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Cloning and Expression Analysis of a Vacuolar H+ PPase Gene from Tamarix hispida

刚毛柽柳液泡膜H+ PPase基因的克隆与胁迫下的表达分析



全 文 :书!"#$%&
,2016,36(5):0881-0887
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NAz{€3022bp,‚ƒ„9…~2298bp,†‡765tˆ,‰,†‡Š‹ŒgRSŽ80.37kD,G‘3
5.25。犜犺犞犘1†‡Š‹y’“”•–,—˜13t™wš。ˆ,‰›z{œgžŸ ,犜犺犞犘1¡˜¢£yuv
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犞犘18jk¿«ÀÁÂÃĶŸÅƵÇ,µÇŽ°¥ÇÈgÉy20.9Ê,Ë 犜犺犞犘1ÌÍ8hijkDÎÏ
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犆犾狅狀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犪犞犪犮狌狅犾犪狉犎+犘犘犪狊犲
犌犲狀犲犳狉狅犿犜犪犿犪狉犻狓犺犻狊狆犻犱犪
ZHANGChunrui1,JIAYuanyuan1,WANGYanmin1、2,WANGYucheng1,
YANGChuanping1,WANGChao1
(1StateKeyLaboratoryofTreeGeneticsandBreeding,NortheastForestryUniversity,Harbin150040,China;2Heilongjiang
ForestryAcademyofScience,Harbin150081,China)
犃犫狊狋狉犪犮狋:AfullengthcDNAofavacuolarH+PPasegene(named犜犺犞犘1)wasisolatedfromthetran
scriptomecDNAlibrarysof犜犪犿犪狉犻狓犺犻狊狆犻犱犪.犜犺犞犘1was3022bpinlength,includinganopenreading
frameof2298bpwhichwaspredictedtoencodeapolypeptideof765aminoacids.Theestimatedmolecular
weightandisoelectricpointsoftheputativeproteinwere80.37kDand5.25,respectively.Hydrophobicity
analysisandtransmembranedomainpredictionindicatedthatthe犜犺犞犘1contained13potentialtransmem
branedomainswithstronghydrophobicity.Theaminoacidssequenceof犜犺犞犘1containsthreeconserva
tivedomains(CS1,CS2andCS3),whichshows93%identitiesinaminoacidssequencetovacuolarH+
PPasegenesfrom犚犲犪狌犿狌狉犻犪狋狉犻犵狔狀犪.Phylogeneticanalysisindicatesthat犜犺犞犘1belongstoclassItype
vacuolarH+PPasegene.QuantitativerealtimePCRassayrevealedthatthemRNAlevelof犜犺犞犘1was
significantlyupregulatedbymorethan20foldhigherthanthatofcontrolunderNaClandPEGtreatments
in犜犪犿犪狉犻狓犺犻狊狆犻犱犪,suggestingthat犜犺犞犘1mightplayanimportantroleinsaltanddroughttoleranceof
犜.犺犻狊狆犻犱犪.
犓犲狔狑狅狉犱狊:犜犪犿犪狉犻狓犺犻狊狆犻犱犪;vacuolarH+PPase;stressresponses;geneexpression
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gorf/gorf.html)Ro犜犺犞犘1y‚ƒ9‡…;˜Ô
ExPASy (http://www.expasy.org/tools/prot
param.html)8ÛÝÞßÍ犜犺犞犘1†‡yˆ,‰
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犞犘1y ( à Š ‹ z { ³ Ž › z { œ g;˜ Ô
MEGA5.0ÝÞÁNeighborJoiningúûáâ±²
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predictprotein.org/)ßÍ犜犺犞犘1,x†‡yŠ‹
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˜Ô8ÛÜ¡ TMpred(http://
www.genome.cbs.dtu.dk/services/TMHMM/)
ßÍ 犜犺犞犘1ÌÍy™wš;˜Ô Protscale(ht
tp://web.expasy.org/cgibin/protscale/protscale.pl)
288 ! " # $ % &                   36æ
C1 Sbcd犚犜犘犆犚ef`a
Table1 PrimersusedforqRTPCRanalysis
,x
Gene
íüç$z{
Forwardprimersequence(5′→3′)
Ëüç$z{
Reverseprimersequence(5′→3′)
犜犺犞犘1 GGTCTCGCTCTTGGTTAC CACGGCTCACATAGGCAC
α狋狌犫狌犾犻狀 CACCCACCGTTGTTCCAG ACCGTCGTCATCTTCACC
β犪犮狋犻狀 AAACAATGGCTGATGCTG ACAATACCGTGCTCAATAGG
β狋狌犫狌犾犻狀 GGAAGCCATAGAAAGACC CAACAAATGTGGGATGCT
8ÛRo犜犺犞犘1Š‹y’“”。ÖÔ WoLF
PSORTÝÞ(http://wolfpsort.seq.cbrc.jp/)³Žè
àáqéßÍ

1.2.3 Sbcd犚犜犘犆犚 ¿“犜犺犞犘1€cD
NA z { ê ë q Ž ç $,ô h i j k βactin
(FJ618517)、βtubulin (FJ618518)« βtubulin
(FJ618519),xJ~"ì,x,ç$z{íµ1。
˜Ô MJOpticon[¹qŽPCRî(BioRad,Her
cules,CA)Ro犜犺犞犘1yµÇïð。[¹º»q
Ž RTPCR ñÔòóô TransStartTop Green
qPCRSuperMix(TransGen)³Ž,ËÖc±:2×
TransStartTopGreenqPCRSuperMix10μL,Åõ
ç$«¾õç$
(10μmol/L)#1μL,öÏây†
ÐcDNA2μL,r÷EøçS“ùúcoû20μL。
ËÖÑz~
:94℃ßb”30s;94 ℃b”12s,
58℃üý30s,72℃þÿ45s,79℃9Ð1s,45t
!"

#PCRË֝$â,þËÖ®¦ô0.5℃/s
yŦ@55℃%È99℃。½t¿ÀZe3¾,Ô
2-ΔΔCtúû³Ž,xyŒgqŽRo[1415]。
2 ž«Ro
2.1 犜犺犞犘1,;^_犮犇犖犃HfghijklmnB“Ro

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‡ H+PPase,xyz{。L¸lmnÍzrsy
,xz{ØL&©ªÙñå

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ž¬lmnÍzÙy†‡z{,€Œ(

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á/Ro

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›·¸ H+lý:F;<‰%
M(¤

¡˜¢£y H+PPaseá/(01),þ
,x|}~犜犺犞犘1(GenBank1m2KU880710)。
犜犺犞犘1,xcDNA€~3022bp,m—2298bp
,3y‚ƒ9‡…

†‡765tˆ,‰。4Í犜犺
犞犘1†‡Š‹yRSŽ~80.37kD,G‘3
~5.25,RS‡~C3670H5772N900O1053S32。
¡ÔPredictproteinÝÞßÍ犜犺犞犘1†‡Š
‹yäåá

02),žµ¶:Š‹yäå
áÁ
,α56«:78æ9:>?0R,R=:
60.92%«33.86%。’“áß͵¶,犜犺犞犘1
†‡Š‹¡˜¶Ÿy’“š

;ÂÃR<¸3t
=>

03)。™wáß͟ ,Š‹m—13t
™w56á

04),èàáqéß͟ ›qé
¸uvwÅ

µ¶犜犺犞犘1Ø®tuvw™wŠ‹。
2.2 犜犺犞犘1jklm?nopqr
þ犜犺犞犘1yˆ,‰z{¬10T#$uvw
H+PPaseyˆ,‰z{³Ž(à”œ•,žŸ
 犜犺犞犘1¬›?#$yVP1ˆ,‰z{®¯”•
¥

~93%~89%。犜犺犞犘1¡˜ H+PPase(¤
¥¦§¨y3tá/(CS1,CS2« CS3),›Á
CS1Ám—§¨yDVGADLVGKVEˆ,‰z{,
¬ H+PPase(¤,xá®¯(06)[1617]。
˜Ô MEGA5.0ÝÞg…(#$y H+PPase
,xˆ,‰z{áâ±²³´ã

05),žŸ 
犜犺犞犘1¬W@犆犿犞犘1I$uvwH+PPaseA
BC±•D

ñ¬VWX VP2Ⅱ$uvw H+
PPaseABC±•E,µ¶犜犺犞犘1Øjky®tI
$uvw H+PPase。
2.3 stf@AB犜犺犞犘1,;~+³®—;<犜犺犞犘1,xgEÛ¼½yÕ
Öïð

¡Ô[¹RTPCRúûRo›8 NaCl«
PEG¼½¾yµÇ†‡。犜犺犞犘18¥ÐF×μ
½¾ÂÃÄ~µÇÅÆ

F8#¼½¹œ3«…(
n’ÁyµÇ†‡˜eGH

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¾
,犜犺犞犘18jk¿0µÇŽIÅÅ%,8¼½6h
µÇŽÇÈ°¥

µÇÅÆ12.2Ê,f~¼½¹œ
yþ
,犜犺犞犘1µÇŽ8JK)*;8vÅ0n’
Á
,犜犺犞犘1µÇŽ83«12hÃÄ2t¥L,µÇ
R=ÅÆ20.9«17.9Ê。
8PEG6000†V×μ½¾,犜犺犞犘18jk
¿0«vÅn’µÇÂÃÄMÅ%N¾)yO.

8¿0n’Á
,犜犺犞犘18¼½12hµÇŽÇÈP
L

µÇÅÆ14.2Ê;8vÅ0n’Á,犜犺犞犘1µ
3885          456,:hijkuvw H+PPase,xyYZ¬¼½¾yµÇRo
02 ThVP1Š‹äåáßÍ
Fig.2 SecondarystructurepredictionofThVP1
01 ThVP1§¨á/Ro
Fig.1 ConserveddomainanalysisofThVP1
03 ThVP1Š‹y’“áßÍ
Fig.3 HydrophobicityanalysisofThVP1
04 ThVP1Š‹y™wáßÍ
Fig.4 Transmembranedomainpredictionof
theantiporterproteinThVP1
ǎ86hÇÈ°WQ,µÇÅÆ16.5Ê。
RÅeS
,犜犺犞犘1g¥Ð«×μ½¡˜¶Ÿ
yÕÖ

8jk¿0«vÅn’ÁµÇŽŸlÅ%

µ¶犜犺犞犘1ÌÍì¬jkDÎÏÐ=GfÑ。(
¹ÒÄ犜犺犞犘18vÅ0ŒgµÇŽ¥¸¿0,4
͛>?8jkÀ«TÁÒÓDE Í

UVïµ½Wéˆ,‰yb´
,0.1ïµXtz{Yœ10%y
GH
;ThVP1.hijk;VrVP1.Z­;OsVP1.“[;HbVP1.
\Ôã
;GhVP1.w];NtVP1.^_;AtVP1.VWX;SbVP1.
¥`
;AtVP2.VWX;BvVP1.ab;CmVP1.W@;TaVP1.
&c
;HvVP1.Wc;ZmVP1.de;ZmVP2.de;ThVP2.
ÐX
;VvVP2.fg
05 hT#$VPŠ‹(àz{y±²³´ãRo
Thescalebarexpectedthenumberofsubstitutionpersite,0.1
means10%changeswereobservedbetweentwosequences;
ThVP1.犜犪犿犪狉犻狓犺犻狊狆犻犱犪(KU880710);VrVP1.犞犻犵狀犪狉犪犱犻犪狋犪
(AB009077);OsVP1.犗狉狔狕犪狊犪狋犻狏犪(BAD02277.1);HbVP1.
犎犲狏犲犪犫狉犪狊犻犾犻犲狀狊犻狊(AY514019);GhVP1.犌狅狊狊狔狆犻狌犿犺犻狉狊狌狋狌犿
(ADN96173);NtVP1.犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿(X77915);AtVP1.
犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪(M81892);SbVP1.犛狅狉犵犺狌犿犫犻犮狅犾狅狉
(ADJ67258);AtVP2.犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪(AF182813);
BvVP1.犅犲狋犪狏狌犾犵犪狉犻狊(L32791);CmVP1.犆狌犮狌狉犫犻狋犪犿狅狊犮犺犪狋犪
( BAA3333149);TaVP1.犜狉犻狋犻犮狌犿犪犲狊狋犻狏狌犿(AY296911);HvVP1.
犎狅狉犱犲狌犿狏狌犾犵犪狉犲(ACA63883);ZmVP1.犣犲犪犿犪狔狊(CAG29370);
ZmVP2.犣犲犪犿犪狔狊(ABK51382);ThVP2.犈狌狋狉犲犿犪犺犪犾狅狆犺犻犾狌犿
(BAJ33614);VvVP2.犞犻狋犻狊狏犻狀犻犳犲狉犪(CA041672)
Fig.5 PhylogenetictreeanalysisofVPproteinsfrom
variousplantspecies
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ižyy H+PPaseˆ,‰z{
06 犜犺犞犘1¬›?#$ H+PPase(àz{yœ•
ThethicklinesstandforconserveddomainCS1;dashedboxstandforconserveddomainCS2;thinboxstandforconserveddomainCS3;
Theaminoacidsequencesinthemapwhichfromthetoptothebottomrepresent:犜犪犿犪狉犻狓犺犻狊狆犻犱犪(KU880710),犌犾狔犮犻狀犲犿犪狓
(XP_003542656.1),犘犺犪狊犲狅犾狌狊狏狌犾犵犪狉犻狊(XP_007155080.1),犈狌犮犪犾狔狆狋狌狊犵狉犪狀犱犻狊(XP_010050115.1),犌狅狊狊狔狆犻狌犿犺犻狉狊狌狋狌犿
(ADN96173.1),犜犺犲狅犫狉狅犿犪犮犪犮犪狅(XP_007013600.1),犈犾犪犲犻狊犵狌犻狀犲犲狀狊犻狊(XP_010928300.1),犛狅犾犪狀狌犿狋狌犫犲狉狅狊狌犿
(XP_006359496.1),犕犪犾狌狊犱狅犿犲狊狋犻犮犪(XP_008352108.1)犪狀犱犘狅狆狌犾狌狊狋狉犻犮犺狅犮犪狉狆犪(XP_006381091.1)
Fig.6 Alignmentanalysisof犜犺犞犘1withH+PPasefromotherplants
5885          456,:hijkuvw H+PPase,xyYZ¬¼½¾yµÇRo
07 犜犺犞犘1,x8…(¼½¾yµÇ†‡Ro
Fig.7 Expressionanalysisof犜犺犞犘1geneunder
severalabioticstresses
RSŽ~80~81kD[1821],…($TÁy H+PPase
,x¡˜¥¦yz{(à”
[22]。
‹;<@hijk
ÁYZÈH+PPase,x犜犺犞犘1,›‚ƒ„9…2
298bp,†‡765tˆ,‰,RSŽ~80.37kDa。
犜犺犞犘1¡˜#$ H+PPasez˜yˆ,‰z{DV
GADLVGKVE,¬W­、b­、qr、w]#$yu
vwH+PPase,x¡˜•¥(à”。x0,犜犺犞犘1
8áÅ{¬#$uvwH+PPasey|”。
¥#$Á@8 2 T$£yuvw H+
PPase:Ⅰ£«Ⅱ£。Ⅰ£÷} K+ ~ñ€¦
Ca2+~,Ⅱ£g K+ …_`ñg Ca2+ ‚›_
`
[2324]。
{2T$£yH+PPase8ˆ,‰z{Œ
ƒ”„˜37%~39%,8àáÁ쬅(y=Gf
Ñ

҅gⅠ£ Íy;<›¸Ⅱ£[25]。‹;<Á
yjk犜犺犞犘1¬W­、VWX#$yⅠ£ H+
PPaseyˆ,‰z{Œƒ”•¥(>80%),ABC
±•D

ñ¬VWXⅡ£ H+PPaseˆ,‰z{Œ
ĥ*
(<40%),µ¶ 犜犺犞犘1·¸Ⅰ£uvw
H+PPase。
  †‡…($TÁyH+PPase,x¡˜¥¦y
(à”

F;<Òą(#$y H+PPase,xl
m“舔gм½yÕÖ@8ŠWGH
。Co
lombo«Cerana&ˆÐ¼½Ì/¥‰Š‹Œà
áÁuvw H+PPase”[26];Fukuda [27]Ô
100mmol/LNaClkGWcâ,›¿Á H+PPase
,xlm“èŸl%¥

µ¶NaClŽP H+PPase
”ˆlm“èqr



˜;<ÒÄNa+g®
|#$y H+PPase¡˜òÚJÔ。Nakamura

[28]
ÒÄ100mmol/LNaClkG¾Z­¿0 H+
PPase”zȖòÚ。NaClkG¾ÇÀÁ
]
(犕犲狊犲犿犫狉狔犪狀狋犺犲犿狌犿 犮狉狔狊狋犪犾犾犻狀狌犿 )Á H+
PPasey—Ž«”¾)。{|;<žµ¶
H+PPase”ˆlm“èyb´Ìͬ#$T
$

‘’$£

Ò/´µ“›xܘC

~+;<
uvw H+PPase,x8jkž=$¼½ÖŸÁy
 Í

‹;<¡Ô[¹ RTPCRúûRo犜犺犞犘1
8NaCl«PEG¼½¾yµÇ†‡。8 NaCl«
PEG¼½kGâ,犜犺犞犘1,x8jky¿0«v
Å0yµÇŽÂ¶Ÿqr

|=Ø8vÅ0n’Á

犜犺犞犘1,x8¼½âÃÄ¥”¦µÇ。犜犺犞犘18
×Ϋ¥Ð¼½¾yµÇb´x@8‘’GH”

›8vÅ0yŒgµÇŽ¥¸¿0

4͛>?8
jkÀ«TÁì¬gNa+yùç,@ñ/¥jky
ÏЫDÎÍ7

2001U,Gaxiola[11]&ˆ+fµÇVWXu
vwH+PPase,x犃犞犘1/¥+l,xVWXy
DÎÏÐÍ7



ÐX

Ðv•–

¥—˜

Й
™y H+PPase , x y D E   Í x s È + š
[2931]。
{|;<µ¶˜Ôl´uvw H+PPase
,x/¥#$yÏÐÍ7

ؘÔ,xÜÑúû°
/ÏÐ#$›ÀTy®T˜œž

hijkء
˜Ÿ¶DEÍ7yÐ=#$

¬aÝ#$Œœ

Ð=
#$yçSšù´y~¶Ÿ«˜œ
。犜犺犞犘1g×
ΫÐR¼½¥¦ÕÖ

Ë ›ÌÍ8hijkç
Sšù´fÑÁÒÓZ?JÔ
[32]。
â ;<þI
犜犺犞犘1lßVWX«jkÁ\š›DE Í,ô
~#$DE/T/4˜œy,x¡à

uv!O

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431434.

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