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Characterization of a CBL interacting Protein Kinase Gene TaCIPK16 in Wheat

小麦CBL结合蛋白激酶基因TaCIPK16的克隆及特征分析



全 文 :书!"#$%&
,2016,36(6):1073-1079
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“973”)*(2013CB127700);(+,-%./(31371889);0123456789:;)*(NCET120471);<=%>
%-?3@A)*
(B07049)
0123
:MYOTHWIN(1990-),B,CD,EFGH#$IJ%KL。Email:myothwinpatho@gmail.com
MNOP:QRS,0T,UDSVW,EFGHXYZ[\]^_KL。Email: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{(calcineurinB1ikeprotein,CBL)O|}st~€z{,M‚ƒ}sz{„
x
(CBLinteractingproteinkinase,ClPK)€,G…†tN‡ˆ‰]SŠS‹‚ŒŽO。‘KL†Z[’
“V]XY”•–—˜™CIPK(š1›.œ犜犪犆犐犘犓16,žqRTPCRbŸ、 ¡¢£¤bŸ¥y¦§
¨©bŸª«¬­®¯°±

²³ª«´µ
,犜犪犆犐犘犓16¶·447›¸.w,¹ºd»]„x¼‹€½¾¥u_
€½¾

ƒ¿À

ÁÂÃCIPKz{ÄÅ<ÆÇȱ。 ¡¢£¤ª«nÉÊË,TaCIPK16ƒTaCBL4ÌTaCBL9
͆ÎÏÐO

¨Ñª«´µ
,犜犪犆犐犘犓16ÒÓZ[’]“V´Ô,†XYƒZ[’ÐO‚ŒÕÊÖ×Ø´Ô
ÙÚ

Û܀Ý
,犜犪犆犐犘犓16Þ¯O|ßu_œàƒ¬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:ThecalcineurinBlike(CBL)CBLinteractingproteinkinase(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cloningandRTPCR.ThefullengthcDNAof犜犪犆犐犘犓16was1606bp,whichencoding433a
minoacids.MultisequencealignmentshowedthatTaCIPK16sharehighsimilaritywithOsCIPK16and
AtCIPK16inriceand犃狉犪犫犻犱狅狆狊犻狊,respectively.Analysisoftheproteindomainfeaturesindicatedthat
TaCIPK16containedconservedregulatorydomaininCterminalandproteinkinasesdomaininNterminal.
SubcelularlocalizationassaysrevealedthatTaCIPK16displayedalocalizationpatternsimilartothatofthe
GFPcontrol,indicatingacytoplasmicandnuclearlocalization.Yeasttwohybridassaysshowedthat
TaCIPK16stronglyinteractswithTaCBL4andTaCBL9,respectively.qRTPCRassaysindicatedthat
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]닍,#$ìí
¬îï]N‡Mð
。Ca2+ñN‡òVkF]ó
ôNõ

†#$]SŠÌSö‚Œ÷økF]O


#$†ùåúû]N‡Ìüýþ

¦§]
Ca2+¿ÿ!ŽSÇå]"é[1]。#$Í†3#t
~€z{

ª$ñtu%CaM (Calmodulin)¥
­Ç&z{

tˆ‰±z{„x
(Ca2+dependent
proteinkinases,CDPK)、tuvwx B sz{
(calcineurinBlikeprotein,CBL)[2]。­CBL
()ńx*±

+,Ì-z{CIPK€ìíç
.9¯ŽO
[3]。CIPKñ}›#$°Å]/
¸w0¸wz{„x(š,12ó3s]SnRK3
„x
(SNF1relatedproteinkinase3,SnRK3)[4]。
á34#$ÁÂÃCIPKz{]€½ª«´µ,5
Å]CIPKz{ºÅ N6]„xz{¾ÌC6]
u7¾

8P|CBL°Ø9]NAF€½¾[5]Ì
¯ƒPP2CÇÐO]PPI€½¾[6]。
:;KL´µCIPK(š.œàƒùå<<、
=>?P@güý

Aüý

B¥CK+、Dw<、C
E=­Füý
[7]。
†ÁÂÍ
,AtCIPK23ÞBƒ
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 ƒ SOS2
(CIPK24)z{„x€ìíz{ç.,ª$†
NO¥NÜ2ªu_Çå-z{]´Ô

G…õ#
$¯PQ?RS<<üýTU]VW
[9]。
†¿À


M‚RNAibŸXY犗狊犆犐犘犓23¯ZV[¿À
âg±]R[10]。
]^_ú
,CBLCIPKN‡`a
bcàƒ#$SŠÌŽ1]‚Œ

¿À Os
CIPK15ƒ ¿ 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.4ƒjk
4]Sö%lmnµÊR,
oí#p´q|”


Ostuc

vwŽSxy=Södz
[12]。
]^_ú

&2CIPKz{„x†#$ùåS
$üý‚Œ]O{Ŭ}¨]KL

|5}
~

*±E
(reactiveoxygenspecies,ROS)†#$
â\äå÷Ó&kF]O

F¯Z@÷¦§
€

G…‚ƒ\„’]ë}…†‡

ˆÞO|}
›N‡ª“Vâ\Ç&.œ]´Ô
[13]。
†ÁÂ
Í
,AtCIPK26¯ZÌ}›NADPHE‹xAtR
bohFÐO‰u_­ŠSROS[14]。†¿À,Os
CIPK14/15¯‹ÒÓ\„Ç&ª34(microbe
associatedmolecularpatterns,MAMP)]“V,Œ
OsCIPK14/15]´ÔÒӍƒ,MAMP“VŠS
]ROS{ÒӍƒ,G…Žµ OsCIPK14/15àƒ
¬PAMP“V]â±(PAMPtriggeredimmunity,
PTI)[15]。†,CBL10ÌCIPK6¯Zßu_
¦§IJäå̦§Œ²±‘

†’“

CIPK6ƒ CBL10]ÐOàƒ ETI(effectortrig
geredimmunity)‚ŒROS]ŠS[16]。ÅKL´
µ

¿qwN‡Mð{ÒÓ¬CIPK]u7。†Á
ÂÍ
,NPR1¯Z”CIPK11/PKS5vw‹,G…
“V WKY38Ì WKY62]´Ô[17]。
•:

XY&2CIPKz{„x†S$üý
]®¯~_–—

KLXYCIPKz{„x†X
YZ[’ÐO.`]®¯á˜™­OšŠÄÅ
kF]›œ

œ^

KLª~˜™¬}›z{„
x.œ犜犪犆犐犘犓16,žqRTPCRbŸ、 ¡¢
£¤bŸ¥y¦§¨©bŸª«¬­®¯°±

|
ë}…Ë­†XYZ[’ÐO‚Œ]ªš
Šž¨¬.Ÿ

1  ¡ƒ¢£
1.1 QRST@RURV
mn5XYZ[’SŠX#|

Z23
‡
’(CYR23)Ì‘Z31‡’(CYR31),XY¤#|

¥¦169’Ì‘¿§11’。XY¨©、Z[’ª«¥
9#¢£à¬Kang=[18]]¢£。TaqDNA ­
x
(Fermentas,®),dNTP、pMD18T(Takara,¯

),
°±g²³ DNA´Jµ¶·(¸ ¹–,),
Top10ºÒ»¦§(Wolsen,®),¼½¾¿·(Bi
oMiga,®),‚ƒ±ÀÁx(Fermentas,®),T4
DNAÂ9x(Fermentas),BIOZOL(BioFlux,¯

),RNaseinhibitor(Promega,®),MMLV 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/)ÆÇȍ™Ó
ÁÂÃÌ¿À]CIPK²³,†É.œÊÆÇ
4701 ! " # $ % &                   36Ë
ȍ
(http://wheaturgi.versailes.inra.fr/Seq
Repository/Genesannotations)™ÓXYcDNAÆ
ÇÈ

B¿ÀÌÁÂÃ]CIPK²³|#²³,
àÌNCBI“BLAST+usermanual”]¢£,áO
Í]XYcDNAÆÇÈëÎNBLASTÏÐ,™
ÓXYÑÒ]CIPK²³,M‚ MEGA5.0½Ó
ë‹ÔµÕƒOsCIPK16Ö×&`ØÙ]²³,Ú
|ÑÒ]TaCIPK16。wDz³ª«™Ó]ÑÒ.
œ²³Û)@$TaCIPK16F/R(´ 1)。¾¿¿§
11”•Ü RNAäÃÄícDNA,Ä.¢£àÌ
FermentsReverAidFirstStrandcDNAsynthesis
µ¶·ÝOŽµ

BcDNA|3Þ,PCR†ßŒ²
|95℃àé±5min,95℃ é±30s,55℃ áâ
30s,72℃xã1min,ä35›åê;72℃ xã
10min。PCRŠ$æ‹8Â9ÓpGEMTEasy
Í.

çcèé’Ë8¾¿¼½êë²

1.2.2 YZCD ìmn–—ë²]€ÝÌM‚
àë™Ó]ÑÒ.œ²³ëÎÇȱíá

õ
ExPAsyîï]†ðñòComputepI/Mwtool(ht
tp://web.expasy.org/compute_pi/)ëÎ)ó¶·
z{]=ôl̪Ñ

ž DNASTAR ñò,
BLAST(http://www.ncbi.nlm.nih.gov/blast/)
ÌORFFinder(http://www.ncbi.nlm.nih.gov/
gorf/gorf.html)ëÎcDNA ²³]ª«。ž
ClustalW2 (http://www.ebi.ac.uk/Tools/msa/
muscle/)Ì DNAMAN7.0 ñò (version7.0;
LynnonBiosoft,USA)ëβ³í᪫。õ
MEGA5software (version5.0)ñòBõ9£
(NeighborJoiningmethod)ëΕ]z{ƒ­ö§
z{]èk²³íá¥ë‹Ôª«

õInter
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÷ć| KU736850)Ì TaCBL9(Gen
Bank÷ć| KU736852) ¡¢£¤–—@$
TaCBL4BDF/R、TaCBL9BDF/R(´ 1),–—
pGBKT7Í.Ü,Û)TaCIPK16 ¡¢£¤–
—@ $ TaCIPK16ADF/R (´ 1), – — 
pGADT7Í.Ü。øÌ ¡¢£¤ùú(Clontech,
Japan),ì TaCBL4ƒ TaCIPK16?P TaCBL9ƒ
TaCIPK16äà¡’pAH109,ª$†dz
±¨©.SDLeucineTryptophan(LT)ÌSDAde
nineHistidineLeucineTryptophan(º20μg·mL
-1
]
`1 ab`
Table1 Primersusedinthisstudy
@$ûüPrimer @$²³Primersequence(5′→3′)
TaCIPK16F GAGGAGTGGTAGTGATTAGTGA
TaCIPK16R CTTCAAGTTGCTCTGCACGTTAC
TaCBL4BDF CATGCCATGGGCTGCGTGTTGTCATC
TaCBL4BDR GGCTGCAGTTATTTGCTGATTCCGCTGTAA
TaCBL9BDF CATGCCATGGCCTCACGCTTCAGCTT
TaCBL9BDR GGCTGCAGTTAGTCTTCAACCGCGGTATTG
TaCIPK16ADF CCGGAATTCATGTCCGCGAGCCCGG
TaCIPK16ADR AAAACTGCAGGCTACGTCAAGATGTACCAGAAGA
TaCIPK16163F AAGCTTATGGCGAGAGGGCGGGAGGG
TaCIPK16163R GGATCCCGCGACGGCGCCCGG
TaCIPK16qRTF1 GCCGCCGCAGGCCGCGCGGCTGC
TaCIPK16qRTR1 CCTTCTCCCGGTCGAGCACCTTGAT
TaEF1F TGGTGTCATCAAGCCTGGTATGGT
TaEF1R ACTCATGGTGCATCTCAACGGACT
XαGal)(LTHA+ Xαgal)ÜëΨ©。¨©
4d8,žýþ)ÆÞì ¡¦§ÿ™Ó!"#
¿ºÅ107、106、105Ì104›¦§,lLTHA+
Xαgal¨©.ܨ©,24h8ÌÇ。
1.2.4 cdefg ˆÇpCaMV35S:GFPè
–—©lN$

Û)íTaCIPK16y¦§¨©
– — @ $ TaCIPK16163F/R (´ 1), ì
TaCIPK16–— pCaMV35S:GFP ,àÌ Li
=
[19]
]¢£

ƒ%XY„S¼.

à̑¢£ì
rÍ.pCaMV35S:TaCIPK16∶GFP¥&Í.
pCaMV35S∶GFPÃXY„S¼.,¨ ©18h
8

M‚ä­Ê()ëÎ*+ÌÇ

1.2.5 狇犚犜犘犆犚CD wǖ—,U]犜犪犆犐犘犓16
.œÌÀà.œXYxãœ犜犪犈犉1α(GenBank÷
ć Q03033)ª$Û)°Ø¨Ñ@$ TaCIPK16
qRTF1/R1¥TaEF1F/R(´ 1)。-.9#Z[
’80、12、18、24、72Ì120hþ/l]”•¾¿
RNA,äÃÄ|cDNA,B^cDNA|3Þ,qRT
PCRä匲|95℃àé±5min,95℃é±,
10s,55℃ áâ20s,72℃xã40s,ä40›åê。
qRTPCR]äå.`¥Œ²àÌ Feng=]¢
£
[20],
Ø8-2-ΔΔCt£)ó•0.œáÀà.œ
]Çá´ÔÑ

2 €Ýƒª«
2.1 45犜犪犆犐犘犓16,<=>?@YZCD
M‚RTPCRbŸ–—™Ó]cDNA²³Ä
57016;        MYOTHWIN,=:XYCBL€z{„x.œ犜犪犆犐犘犓16]–—¥°1ª«
Å1606›23w(41),¶·1›447¸.w]z
{

z{ªÑ|49.521kD,=ôl|9.09。õ
InterProScanñòª«´µ,­N6¹º}›z
{„x€½¾
,C6ºÅ}›u_€½¾,†u7
¾¹º¯ZƒCBL°Ø€]NAF€½¾¥ƒ
PP2CÇÐO]PPI€½¾(42)。žÁÂÃ
ƒ¿À5Å]CIPK²³¸.w²½Óë‹ÔŽ
q‘z{ƒ¿À] OsCIPK16(LOC_Os09g25090)
¥ÁÂÍ] AtCIPK5(AAF86504)、AtCIPK16
(AAK50348)¥AtCIPK25(AF44226)ö1}›ª
:

43)。5Z²³í᪫´µ,‘z{²³ƒ
1~2.PCR†ßŠ$;M.Marker
41 犜犪犆犐犘犓16.œcDNA]PCR†ß•5
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:kç‘7l]ÞNÆ
43 TaCIPK16ƒ¿À、ÁÂÍCIPK(š]ë‹Ôª«
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{„x€½¾;<ð´ËC6u_€½¾;<=´ËNAF€½¾;;ð=´ËPPI€½¾
42 TaCIPK16ƒ¿À、ÁÂÃö§.œ]è²³íá
Theconservedproteinkinasedomainandregulatorydomainaremarkedwithblackfullineanddashline,respectively.
TheconservedNAFandPPImotifsaremarkedwithblackboxanddashbox
Fig.2 MultisequencealignmentofTaCIPK16withhomologousproteinsinriceand犃狉犪犫犻犱狅狆狊犻狊
6701 ! " # $ % &                   36Ë
OsCIPK16ÇÈÆÔÓ88.04%,ƒÁÂÃ(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

ìTaCIPK16ƒTaCBL4?PTaCIPK16
ƒTaCBL9äöࡍ。†AÒ¨©.SD
LeucineTryptophanÜ ¡¯ZßBSö,Žµ2
#r¼½í®ÃC ¡

44,A)。†AÒ¨
©.SDAdenineHistidineLeucineTryptophan(º20
ºÅkʼ½]AH109 ¡’p†ÒD¨©.SDLeucineTryptophan(LT)ÌSDAdenineHistidineLeucineTryptophan(E20μg/mL
]Xαgal)¨ ©;A、B.F±áÌ,Takala ¡µ¶·]SV40largeTantigenÌmurinep53ÐO€Ý:C、D.TaCIPK16ÌTaCBL4]ÐO
€Ý
;E、F.TaCIPK16ÌTaCBL9]ÐO€Ý;G、H.G±áÌ,Takala ¡µ¶·SV40largeTantigenƒhumanlamin]ÐO€Ý
44 TaCIPK16ƒTaCBL4、TaCBL9]ÐOª«
CelsofyeaststrainAH109harboringtheindicatedplasmidcombinationsweregrownonselectivemediaSDLeucineTryptophan
(LT)andSDAdenineHistidineLeucineTryptophan(containing20μg/mLXαgal);A,B.Positivecontrol,theinteraction
betweenSV40largeTantigenandmurinep53;C,D.TheinteractionofTaCIPK16andTaCBL4:E,F.Theinteractionbetween
TaCIPK16andTaCBL9;G,H.Negativecontrol,theinteractionbetweenSV40largeTantigenandhumanlaminC.
Fig.4 AnalysisofinteractionsbetweenTaCIPK16andTaCBL4/TaCBL9
A~D.F±áÌ,pCaMV35S:GFPË]XY„S¼¦§;E~H.pCaMV35S:TaCIPK16:GFPË]XY„S¼¦§;A、E.Ò
„Ž]GFPHI̔J.+ŽHIʁ€Ý;B、F.”J.+ŽHI;C、G.҄Ž]GFPHI;D、H.{IO]„S¼¦§
45 XY„S¼.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,=:XYCBL€z{„x.œ犜犪犆犐犘犓16]–—¥°1ª«
46 犜犪犆犐犘犓16.œ†XYZ[’ÐO‚Œ]
ÃÄ´Ô°1
Fig.6 Expressionpatternof犜犪犆犐犘犓16duringthe
compatibleandincompatibleinteractionbetweenwheatandPst
μg·mL
-1XαGal) ¡¯ZSöˆíKL,ƒF
± á Ì Ç È

Ž µ TaCIPK16 ƒ TaCBL4 Ì
TaCBL9n͆ÐO(44,B)。ì ¡¨©Mÿ™
Ó!"#ºÅ104› ¡¦§þ,N¯*+Ó ¡
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