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Clone and Expression Analysis of GGPPS1 Gene from Rehmannia glutinosa

地黄GGPPS1基因克隆及表达分析



全 文 :书!"#$%&
,2016,36(5):0888-0895
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> ?:klmnop,TqQrlmstuvw,x0yz{|$,}~lm€‚:€‚:ƒ„…†‡
(geranylgeranylpyrophosphatesynthase,GGPPS):ˆ‰cDNA Š‹,Œn 犚犵犌犌犘犘犛1,GenBankŽtn
KU258808。i‘A$U’%Qr‰:“”,•–—˜™š、›œkužyz{™šQr。Ÿ ¡¢:(1)
犚犵犌犌犘犘犛1:ˆ£¤¥¦§n987bp,¨ ©328ª«:…。(2)A$U’%QrŸ ¡¢,RgGGPPS1¬­®¯2
ª°®±²«…‰:Š
(DDXXXXDD³DDXXD),e´µ¶·R¸#$4‰GGPPS¬­¹º{»¼。(3)½P
¾¿‰—˜™šÀÁpET32a犚犵犌犌犘犘犛1‘@ÂÃÄBL21(DE3)ÄÅ4ÆǙšRgGGPPS1Èu¬­,ÉP
Ni2+ʳËrÌÍ›œ‰RgGGPPS1Èu¬­。(4)ÎÏÐÑPCRŸ ¡¢,犚犵犌犌犘犘犛1:ˆ‘Ò4™šÑ
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lm
;犚犵犌犌犘犘犛1:ˆ;A$U’%Qr;—˜™š;™šQr
CD:Q785;Q786 !FGHI:A
犆犾狅狀犲犪狀犱犈狓狆狉犲狊狊犻狅狀犃狀犪犾狔狊犻狊狅犳犌犌犘犘犛1犌犲狀犲犳狉狅犿犚犲犺犿犪狀狀犻犪犵犾狌狋犻狀狅狊犪
ZHAOLe1,2,SHIJingjing1,2,MALigang1,2,HEQingxiang1,FENGWeisheng1,2,
ZHENGXiaoke1,2,ZHUYunhao1,2
(1SchoolofPharmacy,HenanUniversityofTraditionalChineseMedicine,Zhengzhou450046,China;2ColaborativeInnova
tionCenterforRespiratoryDiseaseDiagnosisandTreatment&ChineseMedicineDevelopmentofHenanProvince,Zhengzhou
450046,China)
犃犫狊狋狉犪犮狋:犚犲犺犿犪狀狀犻犪犵犾狌狋犻狀狅狊犪wasusedasexperimentalmaterialinthisstudy.Throughanalyzingthe
transcriptomedataof犚.犵犾狌狋犻狀狅狊犪anddesigningspecificprimers,weisolatedthecDNAsequenceofgera
nylgeranylpyrophosphatesynthase(GGPPS)from犚.犵犾狌狋犻狀狅狊犪andnamedas犚犵犌犌犘犘犛1(GenBankac
cessionNo.KU258808).Meanwhile,onthebasisofbioinformaticanalysis,weperformedtheprokaryotic
expression,purificationandtissuespecificexpressionanalysis.Theresultsindicatedthat:(1)犚犵犌犌
犘犘犛1hasanopenreadingframe(ORF)of987bp,whichencodedaproteinof328aminoacidresidues.
(2)BioinformaticanalysisindicatedthatRgGGPPS1proteincontainsthetwoconservedmotifs(DDXXXX
DD)and(DDXXD);RgGGPPS1proteinshowedthehighesthomology,92%identity,withGGPPSpro
teinfrom犛犲狊犪犿狌犿犻狀犱犻犮狌犿.(3)ByutilizingtheconstructionofprokaryoticexpressionvectorpET32a
犚犵犌犌犘犘犛1,wesuccessfulyexpressedtherecombinantRgGGPPS1proteinin犈.犮狅犾犻BL21(DE3)cels.
Furthermore,therecombinantRgGGPPS1proteinwaspurifiedthroughNi2+affinitychromatography.(4)
RealtimePCRanalysisindicatedthat犚犵犌犌犘犘犛1wasexpressedinhightranscriptlevelinroots,lowlev
elsinleavesandstems.Theresultsofthisstudyprovidedthefundamentalinformationabout犚犵犌犌犘犘犛1
geneforfolowupresearchofitsfunctioninvolvediniridoidglycosidebiosynthesispathway.
犓犲狔狑狅狉犱狊:犚犲犺犿犪狀狀犻犪犵犾狌狋犻狀狅狊犪;犚犵犌犌犘犘犛1gene;bioinformaticanalysis;prokaryoticexpression;ex
pressionanalysis
  lm(犚犲犺犿犪狀狀犻犪犵犾狌狋犻狀狅狊犪Libosch)áâã,
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CAAGAACATAACC3’)³fÂ|$RgGGPPS1
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m

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ŠÌ͉Š‹nÙÍ NCBIORFFinderÚÛ£¤
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¾á

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çúòTq TMHMMServerv.2.0ò;¢P
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한é
,bootstrap±ƒ‰ÈêXvn1000X。
1.2.4 犚犵犌犌犘犘犛1,6Z[:;\]T^_P`
a:; Òw犚犵犌犌犘犘犛1:ˆ‰²ŠŸ ,x01
7犚犵犌犌犘犘犛1:ˆ‰—˜™š|$,ÁÂ|$
RgGGPPS1ExpF(5’CGGAATTCATGATTTTCT
CAACAG3’,ë1ÞìQn犈犮狅RI‡íåî)³fÂ
|$RgGGPPS1ExpR(5’CCGCTCGAGTCAAACT
GGTGCGGCA3’,ë1ÞìQn犡犺狅I‡íåî),k
²ŠÁï‰pMD19T犚犵犌犌犘犘犛1Eðn½¾,P
PrimerSTARHS犜犪狇(Takara~ª)‹Œ犚犵犌犌
犘犘犛1:ˆ‰ORFŠ‹,PCR`Šn:95℃ÃÄ{
2min;95℃Ä{10s,55℃ÅÆ45s,72℃ÇÈ
1min,30ªÉØ;72℃ÇÈ8min。PCR$$9¬
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Qò•–·‡í

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ÃÄBL21(DE3)ÑÒÓÔÕ("’·¸;~ª),×
õ}~n£Eð9·‡íöА

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ÐPÍLBšÁRø:4(®«ùúi2),þÿR
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(OD600!0.6),ñ‘0.4mmol/
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PPS1Èu¬­‰#$™š‡À,‹@RøÁ,#
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(pH7.3)È(ÄÁ,)*+,@ÂÃÄÔÕ。@
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1.2.6 犚犵犌犌犘犘犛1,6:;hi<= lm¼u
ž‰cDNA;Š50¾

k犚犵犜犐犘41:ˆ[15]n=ã,GenBankŽt
n KT306007(ÁÂ|$5’ATTGGGTAGATT
GCCAGGAG3’;fÂ|$5’CCATTGTCAGC
CAATTCATC3’),±²犚犵犌犌犘犘犛1:ˆ‘lm
QR‘P

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犚犵犌犌犘犘犛1 : ˆ RTPCR Á  | $ (5’
CCCATCTAGAAGAGGCAAGC3’),f  | $
(5’GAACAATGCGTCTCCTGCTA3’),>ª™
‘Èê 3 X,RTPCR fgÁ:n:10μL2×
QuantiNovaSYBRGreenPCR MasterMix(QIA
GEN)、ÁfÂ|$¼0.4μL、cDNA ½¾2μL、?
ddH2O!20μL,fg`Šn:95℃ÃÄ{3min,
95℃Ä{10s,56℃ÅÆ20s,72℃ÇÈ30s,35
ªÉØ

Òw@.AÞBC RTPCR$$‰yz
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Pí0Qr¿ÀSPSS13.0•–õˆ2@DQr,
klm“AP҉ CtE‰FBvxnl,•–
犚犵犌犌犘犘犛1:ˆ™š½¸Qr。
2 Ÿ eQr
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1100bp,¯ °Ÿ sJ1&¢,²ŠÌÍlm
犚犵犌犌犘犘犛1:ˆ³´‰ORFŠ‹,@Ìn987bp,
¨©328ª«:…,犚犵犌犌犘犘犛1:ˆ‰Š‹U’T
9nÙÍNCBIGenBank,ŽtnKU258808。
2.2 犚犵犌犌犘犘犛1,6#IdeTUVWXY<=
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M.DL2000;1.犚犵犌犌犘犘犛1
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Fig.1 PCRamplificationof犚犵犌犌犘犘犛1gene
098 ! " # $ % &                   36L
²lmRgGGPPS1¬­‰QRÑ35.90kD,¶¯
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BlastÝ7Ÿ ¡¢,RgGGPPS1¬­e´µ
(犛犲狊犪犿狌犿犻狀犱犻犮狌犿,SiGGPPS,XP_011092951.1)、

(犛犪犾狏犻犪犿犻犾狋犻狅狉狉犺犻狕犪,SmGGPPS,AEZ55680.
1)、v w (犛狅犾犪狀狌犿 狆犲狀狀犲犾犾犻犻,SpGGPPS,XP_
015087865.1)、yç(犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿,NtGG
PPS,AIC77784.1)、. Z (犘犪狀犪狓狀狅狋狅犵犻狀狊犲狀犵,
PnGGPPS,AIZ00598.1)³t3u (犃狉犪犫犻犱狅狆狊犻狊
狋犺犪犾犻犪狀犪,AtGGPPS,AAA81879.1)¶#$‰ GG
PPS¬­Š‹ÖÍ{»¼,Qòn92%、87%、
77%、79%、76%³65%。PDNAMAN¿À7
7P#$GGPPS¬­«:…Š‹•–劋Ý7,
Ÿ 

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\4ì³C[Hà{»],¯ 2ª°®±²«…‰

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PPS1¬­‰«:…Š‹4^¯2ªHà:Š,Q
òáDDLPCMDD³DDILE(J3)。
#$

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lmn©RP2o,pqvr™¢˜Ü…ås,tqvr™¢«:…ås,ÌuvÁn犚犵犌犌犘犘犛1:ˆ‰}~|$Š‹
J2 犚犵犌犌犘犘犛1:ˆ‰cDNAŠ‹7g‰«:…Š‹
Themarker“”representsterminationcodon,theleftnumberindicatesnucleotideposition,therightnumberindicates
aminoacidposition,theitaliclowercaselettersrepresentcloningprimersequencesof犚犵犌犌犘犘犛1gene
Fig.2 Nucleotidesequenceandaminoacidsequenceof犚犵犌犌犘犘犛1genecDNA
1985…              G H,¶:lm犌犌犘犘犛1:ˆ}~™šQr
2ª°®±²«…‰Hà:ŠPwx§2g;Si.´µ;Sm.xã;Sp.vw;Nt.yç;Pn..Z;At.t3y
J3 lmRgGGPPS1¬­eéz#$4GGPPS¬­‰åŠ‹Ý7Qr
ThetwoAsprichconservedmotifsareboxedinblack;Si.犛犲狊犪犿狌犿犻狀犱犻犮狌犿;Sm.犛犪犾狏犻犪犿犻犾狋犻狅狉狉犺犻狕犪;Sp.犛狅犾犪狀狌犿狆犲狀狀犲犾犾犻犻;
Nt.犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿;Pn.犘犪狀犪狓狀狅狋狅犵犻狀狊犲狀犵;At.犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪
Fig.3 MultiplesequencealignmentofRgGGPPS1andGGPPSproteinsfromotherplantspecies
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