全 文 :书!"#$%&
,2016,36(6):1073-1079
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
!"#$:10004025(2016)06107307 犱狅犻:10.7606/j.issn.10004025.2016.06.1073
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:
(
“973”)*(2013CB127700);(+,-%./(31371889);0123456789:;)*(NCET120471);<=%>
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(B07049)
0123
:MYOTHWIN(1990-),B,CD,EFGH#$IJ%KL。Email:myothwinpatho@gmail.com
MNOP:QRS,0T,UDSVW,EFGHXYZ[\]^_KL。Email:kangzs@nwsuaf.edu.cn
45犆犅犔6789:;,<犜犪犆犐犘犓16=
>?@ABCD
MYOTHWIN,! ",# $,%&,( ),*+,
(
!"`a-bc% #$de%f
,
ghO$ijS$%(klmno
,
p!qr712100)
E F:stuvwxy.Bz{(calcineurinB1ikeprotein,CBL)O|}st~z{,M}sz{
x
(CBLinteractingproteinkinase,ClPK),G
tN]SSO。KLZ[
V]XYCIPK(1.犜犪犆犐犘犓16,qRTPCRb、 ¡¢£¤b¥y¦§
¨©bª«¬®¯°±
。
²³ª«´µ
,犜犪犆犐犘犓16¶·447¸.w,¹ºd»]x¼½¾¥u_
½¾
,
¿À
、
ÁÂÃCIPKz{ÄÅ<ÆÇȱ。 ¡¢£¤ª«nÉÊË,TaCIPK16TaCBL4ÌTaCBL9
ÍÎÏÐO
。
¨Ñª«´µ
,犜犪犆犐犘犓16ÒÓZ[]V´Ô,XYZ[ÐOÕÊÖ×Ø´Ô
ÙÚ
。
ÛÜÝ
,犜犪犆犐犘犓16Þ¯O|ßu_à¬XYáZ[]â\^ãäå。
GHI
:
XY
,CBL,z{x,TaCIPK16,Z[
JKCL$
:Q785;Q786 !MNOP:A
犆犺犪狉犪犮狋犲狉犻狕犪狋犻狅狀狅犳犪犆犅犔犻狀狋犲狉犪犮狋犻狀犵犘狉狅狋犲犻狀犓犻狀犪狊犲犌犲狀犲
犜犪犆犐犘犓16犻狀犠犺犲犪狋
MYOTHWIN,LIUPeng,MAWei,XUEQinghe,GUOJun,KANGZhensheng
(StateKeyLaboratoryofCropStressBiologyforAridAreas,ColegeofPlantProtection,NorthwestA&FUniversity,Yan
gling,Shaanxi712100,China)
犃犫狊狋狉犪犮狋:ThecalcineurinBlike(CBL)CBLinteractingproteinkinase(CIPK)networkplaysapivotalrole
inregulatingthephysiologicalaswelasdevelopmentalprocessesinplants.Inthisstudy,weobtaineda
CIPKgene,犜犪犆犐犘犓16,fromthewheatleavesinfectedby犘狌犮犮犻狀犻犪狊狋狉犻犻犳狅狉犿犻狊f.sp.狋狉犻狋犻犮犻(犘狊狋)using
犻狀狊犻犾犻犮狅cloningandRTPCR.ThefullengthcDNAof犜犪犆犐犘犓16was1606bp,whichencoding433a
minoacids.MultisequencealignmentshowedthatTaCIPK16sharehighsimilaritywithOsCIPK16and
AtCIPK16inriceand犃狉犪犫犻犱狅狆狊犻狊,respectively.Analysisoftheproteindomainfeaturesindicatedthat
TaCIPK16containedconservedregulatorydomaininCterminalandproteinkinasesdomaininNterminal.
SubcelularlocalizationassaysrevealedthatTaCIPK16displayedalocalizationpatternsimilartothatofthe
GFPcontrol,indicatingacytoplasmicandnuclearlocalization.Yeasttwohybridassaysshowedthat
TaCIPK16stronglyinteractswithTaCBL4andTaCBL9,respectively.qRTPCRassaysindicatedthat
犜犪犆犐犘犓16wasinducedby犘狊狋infectionanddifferentialyexpressedduringincompatibleandcompatible
interactionsbetweenwheatand犘狊狋.Ourresultssuggestthat犜犪犆犐犘犓16hasapositiveroleinregulating
wheatresistanceagainst犘狊狋.
犓犲狔狑狅狉犱狊:wheat;CBL;proteinkinase;TaCIPK16;犘狌犮犮犻狀犻犪狊狋狉犻犻犳狅狉犿犻狊f.sp.狋狉犻狋犻犮犻
æåç£èé+,êj]ë,#$ìí
¬îï]NMð
。Ca2+ñNòVkF]ó
ôNõ
,
#$]SÌSö÷økF]O
。
#$ùåúû]NÌüýþ
,
¦§]
Ca2+¿ÿ!SÇå]"é[1]。#$Í3#t
~z{
,
ª$ñtu%CaM (Calmodulin)¥
Ç&z{
、
t±z{x
(Ca2+dependent
proteinkinases,CDPK)、tuvwx B sz{
(calcineurinBlikeprotein,CBL)[2]。CBL
()Åx*±
,
+,Ì-z{CIPKìíç
.9¯O
[3]。CIPKñ}#$°Å]/
¸w0¸wz{x(,12ó3s]SnRK3
x
(SNF1relatedproteinkinase3,SnRK3)[4]。
á34#$ÁÂÃCIPKz{]½ª«´µ,5
Å]CIPKz{ºÅ N6]xz{¾ÌC6]
u7¾
,
8P|CBL°Ø9]NAF½¾[5]Ì
¯PP2CÇÐO]PPI½¾[6]。
:;KL´µCIPK(.àùå<<、
=>?P@güý
,
Aüý
,
B¥CK+、Dw<、C
E=Füý
[7]。
ÁÂÃ
,AtCIPK23ÞB
AtCBL1ÐO,vw* K+MG (AKT1)Hë K+
]I J
[8]。
K Á Â Ã L Ò < < ü ý þ
,SOS3
(CBL4)¥ CBL10 Ca2+, M 8 SOS2
(CIPK24)z{xìíz{ç.,ª$
NO¥NÜ2ªu_Çå-z{]´Ô
,
G
õ#
$¯PQ?RS<<üýTU]VW
[9]。
¿À
,
MRNAibXY犗狊犆犐犘犓23¯ZV[¿À
âg±]R[10]。
]^_ú
,CBLCIPKN`a
bcà#$SÌ1]
。
¿À Os
CIPK15 ¿ À ] d E e ± Å &,Þ ¯ u 7
SnRK1A f d E ù å Ì g N ä å[11]。
CIPK6®¯dh]ié.犮犻狆犽6,j.4jk
4]Sö%lmnµÊR,
oí#p´q|
r
、
Ostuc
、
vwSxy=Södz
[12]。
]^_ú
,
&2CIPKz{x#$ùåS
$üý]O{Ŭ}¨]KL
。
|5}
~
,
*±E
(reactiveoxygenspecies,ROS)#$
â\äå÷Ó&kF]O
,
F¯Z@÷¦§
,
G
\]ë}
,
ÞO|}
NªVâ\Ç&.]´Ô
[13]。
ÁÂ
Ã
,AtCIPK26¯ZÌ}NADPHExAtR
bohFÐOu_SROS[14]。¿À,Os
CIPK14/15¯ÒÓ\Ç&ª34(microbe
associatedmolecularpatterns,MAMP)]V,
OsCIPK14/15]´ÔÒÓ,MAMPVS
]ROS{ÒÓ,G
µ OsCIPK14/15à
¬PAMPV]â±(PAMPtriggeredimmunity,
PTI)[15]。,CBL10ÌCIPK6¯Zßu_
¦§IJäå̦§²±
。
,
CIPK6 CBL10]ÐOà ETI(effectortrig
geredimmunity)ROS]S[16]。ÅKL´
µ
,
¿qwNMð{ÒÓ¬CIPK]u7。Á
ÂÃ
,NPR1¯ZCIPK11/PKS5vw,G
V WKY38Ì WKY62]´Ô[17]。
:
,
XY&2CIPKz{xS$üý
]®¯~_
。
KLXYCIPKz{xX
YZ[ÐO.`]®¯áOÄÅ
kF]
。
^
,
KLª~¬}z{
x.犜犪犆犐犘犓16,qRTPCRb、 ¡¢
£¤b¥y¦§¨©bª«¬®¯°±
,
|
ë}
ËXYZ[ÐO]ª
¨¬.
。
1 ¡¢£
1.1 QRST@RURV
mn5XYZ[SX#|
‘
Z23
’(CYR23)Ì‘Z31’(CYR31),XY¤#|
‘
¥¦169’Ì‘¿§11’。XY¨©、Z[ª«¥
9#¢£à¬Kang=[18]]¢£。TaqDNA
x
(Fermentas,®),dNTP、pMD18T(Takara,¯
),
°±g²³ DNA´Jµ¶·(¸ ¹,),
Top10ºÒ»¦§(Wolsen,®),¼½¾¿·(Bi
oMiga,®),±ÀÁx(Fermentas,®),T4
DNAÂ9x(Fermentas),BIOZOL(BioFlux,¯
),RNaseinhibitor(Promega,®),MMLV ä
Ãĵ¶·
(Fermentas,®),SYBRGreen Ì
RoxreferenceÅ¡(Invitrogen,®)。
1.2 W X
1.2.1 犜犪犆犐犘犓16,<=>? TAIRv10(ht
tp://www.arabidopsis.org/)Ì RGAPv7 (ht
tp://rice.plantbiology.msu.edu/)ÆÇÈÓ
ÁÂÃÌ¿À]CIPK²³,É.ÊÆÇ
4701 ! " # $ % & 36Ë
È
(http://wheaturgi.versailes.inra.fr/Seq
Repository/Genesannotations)ÓXYcDNAÆ
ÇÈ
,
B¿ÀÌÁÂÃ]CIPK²³|#²³,
àÌNCBI“BLAST+usermanual”]¢£,áO
Í]XYcDNAÆÇÈëÎNBLASTÏÐ,
ÓXYÑÒ]CIPK²³,M MEGA5.0½Ó
ëÔµÕOsCIPK16Ö×&`ØÙ]²³,Ú
|ÑÒ]TaCIPK16。wDz³ª«Ó]ÑÒ.
²³Û)@$TaCIPK16F/R(´ 1)。¾¿¿§
11Ü RNAäÃÄícDNA,Ä.¢£àÌ
FermentsReverAidFirstStrandcDNAsynthesis
µ¶·ÝOµ
。
BcDNA|3Þ,PCRß²
|95℃àé±5min,95℃ é±30s,55℃ áâ
30s,72℃xã1min,ä35åê;72℃ xã
10min。PCR$æ8Â9ÓpGEMTEasy
Í.
,
çcèéÃ8¾¿¼½êë²
。
1.2.2 YZCD ìmnë²]ÝÌM
àëÓ]ÑÒ.²³ëÎÇȱíá
。
õ
ExPAsyîï]ðñòComputepI/Mwtool(ht
tp://web.expasy.org/compute_pi/)ëÎ)ó¶·
z{]=ôl̪Ñ
;
DNASTAR ñò,
BLAST(http://www.ncbi.nlm.nih.gov/blast/)
ÌORFFinder(http://www.ncbi.nlm.nih.gov/
gorf/gorf.html)ëÎcDNA ²³]ª«。
ClustalW2 (http://www.ebi.ac.uk/Tools/msa/
muscle/)Ì DNAMAN7.0 ñò (version7.0;
LynnonBiosoft,USA)ëβ³í᪫。õ
MEGA5software (version5.0)ñòBõ9£
(NeighborJoiningmethod)ëÎ]z{ö§
z{]èk²³íá¥ëÔª«
。
õInter
ProScan(http://www.ebi.ac.uk/Tools/pfa/ipr
scan/)ÌPROSITEScan(http://prosite.expasy.
org/scanprosite/)Uàëz{]d»z{½¾。
1.2.3 [\]^_CD Û)í TaCBL4
(GenBank÷Ä| KU736850)Ì TaCBL9(Gen
Bank÷Ä| KU736852) ¡¢£¤@$
TaCBL4BDF/R、TaCBL9BDF/R(´ 1),
pGBKT7Í.Ü,Û)TaCIPK16 ¡¢£¤
@ $ TaCIPK16ADF/R (´ 1),
pGADT7Í.Ü。øÌ ¡¢£¤ùú(Clontech,
Japan),ì TaCBL4 TaCIPK16?P TaCBL9
TaCIPK16äà ¡pAH109,ª$dz
±¨©.SDLeucineTryptophan(LT)ÌSDAde
nineHistidineLeucineTryptophan(º20μg·mL
-1
]
`1 ab`
Table1 Primersusedinthisstudy
@$ûüPrimer @$²³Primersequence(5′→3′)
TaCIPK16F GAGGAGTGGTAGTGATTAGTGA
TaCIPK16R CTTCAAGTTGCTCTGCACGTTAC
TaCBL4BDF CATGCCATGGGCTGCGTGTTGTCATC
TaCBL4BDR GGCTGCAGTTATTTGCTGATTCCGCTGTAA
TaCBL9BDF CATGCCATGGCCTCACGCTTCAGCTT
TaCBL9BDR GGCTGCAGTTAGTCTTCAACCGCGGTATTG
TaCIPK16ADF CCGGAATTCATGTCCGCGAGCCCGG
TaCIPK16ADR AAAACTGCAGGCTACGTCAAGATGTACCAGAAGA
TaCIPK16163F AAGCTTATGGCGAGAGGGCGGGAGGG
TaCIPK16163R GGATCCCGCGACGGCGCCCGG
TaCIPK16qRTF1 GCCGCCGCAGGCCGCGCGGCTGC
TaCIPK16qRTR1 CCTTCTCCCGGTCGAGCACCTTGAT
TaEF1F TGGTGTCATCAAGCCTGGTATGGT
TaEF1R ACTCATGGTGCATCTCAACGGACT
XαGal)(LTHA+ Xαgal)ÜëΨ©。¨©
4d8,ýþ)ÆÞì ¡¦§ÿÓ!"#
¿ºÅ107、106、105Ì104¦§,lLTHA+
Xαgal¨©.ܨ©,24h8ÌÇ。
1.2.4 cdefg ÇpCaMV35S:GFPè
©lN$
,
Û)íTaCIPK16y¦§¨©
@ $ TaCIPK16163F/R (´ 1), ì
TaCIPK16 pCaMV35S:GFP ,àÌ Li
=
[19]
]¢£
,
%XYS¼.
,
àÌ¢£ì
rÍ.pCaMV35S:TaCIPK16∶GFP¥&Í.
pCaMV35S∶GFPÃXYS¼.,¨ ©18h
8
,
MäÊ()ëÎ*+ÌÇ
。
1.2.5 狇犚犜犘犆犚CD wÇ,U]犜犪犆犐犘犓16
.ÌÀà.XYxã犜犪犈犉1α(GenBank÷
Ä Q03033)ª$Û)°Ø¨Ñ@$ TaCIPK16
qRTF1/R1¥TaEF1F/R(´ 1)。-.9#Z[
80、12、18、24、72Ì120hþ/l]¾¿
RNA,äÃÄ|cDNA,B^cDNA|3Þ,qRT
PCRäå²|95℃àé±5min,95℃é±,
10s,55℃ áâ20s,72℃xã40s,ä40åê。
qRTPCR]äå.`¥²àÌ Feng=]¢
£
[20],
Ø8-2-ΔΔCt£)ó0.áÀà.
]Çá´ÔÑ
。
2 ݪ«
2.1 45犜犪犆犐犘犓16,<=>?@YZCD
MRTPCRbÓ]cDNA²³Ä
57016; MYOTHWIN,=:XYCBLz{x.犜犪犆犐犘犓16]¥°1ª«
Å160623w(41),¶·1447¸.w]z
{
,
z{ªÑ|49.521kD,=ôl|9.09。õ
InterProScanñòª«´µ,N6¹º}z
{x½¾
,C6ºÅ}u_½¾,u7
¾¹º¯ZCBL°Ø]NAF½¾¥
PP2CÇÐO]PPI½¾(42)。ÁÂÃ
¿À5Å]CIPK²³¸.w²½ÓëÔ
qz{¿À] OsCIPK16(LOC_Os09g25090)
¥ÁÂÃ] AtCIPK5(AAF86504)、AtCIPK16
(AAK50348)¥AtCIPK25(AF44226)ö1}ª
:
(
43)。5Z²³í᪫´µ,z{²³
1~2.PCRß$;M.Marker
41 犜犪犆犐犘犓16.cDNA]PCRß5
1-2.PCRproduct;M.Marker
Fig.1 Electrophoresisof犜犪犆犐犘犓16cDNAfragment
At.ÁÂÃCIPK²³;Os.¿ÀCIPK²³;678,mn
]TaCIPK16²³;7lÜ]Æ9´ËBootstrapnÉ
.21000:kç7l]ÞNÆ
43 TaCIPK16¿À、ÁÂÃCIPK(]ëÔª«
At,CIPKsequencesof犃狉犪犫犻犱狅狆狊犻狊downloadfromTAIRv10;
Os,CIPKsequencesofricedownloadfromRGAPv7;Thearrow
indicatesTaCIPK16;Thenumbersatthenodesrepresentsthe
reliabilitybasedon1000replicates
Fig.3 PhylogeneticanalysisofTaCIPK16withCIPK
familyfromriceand犃狉犪犫犻犱狅狆狊犻狊
;ð´ËN6z{x½¾;<ð´ËC6u_½¾;<=´ËNAF½¾;;ð=´ËPPI½¾
42 TaCIPK16¿À、ÁÂÃö§.]è²³íá
Theconservedproteinkinasedomainandregulatorydomainaremarkedwithblackfullineanddashline,respectively.
TheconservedNAFandPPImotifsaremarkedwithblackboxanddashbox
Fig.2 MultisequencealignmentofTaCIPK16withhomologousproteinsinriceand犃狉犪犫犻犱狅狆狊犻狊
6701 ! " # $ % & 36Ë
OsCIPK16ÇÈÆÔÓ88.04%,ÁÂÃ(犃狉犪犫犻
犱狅狆狊犻狊狋犺犪犾犻犪狀犪) AtCIPK16 Ç È Æ Ô Ó
76.43%,>?û|TaCIPK16。
2.2 [\]^CD
|¬nÉ TaCIPK16ÌTaCBL4?TaCBL9ñ@
ÅÇÐO
,
ìTaCIPK16TaCBL4?PTaCIPK16
TaCBL9äöà ¡。AÒ¨©.SD
LeucineTryptophanÜ ¡¯ZßBSö,µ2
#r¼½í®ÃC ¡
(
44,A)。AÒ¨
©.SDAdenineHistidineLeucineTryptophan(º20
ºÅkʼ½]AH109 ¡pÒD¨©.SDLeucineTryptophan(LT)ÌSDAdenineHistidineLeucineTryptophan(E20μg/mL
]Xαgal)¨ ©;A、B.F±áÌ,Takala ¡µ¶·]SV40largeTantigenÌmurinep53ÐOÝ:C、D.TaCIPK16ÌTaCBL4]ÐO
Ý
;E、F.TaCIPK16ÌTaCBL9]ÐOÝ;G、H.G±áÌ,Takala ¡µ¶·SV40largeTantigenhumanlamin]ÐOÝ
44 TaCIPK16TaCBL4、TaCBL9]ÐOª«
CelsofyeaststrainAH109harboringtheindicatedplasmidcombinationsweregrownonselectivemediaSDLeucineTryptophan
(LT)andSDAdenineHistidineLeucineTryptophan(containing20μg/mLXαgal);A,B.Positivecontrol,theinteraction
betweenSV40largeTantigenandmurinep53;C,D.TheinteractionofTaCIPK16andTaCBL4:E,F.Theinteractionbetween
TaCIPK16andTaCBL9;G,H.Negativecontrol,theinteractionbetweenSV40largeTantigenandhumanlaminC.
Fig.4 AnalysisofinteractionsbetweenTaCIPK16andTaCBL4/TaCBL9
A~D.F±áÌ,pCaMV35S:GFPÃ]XYS¼¦§;E~H.pCaMV35S:TaCIPK16:GFPÃ]XYS¼¦§;A、E.Ò
]GFPHIÌJ.+HIÊÝ;B、F.J.+HI;C、G.Ò]GFPHI;D、H.{IO]S¼¦§
45 XYS¼.TaCIPK16y¦§¨©ª«(Bar=5μm)
A~D.Positivecontrol,pCaMV35S:GFP;E~H.pCaMV35S:TaCIPK16:GFP;A,E.overlay;B,F.Chloroplast;C,G.GFP;
D,H.Brightfield.
Fig.5 SubcelularlocalizationofTaCIPK16inwheatprotoplasts
77016; MYOTHWIN,=:XYCBLz{x.犜犪犆犐犘犓16]¥°1ª«
46 犜犪犆犐犘犓16.XYZ[ÐO]
ÃÄ´Ô°1
Fig.6 Expressionpatternof犜犪犆犐犘犓16duringthe
compatibleandincompatibleinteractionbetweenwheatandPst
μg·mL
-1XαGal) ¡¯ZSöíKL,F
± á Ì Ç È
,
µ TaCIPK16 TaCBL4 Ì
TaCBL9nÍÐO(44,B)。ì ¡¨©Mÿ
Ó!"#ºÅ104 ¡¦§þ,N¯*+Ó ¡
]Sö
,´
µTaCIPK16TaCBL4ÌTaCBL9Ä
ÅÎÏ]ÐO
。
2.3 cdefgCD
|nÉTaCIPK16XY¦§]ªOPQ,ì
rÍ.pCaMV35S∶TaCIPK16∶GFPB¥&Í.
pCaMV35S∶GFPÃRXYS¼.。Ý´
µ
,TaCIPK16XY¦§ªOF±áÌGFP]
PQÇö
,
fXY¦§nŪO
(
45)。
2.4 45犜犪犆犐犘犓16,<=hifj 犚犜犘犆犚
CD
mþ¨ÑRTPCRª«Ý´µ,犜犪犆犐犘犓16
ÒÓZ[V´Ô
。
SÖÌÐO
(CYR23),
犜犪犆犐犘犓16ÃÄ¿ÿ9#86~36hC20h´
Ô¿ÿ
,
ÕOu´Ô
;
KÓ48h´ÔѵÊÜu,
ÔÓT9
(
Çá´ÔÑ|10.8),B8]þ/l,
´ÔÑUçÓVW¿ÿ
(
4 6)。ÖÌÐO
(CYR31),犜犪犆犐犘犓16]Çá´ÔÙÚSÖ
Ì]´ÔÙÚÇö
,
X948h8S(]Üu
´ÔÑ
(
Çá´ÔÑ|2)ÊÖC2SÖÌÐO
´ÔÑ
(
46)。Y^Z[,犜犪犆犐犘犓16Þ¯à¬
XYáZ[]â\^ãäå
。
3 \ ]
^Å]KL
,
#$CIPK.(ñ}
_c](
,
(À`ö]CIPKía]®¯Í
°Ø±
,
ÞBùå}#?Pè#S$?SS$üý
。
KL¬1XY¶·CIPKz{x].
,
ÁÂÃÌ¿À] AtCIPK16 Ì Os
CIPK16ÄÅ<Æ]Çȱ,bñ N6]¼
½¾ÌC6]u7½¾,ZëTaCIPK16¯Zº
~Ca2+ N,®¯¿ À Ì Á  à ]
CIPK16z{®¯<Æd»。
CBLz{cFCIPKz{xìíz{
ç.
,¯
*¦§À}`³]SS]é
。
B:KL´µ
,`
ö]CBLz{`ö]CIPKz
{¯ZS`ö]NMð
,
`ö]N
Mð_/
,CBLCIPKz{ç.{÷Óde]
O
[21]。
^
,
µÕCIPKCBL]ÐOîfñK
LCIPKz{O].。KLM ¡
¢£b
,
µÕ TaCIPK16 TaCBL4Ì TaCBL9
ÅÎÏ]ÐO&`
。
Y^Z[
,TaCIPK16¯Zù
åè#êjá#$]g
,
¯XYCBLìí
`öz{ç.°Ø®¯
。
ª«CIPK¦§]ªOPQÅh2i
CBLCIPKz{ç.]®¯¥°Ø]t~N
Mð
。
ÁÂÃCIPK(]c2ª.
ªO¦§2¦§¼
[22]。
jk
,Zm
CIPK16EF¨©¦§2̦§lÜ,¦§¼
ºÑÇá_
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