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Clonging and Expression Analysis of γVPE Gene Related to Powdery Mildew Resistance in Grapevine

葡萄抗白粉病相关基因γVPE的克隆与表达分析



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,2016,36(8):1507-1514
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‘’“g”,•
(γ犞犘犈),–—˜™š犞狆γ犞犘犈 ‡犞狏犆γ犞犘犈。Ž€2›γ犞犘犈,•cDNAœžš1624bp,
ORFš1482bp,Ÿ 493›¡,¢。¡,¢£¤¥¦§–¨©.,‘…†351’、‘‹Œ’、‘ª«…’‡‘¬§­’
ab€γ犞犘犈,•®$¯°±²³€3›´µ¡,¢¶·€¸¡¢(Ser395)ž¹šº¡¢(Ala),f»¼#$€
犞犘犈 ,•®$±²¯°³½¾¿À。rÁÂÃÄÅPCRÆÇ,ȅÉÊË_̀¿ÀÁÍÎ,γ犞犘犈,•Èˆ
ab‡~ab€ÆÏÐÑ¿À

~Ò6犞狆γ犞犘犈 ,•€ÆÏÅÈË_̀ÓÍ(4h‡48h)‡ÌÍ(168
h)ž½¾Ô“,ՈÒ6犞狏犆γ犞犘犈 ,•ÈË_Ì4hÆÏÅÖ×,ØÌÙÚ。γ犞犘犈,•È…ÉÊË_Ì¿
ÀÁÍÎÆÏŀ¹Û

ÆÇγ犞犘犈,•È·ÄܝÝfab€~Þß´。ST¯Mšà·áâ=γ犞犘犈,•È
~ã܁€–äåæçĐ,è

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ab
;γ犞犘犈;,•Ž;…É;Æϖ¨
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犆犾狅狀犵犻狀犵犪狀犱犈狓狆狉犲狊狊犻狅狀犃狀犪犾狔狊犻狊狅犳γ犞犘犈犌犲狀犲犚犲犾犪狋犲犱
狋狅犘狅狑犱犲狉狔犕犻犾犱犲狑犚犲狊犻狊狋犪狀犮犲犻狀犌狉犪狆犲狏犻狀犲
WANGYanqing,ZHANGXiaoying,SONGJianing,WANGYuejin,ZHANGChaohong
(ColegeofHorticulture,NorthwestA&FUniversity,StateKeyLaboratoryofCropStressBiologyinAridAreas,KeyLabora
toryBiologyandGeneticImprovementofHorticultureCrops,MinistryofAgriculture,Yangling,Shaanxi712100,China)
犃犫狊狋狉犪犮狋:Vacuolarprocessingenzyme(γ犞犘犈)wasclonedfromChinesewild犞犻狋犻狊狆狊犲狌犱狅狉犲狋犻犮狌犾犪狋犪acces
sion.‘Baihe351’and犞犻狋犻狊狏犻狀犻犳犲狉犪cv.‘Carinena’byRTPCR.Theywerenamed犞狆γ犞犘犈 and
犞狏犆γ犞犘犈.Thefullengthof犞狆γ犞犘犈and犞狏犆γ犞犘犈cDNAare1624bp,andtheopenreadingframeare
1482bp,encoding493aminoacid.ThealignmentoftheaminoacidsequencerevealedthatSer395,one
keyaminoacidinsubstratepocket,wasreplacedbyAlain犞犻狋犻狊狆狊犲狌犱狅狉犲狋犻犮狌犾犪狋犪accession.‘Baihe351’,
犞犻狋犻狊狏犻狀犻犳犲狉犪cv.‘Carinena’,犞犻狋犻狊狏犻狀犻犳犲狉犪cv.‘Thompsonseedless’,and犞犻狋犻狊狏犻狀犻犳犲狉犪cv.‘Pinot
Noir’.Thedifferentexpressionprofileof犞狆γ犞犘犈 geneand犞狏犆γ犞犘犈 geneindifferentperiodsafter
powderymildewinducedwereanalyzedbyRealtimefluorescentquantitativePCR.Afterinductiontheex
pressionof犞狆γ犞犘犈geneincreaseslightlyin4h,48h,168handthe犞狏犆γ犞犘犈geneshowspredominant
expressionin4hafterthatitdecreasesslowly.Thestudyshowsthatγ犞犘犈isrelatedtoresistanttopow
derymildew.Thestudyprovidesreferencevaluetothemolecularmechanismofγ犞犘犈geneindiseasere
sistance.
犓犲狔狑狅狉犱狊:grape,γ犞犘犈,geneclone,expressionanalysis,powderymildew
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‹óôUã)ŒÛ.W&Ž
Èl…Ó5Ɛ€st‘’“,‡st¡¢“,
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狋犻狊犮犻狀犲狉犲犪)‡¦§¨©„(犜狌狉狀犻狆犿狅狊犪犻犮狏犻狉狌狊)
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Û
[21]。
¸sÆÇ

ªh« VPE €¹º¹]^5
(α犞犘犈/β犞犘犈/γ犞犘犈/δ犞犘犈)»¼½ VPEl…
€#ÞO¼½ƒcaspase1l…€#Þ[18]。=³,
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VPE®$‡·›Kmš44.2μmol/L€caspase1
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ÞÔƀÇÿÈ©RÈγ犞犘犈,•Mº¹5Ý,Õ»
¼€犞犘犈,•óôÉàÁÊ¥ä€êë[22]。


‡Èab,•)Ê©.3›犞犘犈
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:β犞犘犈、γ犞犘犈 ‡δ犞犘犈,%–—È‘ª«…’‡

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•f犞犘犈 (̀»¼ëËߧ,®$¯°±²³
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(Ala)Î/[22]。ÏSTÈ=,èÝ,K‚Rƒ
„ab

…†351’Q‰Šab‘‹Œ’МcD
NAÑҁŽÓԐγ犞犘犈,•,%UãR$V
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oreticulataaccession.‘Baihe351’)‡‰Šab‘‹
Œ
’(犞犻狋犻狊狏犻狀犻犳犲狉犪cv.Carinena)P#˜!"0
h+3i%jk%lPcdeå

ab…ÉÊ
{犈狉狔狊犻狆犺犲狀犲犮犪狋狅狉Schw.[syn.犝狀犮犻狀狌犾犪狀犲犮犪狋狅狉
(Schw.)Burr.]}Î)de偉Šab‘æçè’
(犞犻狋犻狊狏犻狀犻犳犲狉犪cv.‘CabernetSauvignon’)。
1.2 T U
1.2.1 78VWX@YZ |}éêã[23],qæ
çè©ê݀…ÉÊëP˜Rœì×íý€

…†
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168hÊ8›Á̀ab©ê,q|ð€©ê ‘
ñòóôõÌö˜-80℃÷k。
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}OmegaPlantRNAKit¸µø–—ÛΠ…É
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ÓԅÉÊË_Ì¿ÀÁÍ΁‚Rƒ„a
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…†351’、‰Šab‘‹Œ’€cDNA。
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ab
(犞犻狋犻狊狏犻狀犻犳犲狉犪犮狏.Thompson
Seedless)(ûüýšKU240054)γ犞犘犈,•€¤¥
š²Ý

ò} Primerpremier5.0 þÿ8!d$
γVPEF (5′GGCTCCGATGACTATCTTTCCG
3′)‡ γVPER(5′GGAGCAACCAGTAGAAG
CAGCA3′),<…ÉÊË_€‘…†351’f‘‹Œ

’cDNAšÐ",ò}LA犜犪狇”(TaRaKa)GÔ
γ犞犘犈,•。RTPCRRÿ56š:Ð"50ng,d
$#1μL(10μmol/L),10×LAPCRBuffer($
Mg2+)3μL,dNTP2.5μL,LA犜犪狇DNA%°”
1.5U,“ddH2O&(30μL。RTPCRRÿܤ
š
:94℃)¹Þ5min;94℃¹Þ30s,58℃*+
30s,72℃,-2min30s,34›.$;72℃,-
10min,4℃÷k。PCR2$ 1.5% /0123
45€Ì

qPCR2$67%8ë(Ž95
pMD19TÝ,:q8ë2$•Û(i;<Ê(犈.
犮狅犾犻)ˆ8J34Top10,Uã=…I>?@΅
IMÊAà}ÊAPCR€,ÓÔBÞÊÒÌC(
D+EF¤

1.2.3 γ犞犘犈 ,<=[\CD q¤ÓԀ
γ犞犘犈,•€cDNA¤¥ÈNCBIÂGҁà}«
H¢‡¡,¢¤¥§¦

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www.ncbi.nlm.nih.gov/projects/gorf/)I2B
J¤¥€ÖiyKLMN

ò}Expasy€Pro
tparamܤ(http://web.expasy.org/protparam/)–
¨γ犞犘犈,•€æÛÞc,UãProtScaleܤ(ht
tp://web.expasy.org/protscale/)¦γVPEl…€O
PÞà}–¨

} TMHMM2.0ܤ (http://
www.cbs.dtu.dk/services/TMHMM2.0/)–¨
γVPEl…LQV½RS?³。} MEGA6.0þ
ÿà}àÛN–¨

}DNAMANþÿ,q‘…†
351’‡‘‹Œ’γ犞犘犈,•€¡,¢¤¥fªh
«
、‘
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’犞犘犈 ,•(̾½ëˀ
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} SWISSMODEL
(http://swissmodel.expasy.org/)Àe7ÐãT7
VpγVPEl…€UV¯TW,%ÈWJX®$¯
°±²€3›´µ¡,¢Aq€Aö。
1.2.4 4578V]^_γ犞犘犈,<=ABCD
 YGγ犞犘犈,•€cDNA¤¥8!rÁÂÃÄ
Å d $
,qRTPCRγVPEF (5′CAACCTGCG
GACTGAAACG3′)‡qRTPCRγVPER(5′TGTT
GAGACTTAGATCACCATATTGC3′)。< ‘… †
351’‡‘‹Œ’#Ò8›Á̀©êcDNAšÐ
"

PCR18SrRNAF(5′CAACAAACCCCGACTTCTG
3′)‡qRTPCR18SrRNAR(5′TGTCACTACCTC
CCCGTGTC3′),YGrÁÂÃÄŸµøSYBR
PremixEx犜犪狇 TMⅡ(TaRaKa)ÄÇZà}qRT
PCR。qRTPCRR[56š:cDNA1μL,d$#
0.8μL(10μmol/L),2×SYBR10μL,ddH2O
7.4μL。PCRRÿܤ:95℃ 3min;95 ℃ 5s,
58℃30s,45›.$;58℃15s,75›.$。\›
Rÿpî3n,} TheiQ5RealTimePCRDetic
tionSystem][à}rÁÂÃÄÅPCRRÿ,}
Excel^æÂG%RëWÆ。
2 ¯Mf–¨
2.1 456789:;,<γ犞犘犈=>?
<γVPEF‡γVPERšd$,< …ÉÊË


…†351‘‡’‹Œ‘€©êcDNAšÐ",
}LA犜犪狇”GÔγ犞犘犈 ,•。ÓԜ_1700bp
Bې`

W1)。¤¯M¬=,‘…†351’‡‘‹
Œ

abγ犞犘犈 ,•cDNA ¤¥œžš
1624bp,ORFš1482bp,Ÿ 493›¡,¢,–
—˜™š犞狆γ犞犘犈 ‡犞狏犆γ犞犘犈。-b¿ÀPc€γ犞犘犈,•¤¥€÷dÞe×。
90518Í             CfE,::ab~…Éß´,•γ犞犘犈€ŽfÆϖ¨
2.2 45γ犞犘犈,<[\CD
  ProtParam Ü ¤ ) ,‘… †351’€
犞狆γ犞犘犈 ,•Ÿ 493›¡,¢,–äÅ_š54
041.6Da,æg:4qš5.28,¿hÄiš43.51,

‹Œ
’犞狏犆γ犞犘犈,•Ÿ 493›¡,¢,–äÅ
_š54056.6Da,æg:4qš5.17,¿hÄiš
43.62,‘…†351’f‘‹Œ’Ô2€¯M߃”,@
γVPEš¿hĀ¢Þl…。}ProtScale
W1 abγ犞犘犈,•€GÔ
M1.DL2000;1.犞狆γ犞犘犈;M2.DNAmarkerⅢ;2.犞狏犆γ犞犘犈
Fig.1 Amplificationofγ犞犘犈geneingrape
ܤ¦γVPEl…à}OPޖ¨,¯ MÆÇ{l
…j½£›kP‡OP?³

È10aalmkÈ·
›Æ€kP?³

  TMHMM 2.0 ܤ–¨
γVPEl…€RS?³,¯ MÆÇ{l…¿kÈR
S¯T³
,γVPEšnÀ€oRSl…。
ò} MEGA6.0à}p5£¤¥¦§,T76
q©OàÛN

W2)。rGªh«犞犘犈 ,•f»
¼$P‡sÄ2€犞犘犈 ,•€ÀeÞ,q»–
š3›t(Ì,SYZÀ、PäÀ‡ïuÀ,©.‘…
†351’、‘‹Œ’ab€γ犞犘犈 ,•^˜À·–
v

b˜YZÀt(Ì
;3›Ú:#$vw、xy‡
z{ÈÈ犛犿狅犞犘犈1、犛犿狅犞犘犈2,•f×:#$
PäÀt(Ìk|´6P}

»~犞犘犈 ,•ž
ïf×:#$€犞犘犈 ,•–‚À·t(́,ÄÇ
犞犘犈 ,•ÈÚ:#$f×:#$€àÛãÜ-
óvPi

|}DNAMANþÿ,qÓԀ‘…†351’、
▲.‘…†351’犞狆γ犞犘犈 ,•;●.‘‹Œ’犞狏犆γ犞犘犈 ,•;–v݀€Æ=1000npîK‚°%ƒ€ƒ–Â
W2 ab犞狆γ犞犘犈 ‡犞狏犆γ犞犘犈 ,•€àÛN–¨
▲.犞狆γ犞犘犈genein犞犻狋犻狊狆狊犲狌犱狅狉犲狋犻犮狌犾犪狋犪accession.‘Baihe351’;●.犞狏犆γ犞犘犈geneinV犻狋犻狊狏犻狀犻犳犲狉犪cv.‘Carinena’;Thenumbers
onthebranchesrepresentthepercentagesoftimesthatthespeciesaregroupedtogetherinbootstrapanalysisfor1000replicates
Fig.2 Phylogenetictreesof犞狆γ犞犘犈geneand犞狏犆γ犞犘犈geneofgrape
0151 ! " # $ % &                   36ä
At.ªh«;VvPN.¬§­;VvTS.ª«…;Vp.…†351;VvC.‹Œ;▲.2›Û„…5;◆.3›´µ¡,¢;.Æ=º¹Aq
W3 ab犞狆犞犘犈、犞狏犆犞犘犈 ,•‡»¼犞犘犈 ,•€¡,¢¤¥§¦–¨
At.犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪;VvPN.犞犻狋犻狊狏犻狀犻犳犲狉犪.PinotNoir;VvTS.犞犻狋犻狊狏犻狀犻犳犲狉犪.ThompsonSeedless;Vp.犞犻狋犻狊狆狊犲狌犱狅狉犲狋犻犮狌犾犪狋犪
accession.Baihe351;VvC.犞犻狋犻狊狏犻狀犻犳犲狉犪cv.Carinena;▲.Catalyticdyad;◆.Threekeyaminoacids;.Themutationsite
Fig.3 Alignmentoftheaminoacidsequencesof犞狆γ犞犘犈,犞狏犆γ犞犘犈geneingrapewithother犞犘犈genes

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Fig.5 Expressionprofileof犞狆γ犞犘犈 (A)and犞狏犆γ犞犘犈 (B)geneafterpowderymildewinducedindifferentperiodsingrape
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de!L

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