí

l9,A);
åæçè6dd犛犮犃犅犘47i[ª¼Á«—Úƒ,
ž¤í[3¼,µy«ÀdDöÁ‘12d,ª¼Á¿
çÉ\)FG

l9,B)。
3 # ¦
Hz{|}~,-³ÂÞ[Ï ABP1,
s¹“( ¾cuä¤\£

»¤]^¬ÇD#$
>Ød¨ª¼

ƒ„íghÚÏÃ×DÖ×>

´
ÄqѨÅ&¾

D®¯ABP4êQìÝHz,[
eìݤHz{[ëì

QÞ]€\îSTÙQ
ABP1DghÚST[1415],†D¹“#$Úƽ
ABP4[,-&¾。
È,-.9ScABP4IJÆÇÉÄÅÊËÌ|
º¥ÄÅÍ

†ÏõÉÈvwÇÑۇØÊËÖ×
}È·¸ª˜(Àçèé¤(çèdD[¹×—º>
(犘<0.05)
l9 犛犮犃犅犘47iD¤(çèé[ª¼
Thedifferentlowercaselettersindicatesignificantdifferenceofthesametreatmentduringdifferent
treatmenttimes(犘 <0.05)
Fig.9 qRTPCRanalysisof犛犮犃犅犘4expressionindifferenttreatments
17850             @AB,…:MNHz{|}~犛犮犃犅犘47i[ŽQª¼
>~

x?ScABP4DÄÅʍ[ÄÆÇ¡Ñ
ɧÀQ"#
。ScABP4ÏUÓ~,ö÷¬Õh
QABP1Àâ,Ö×ÉØÊË­,\ÿUÓ_`DÊ
ÍÚ.†T‡

Felner[2627].9,ABP4DÞ¥Hz{óe[
Hzë썚tJT‡

DÊË[®¯‹Œ
犃犅犘47iDÞí¥ï)Hz{óeéڃª¼。
È,-犛犮犃犅犘4Dï)Hz{çèéڃª¼,
QFelner[|¦ÀŒ,ö÷ï)Hz{óe=H
ScABP4Q-|},?̧ÜéÀQ[̈́。†
犛犮犃犅犘4DåæçèéCڃª¼,QFelner[|
¦ä¤

ö÷ABP( ¾c[ghÕhKÑ$ŒÖ
×>

D®¯Î¨Ý
,ABP1¥ ABP4¦ëìÞ
]€ÙDé4Ï7zÚghSÐ

†ÏD4ßÑ[
HzÚ
,ABP1ـÒÓ,sDâüԝ.‚Ú,
ABP4¤ABP1Õhš›ƒ„T‡[15]。MNÏ)¼
>#$

MNABP( ¾c[¥3¥ghÕhÕ
žXÖ

#$,{Õ?ƒ„#$Hz.‚

êQJ
+]€qe
。ABAÏÀŒðñ,{,¬hóe#$
×>7i[ª¼
;JAhóe#$=H¤øØÑÙ、
×ÃÄ¥×Ú¨T‡[¨}$

¤øØHè12=
H¤‰të
;SADÛÜiìh[eÄōÝ,ó
eO!ä²~
(pathogenesisrelatedprotein,PR)
7i[ª¼¥ÀÞßà7i[ª¼

D7i[h2
Vø\ŠÑ¢Œiìjk|}Çu

Yې7iÕ
?AR¤(]€[eiVóeª¼



YÞ,
{]€\î-DäST‡

²(ëìJ+á,
[28]。
3S

»¼Ñ犃犅犘 7iDÚâ,{ðñ骼[
ä²&¾

È,-ãM.9DMNìݍ犛犮犃犅犘4
RSA、JA¥ABAóeڃª¼,ö÷YÞ,{ç
è¤MNìÝä¾*+

sMNÝçÉåæ[Hz
.‚ç/

iµ}¾Õ¢Hz{R>

ƒOØ)Hz
{[!Á¥U¶

?µè×YÞ,{[›T‡

é
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、Cu/ZnSOD…~
Ê[tJîiV

êQïX:ð[¯¨¡Pi
ì

Dâ¬>Ê>4{éêQÞ}T‡[+Vq
Iÿ!

ñÁ[é¤#$[Hz.‚Ñá,¥w§


ÿÁu¤#$Ñø

òÏ#$€ÒÓ®Áj
{

)bé‹¤#$=HÙøT‡
[2930]。
DÈ,

,CuCl2 çèúš犛犮犃犅犘4ڃª¼,CdCl2 ç
èúš犛犮犃犅犘4郪¼,Ô«犛犮犃犅犘47iëì
’t8ýðñ

†D¤(t8ýðñé¹×ª¼

TÑ&¾—˜

)b[òhòôXHz{[}
¾
[30],
ö÷犛犮犃犅犘4TžHz{R>s郪¼;
CÑ,-ª«

éðñéóeØÊË~N97i
ڃª¼
[31],ScABP4ÏØÊˍ[UÓ~,ŽÕ
hiµsڃª¼

#$¤ïóJ+]€[ìþW
XXÖ

È,-ÉQ,-’犛犮犃犅犘4D¤(çèé
[ª¼

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?í¤Hz{ƒ„[
UVWX¡ÑɧÀQ,-

yz!M

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