全 文 :书!"#:10004025(2016)06107307 犱狅犻:10.7606/j.issn.10004025.2016.06.1073
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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{
,
ªñtu%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]。 ÁÂà ,AtCIPK23ÞB AtCBL1ÐO,vw* K+MG (AKT1)Hë K+ ]I J [8]。 K Á  à 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 Âà ,NPR1¯ZCIPK11/PKS5vw,G
V WKY38Ì WKY62]´Ô[17]。 : , XY&2CIPKz{xSüý
]®¯~_
。
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。PCRæ8Â9ÓpGEMTEasy 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{]=ôlÌªÑ ; 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,ªdz
±¨©.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)ªÛ)°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?SSüý 。 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_
[2]。
mn
,TaCIPK16{ª
O2f¦§
,
Z[TaCIPK16ZmCIPK16®
¯ÄÅd»±
。
öþ
,
¦§z{]¨©PQÌ
®¯&`mÁÇ&
,
^Z릧 TaCBL4
TaCIPK16TaCBL9TaCIPK16z{ç.¯Z
*è#z{5@÷]NMð
。
犜犪犆犐犘犓16.Z[VOÕÜu´Ô
ÙÚ
,
µà¬Z[V]XYâ\^ãä
å
,
XÖÌäåÌSÖÌäå]´ÔÑ×ص
TaCIPK16nÞ¯à¬XYR.oV]â\N
Mð
,
XYZ[Êí]SÖÌÐO÷
kFO
。
wÇ Wang=[23]&G,SÖÌÐOäå
,
9#Z[:;12~24h,ñXY¦§*±Ep
q524hþÔÓT9,
924h_8*±
E]ºÑ9rWsC
,
Ó9848h,*±E]ºÑ
ÔÓØC
。
mn9812~36hTaCIPK16
]´Ô¿ÿÒÓ
,
9848hTaCIPK16Ò
ÓV´Ô¿ÿÓØc
。
ÅKL´µ
,
XY
TaCIPK29àu_ ROSNMð[24]。^Zë
TaCIPK16Þ¯XYZ[SÖÌÐOu
_ROSNMðB^oVXY]â\^ãäå。
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