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Prokaryotic Expression of Cucumber CsERECTA Gene and Preparation of Its Polyclonal Antibody

黄瓜CsERECTA基因的原核表达及其多克隆抗体制备



全 文 :书!"#$%&
,2016,36(5):1031-1038
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。Email:liyuhong73@126.com
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<=>?@ABCD
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(1!"89+:;% bc%d,e!fg712100;2!"89+:;% Ih+%%d,e!fg712100)
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ˆ‰Š

D‹Œ„Ž‘’“”sK>•

–1HU—x‰˜” maltosebindingprotein(MBP)™šT
pET21aCsERECTA›=~œžŸ },¡¢£D;¤¥¦(犈.犮狅犾犻)BL21(DE3)§T.¨žŸ,©ª«¬ž
ŸT­®
、¯
°±IPTG²®³´£µ¶。·•¸¹º»=¼½¾¶¿À MBPCsERECTA›=~,Á•rTEV
~ªÂ³´Ã

¿ÀCsERECTA~©Ä£–~TÅÆǏ}。wȞÉ,^ _CsERECTA~Ê
Ë̱ÍÎ}2Q‡ÏžŸ,Э”ÑÒ~ÊËÌӇÏ;ÔÕD。Ö׫¬­®Ø23℃,«¬¯°Ø6h,
IPTG²®Ø0.5mmol·L-1。U— WesternblotËÙÚÀ^_ÛaTCsERECTA~,ÜɍÄTÅËǏ}
ݔÞßTàáÓ

ÅÆǏ}TŠâÄسlã1H犆狊犈犚犈犆犜犃Tâäåæ£,ç。
GHI
:^
_
;犆狊犈犚犈犆犜犃,i;œžŸ;ÅÆǏ}
JKLM$
:Q785;Q786 !NOPQ:A !"#$:
犘狉狅犽犪狉狔狅狋犻犮犈狓狆狉犲狊狊犻狅狀狅犳犆狌犮狌犿犫犲狉犆狊犈犚犈犆犜犃犌犲狀犲犪狀犱
犘狉犲狆犪狉犪狋犻狅狀狅犳犐狋狊犘狅犾狔犮犾狅狀犪犾犃狀狋犻犫狅犱狔
WANGQiqi1,LIYuhong1,HULiangliang1,QINYaguang1,ZHAOZiyao1,CHENPeng2
(1ColegeofHorticulture,NorthwestA&FUniversity,Yangling,Shaanxi,712100,China;2ColegeofLifeScience,North
westA&FUniversity,Yangling,Shaanxi,712100,China)
犃犫狊狋狉犪犮狋:犈犚犈犆犜犃geneencodesaleucinerichrepeatreceptorlikeSer/Thrkinase.Itparticipatesinregu
latingmorphogenesisofplantorgansandplaysanimportantroleinthechangeofplanttypeandresistance
toadversity.Inthepresentstudy,pET21aCsERECTA,aprokaryoticexpressionvectorwithmaltose
bindingproteintagswasconstructedandsuccessfulyexpressedin犈.犮狅犾犻BL21(DE3).Expressioncondi
tionswereoptimizedintheoptimaltemperature,inductionduration,andIPTGconcentration.Thefusion
proteinMBPCsERECTAwaspurifiedbynickelchelatingchromatographyandenzymedigestionwascon
ductedbyrTEVprotease.TheCsERECTAproteinwasobtainedanditwasusedtopreparepolyclonalan
tibody.TheresultsshowedthatCsERECTAwasexpressedintheformofsolubleandinclusionbodiesin
犈.犮狅犾犻BL21(DE3),andalargenumberofproteinsweresolubleatlowtemperature.Theoptimaltemper
ature,inductiondurationandIPTGconcentrationwere23℃,6hand0.5mmol·L-1,respectively.West
ernblotshowedthatthepolyclonalantibodyofCsERECTAhasgoodspecificity,becauseendogenous
CsERECTAwasdetected.Thesuccessfulypreparedpolyclonalantibodycanbeusedforfurtherinvestiga
tion,whichestablishthefoundationforinvestigatingthefunctionofthe犆狊犈犚犈犆犜犃geneincucumber.
犓犲狔狑狅狉犱狊:cucumber;犆狊犈犚犈犆犜犃gene;prokaryoticexpression;polyclonalantibodies
  犈犚犈犆犜犃èlmjknopqrstuvT
{|}~€,i
[1]。
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ôuTëP±žõTñ¶ö
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ä1HJKU§DVÏ#$üýþï

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êl@m
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wxyz{Ò
-80℃|Õ。;¤¥¦¦‹BL21(DE3)±œž
Ÿ }pET21aHMT÷0¡f}|Õ;;¤¥¦
~|ˆ,-TOP10Ò"€‚I¶+:ƒ„。
HiProof.|IDNA…=Òf†‡lI
$:S”ˆƒ„

ˆÓÛÁ犅犪犿 HI、犡犺狅I、
犜犪狇DNA …=、‰Š‹ŒŽ(PrimeScriptTM
RTPCRKit)、T4ŒŽ(DNA Ligation
KitVer2.1)、rmarker(DL2000)±~marker
(#SM0431)‘);TaKaRaƒ„;.¾®`’
“C§ÔŒŽ
、DNA”•–ŒŽ、ECL—˜Œ
Ž)"€‚I¶+:ƒ„

™‚—š¶$
™›TœžÒ!Ÿ ¡¢I$:Sƒ„

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Ni2+¦±¼½§(¨ Novagenƒ„);©ª«R、β
Dá¬,­®¯°±²(IPTG)、¬³´µ(Acryl
amide)Ž¶·¸¬³´µ(Bisacrylamide))
Sigmaƒ„;¹R、º»öÂWŒ‘ؼñ
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1.2 VW
1.2.1 犆狊犈犚犈犆犜犃XY7Z[\] D NCBI½
¾¿§ÀÁ GenBank –‹T 犆狊犈犚犈犆犜犃 ,i
(GenBank‹à NC_026658.1)q,ruv。ª
CCMCTCsERECTA~ñÄ·•~`DÅÆ
Ú]ÝSignalP4.1Server§TÇÈÉÊj(Neural
Network,NN)ÆÚVÃËÌÍ;TMHMMServer
v.2.0(http://www.cbs.dtu.dk/services/TM
HMM/)ÆÚÎÏÌ;PlantsP(http://www.ncbi.
nlm.nih.gov/pubmed/11125063)ÆÚ LRRÌR
€Ì

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rT¦ÐӜÑ

eÒ£jk-MÌ317~517Óq
,r

ÔÕÖ$犆狊犈犚犈犆犜犃L(5′TGAGGGATCCGA
GAAGTTGTACCTGCATAGTAATAAGC 3′,×ØÅ
ÙØ犅犪犿HIÃÓÚ)±犆狊犈犚犈犆犜犃R(5′GGC
CGCTCGAGGTTGTTGTTCTCAAGTCTCAGG
GAG3′,×ØÅÙØ犡犺狅IÃÓÚ),Ö$÷"€
Û+ÜÝI$+:”ˆƒ„=Š

ÊCCMC،(,CÞßRNA,·•‰Š‹Œ
Ž

=ŠcDNA。ÁÊcDNAØVà,犆狊犈犚犈犆
犜犃L±犆狊犈犚犈犆犜犃RØÖ$³´?T,iá=,
PCRá=t• HiProof.|IDNA…=³´á
=±âò
,PCR‰ã}ä(25μL)Ø:ï×åÖ$
(10μmol·L
-1)
æ1μL、dNTP(10mmol·L
-1)
0.5μL、5×Qbuffer5μL、HiProofDNAPolymer
ase(5U·μL
-1)0.2μL、VàDNA1μL,ddH2O
çè825μL。PCRá=1min;96℃ùÓ10s,54℃éê20s,72℃30s,
ëìá=32í;Ö{72℃âò10min。PCR¼$
2301 ! " # $ % &                   36î
È1%ïð±ñ”òóÙÚ{,•DNA”•–Œ
Ž•–PCR¼$。
1.2.3 犆狊犈犚犈犆犜犃,689:;fB7[g ¾
¶T犆狊犈犚犈犆犜犃,iôõÈ犅犪犿 HI±犡犺狅I¸
Ã{

RÈö÷¸ÃTpET21aHMTžŸ 
}A  

i ¿ s * ž Ÿ   } pET21aHMT
CsERECTA,Š¶ø;¤¥¦~|ˆ,-TOP10,
ȦùPCRŽ`’PCRÙÚ{,Úufg–žŸ
 }§T,iuvŽEkúT5ûÓ

uvñ½ü
ýØDNAstar±NCBIBLASTn。
1.2.4 犆狊犈犚犈犆犜犃hiXY7jk:;lmno
Lp ÞÚu5ûT¦w,Ä pET21aHMT
CsERECTA`’,þs*`’U—z›jŠ¶ø
BL21(DE3){³´«¬žŸ。ÿÞ!¦ùQÀ
o100μg·mL
-1
q"#«R
(Amp)Tw}LB"
#,§
,37℃、220rpm$%"#—&。íÊ1∶
100á;"#,37℃$%"#8 OD600()Ø0.6
¯*ø1.0mmol·L-1IPTG«¬~žŸ。¦w
È+,-./

]>¯°3s,°0¯°7s,./
40min){,¹s–Uï1±23,ñč÷©³´
SDSPAGEòóÙÚ。
1.2.5 hiXY:;qr7st (1)!"#$%
& Þ4ÓÆÇT¦wöԐQÒ3mo”
AmpIRT5mLw} LB"#,§,ñÄÒ
37℃、28℃±23℃"#8OD600(Ø0.6¯,*ø
1.0mmol·L-1IPTG«¬6h。ñÄÞ500μL¦
w

¹s–U¦}{

*ø100μL1×ï÷56w,
7Ð8 10 min{Þï1³´ SDSPAGE òó
ÙÚ

(2)!"()%& 23℃"#8OD600(Ÿ
0.69:,*ø;²®Ø1mmol·L-1TIPTG,
23℃<­"#,ñÄD0、4、6、8、10h¯Þ÷³´
SDSPAGEòóÙÚ。
(3)IPTG!"*$%& Ò23℃"#8
OD600(Ÿ0.6¯,ñÄ*ø;²®Ø0、0.25、0.5、
0.75±1.0mmol·L-1IPTG,23℃<­"#6h
{ñÄÞ÷³´òóÙÚ

1.2.6 hiXY7ut ‚oX=ö[11]T‘j³´,·•¸¹º»=¼
½§¾¶›=~

(2)+,-.%01 þ200μgT›=~±
30μLTrTEV~(1.5U·μL
-1)、75μLT
rTEVbuffer1(20×)±15μLTrTEVbuffer2
(100×)>=,*Š1.5mLT‰ã}ä,Ò}­
(30℃)—&(14h)‰ã。
1.2.7 xyXY7>?@ABCD þÃ{T
¼$³´SDSPAGE(20cm×20cm)òó,ÃÞ?
™?˜@8ABCDI$:S”ˆƒ„³´
CsERECTATÅÆǏ}Ä。
1.2.8  4 5 z { 犆狊犈犚犈犆犜犃 X Y 7 | }  
CsERECTA~TCÞñÄt•£3Q‘j,Þ÷
Ô±Þ÷ðӑAö

‘j1Ø+y¹sjCÞÏ
~

‚oQianö[12]‘j³´。Þ3gróls¯
0TCCMCuvðÓ*+÷E,wxyz{1FŠ
GH3

*ø6mL ÆITÏ~CÞ56w
(250mmol·L-1JKL,50mmol·L-1TrisHCl,
0.2mmol·L-1EDTA,pH8.0),MÒNï1h{,
4℃5,000g¹s5min,Þï1Ò10000g¹s
10min,Ö{Þï1Ò100000g¹s30min,þ2
3ÌOÒ60μL Ï~ÌOw(5 mmol·L
-1
H2PO4,330mmol·L-1P±,3mmol·L-1 KCl,
pH7.8)§。‘j2ØQrR23jCÞ^_Tß
~

‚oS¸¸ö
[13]
‘j³´

‘j3ØTriton
X100TUjCÞ^_TÏ~,‚o Shpak
ö
[3]
‘j³´

1.2.9 犆狊犈犚犈犆犜犃 XY7 犠犲狊狋犲狉狀犫犾狅狋Lp 
Westernblotñ½‚oXYVö[14]‘j³´。÷
E~SDSPAGEòó{,ȯJòŠW(20mA,
2h)ŠXÀ0.45μmYZTPVDFÏï。*ø
ÄTÅÆǏ}
(1∶2000)[P2h,Á*™‚—š
¶$
(HRP)™›Tœ1gG(1∶10000)}­
[P1h{,•ECL—˜ŒŽ³´—˜,•¶%
ënŠ\W]o

2 wÈRñ½
2.1 45犆狊犈犚犈犆犜犃XY~€Lp
·• SignalP 4.1 Server Æ Ú CCMC §
CsERECTA~`TVÃËÌ,ë¢^32Óq,
rèÖËäT_ÃÓÚ

žÉVÃË`ËäÕDÒ
&32mq,r§。U—TMHHMv2.0ÆÚ^_
CsERECTA~TÎÏ̱-Û、MÌ,wÈë¢
q,r7~29ÓÚa°±q,rÓÚ600bc”2
m Î Ï Ì

U — PlantsP § T Pfscan Æ Ú
CsERECTA~`§TLRṞ€Ì(d1),
wȞÉ
,CCMCTCsERECTA~`LRRÌD
107~513q,ra°,€ÌD657~929q,r


eʂ¾Âwxͱq,rT¦ÐӜÑ

eÒ£TfVÃ˱ÎÏÌa{Tjk-MÌ317
330150           @AB,ö:^ _犆狊犈犚犈犆犜犃,iTœžŸŽÂÅÆǏ}Ä
~517mq,rTôõÔÕ£1ªÖ$(d1§g
hei
)。
^_犆狊犈犚犈犆犜犃,ijklmÎÏT{|}
~€

ŠjTCsERECTA~÷991mq,
rk,xŠ

lmïTñºÔ;“)Ø109kD。D
ïn~wxÆÚT,çï

ÔÕÖ$á=À-M
ÌôõToêØ603bp,jk201mq,rk,,–
haTCsERECTA~ñºÔ)Ø22.8kD,PÒ
rÓ~`

2.2 /7de7?@89:;fB[g
pET21aHMT(pET21aHisMBPTEV) }
èDpET21aT,çïpc{TlmžŸ },Â
ÅÆÇÓÚ§qíÍo£Histag、MBP±TEV
ÃÓÚTAãuv

þhaT犆狊犈犚犈犆犜犃,i
– }{

žŸT›=~

×r§sØ
CsERECTA›=~)˜” His± MBP™š,U
—~`uvÕt–›=~;“)Ø65kD。
CsERECTA›=~§”TEV~ÃÓÚ,
ȗrTEV~Ãa{,ä¿À)Ø42kDT
uv±w=~
(MBP)±22.8kDThaT
w˜ÌÍ.LRRÌ;xőúÌÍ.€Ì;ghyiÙ
ØÖ$TÓÚ
d1 CCMCTCsERECTA~`LRR̎€ÌÆÚ
Grayarea:LRRregion;Diagonalblockarea:kinaseregion;
Thepositionoftheprimersareindicatedbythearrow
Fig.1 LRRregionandkinaseregionpredictionof
CsERECTAproteininCCMC
CsERECTA~。
ÊL^_óô§CÞTRNA‰Š‹ŠTcD
NAØVà,PCRá=9!1?˜,Â;“Ø603
bp,RÆ0Az(d2,óZ1)。PCR¼$•–{Æ
ÇÀpET21aHMT },Š¶TOP10,ÿ!ÆÇ{
{¦ùPCRÙÚ(d2,óZ2)±`’PCRÙÚ

d2,óZ3),ÿe4Ó¦‹þ¦w@TÚu。
uvÚæwȗižÉi¿Ts*ÆÇ }Ío?
™,i

eá=ÀT,iôõ;“Ø603bp,RÆ
0Tôõ;“Aö

²ruv“l|

žÉ
犆狊犈犚犈犆犜犃 ,iTœžŸ }pET21aHMT
CsERECTAx‰Šâ。
2.3 犆狊犈犚犈犆犜犃hiXY‚ƒ„…†J7jk:
;<:;‡ˆ7‰Š
    þ x ‰ Š â T ž Ÿ   } pET21aHMT
CsERECTA¬ø犈.犮狅犾犻BL2l(DE3)§,8ª½I
ê0
(OD600()Ø0.6)¯ *1mmol·L-1IPTG«
¬6h{,–U¦},©Ê}*IPTG«¬T¦Øª
o

³´SDSPAGEÙÚ。wÈRªo(d3,óZ
3)Aì,os*`’T¦wD;)65kD9:Ù(d
3,óZ1±2)9¢1?Þ.²®Tàá?˜,RÆ
0T CsERECTA › = ~  ñ º Ô A ~,ž É
犆狊犈犚犈犆犜犃,iÈD;¤¥¦BL2l(DE3)§›
=žŸ

ªCsERECTA›=~³´žŸ‡ÏTÙ
Ú

wÈíd3ei,D37℃¯,s*žŸ¦}.O
Tï1w§›=~

óZ2)TžŸÔɗ€Ò2
3

óZ1)§TžŸÔ,D28℃¯,s*žŸ¦}
.OTï1w§›=~

óZ5)TžŸÔ”e=
*

2D23℃¯,s*žŸ¦}.OTï1w§›
M.DL2000;1.犆狊犈犚犈犆犜犃,iTPCRá=;2.s*`’pET21aHMTCsERECTAT¦ùPCRæ;
3.s*`’pET21aHMTCsERECTAT`’PCRæ
d2 ^_犆狊犈犚犈犆犜犃,is*`’pET21aHMTCsERECTATæ
M.DL2000;1:AmplifiedbyPCRof犆狊犈犚犈犆犜犃gene;2:ColonyPCRofrecombinantvectorofpET21aHMTCsERECTA;
3:RecombinantplasmidofpET21aHMTCsERECTAidentifiedbyPCRamplification
Fig.2 Identificationof犆狊犈犚犈犆犜犃genewithrecombinantvectorofpET21aHMTCsERECTA
4301 ! " # $ % &                   36î
=~

óZ7)TžŸÔɗ.Ò23(óZ6)§
TžŸÔ

ï1§65kDٔÉpT~?˜,‚ƒ
;ðñT›=~„ÊËÌT‡ÏÕD

…ÜÉ
CsERECTA›=~ÊË̱ÍÎ}rQ‡ÏÕ
D

†23℃Э«¬”·Ò–~ÊËÌT‡Ïž
Ÿ

i%

‡ÔˆÐ«¬­®è‰³~ËÌӞ
ŸT”¨‘jal

«¬CsERECTA›=~ž
ŸTև­®Ø23℃。
2.4 犆狊犈犚犈犆犜犃hiXY:;qr7st
Dª=‡«¬­®³´Š‹{

01Hλ
CsERECTA›=~³´£‡«¬¯°±«¬
²®Tµ¶

ÈSDSPAGEñ½žÉ(d4),s
*¦Dև­®23℃?ý×,›=~žŸÔŽé
«¬¯°Tâê2=*

«¬6hžŸÔÖ;,Ž{
žŸÔl‘DlæВ“É—ù¶

s*¦D
23℃?ý×,*øIPTG ñÄ8;²®Ø0.25、
0.5、0.75±1.0mmol·L-1,«¬"#6h{,È
SDSPAGEòóñ½,žÉ›=~TžŸÔD
IPTG0.5mmol·L-1›=~žŸÔÖ;,Ž{ž
ŸÔ“É—ù¶

÷%ËY
,CsERECTA›=~
TևžŸ?ýØ
:23℃?ý×0.5mmol·L-1T
IPTG«¬6h。
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犫犻犱狅狆狊犻狊犈犚犈犆犜犃geneencodesaputativereceptorproteinki
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[3] SHPAKED,LAKEMANMB,TORIIKU.Dominantneg
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犜犃leucinerichrepeatreceptorlikekinasesignalingpathway
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ticinteractionofthree犈犚犈犆犜犃familyreceptorlikekinases
controls犃狉犪犫犻犱狅狆狊犻狊organgrowthandflowerdevelopmentby
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犪犫犻犱狅狆狊犻狊arerequiredforresistancetothenecrotrophicfungus
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