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Cloning and Expression of HHT Gene in ‘Huanghua’ Pear and Its Bud Mutant ‘Lühuanghua’ Pear (Pyrus pyrifolia Nakai)

‘黄花’梨及其芽变‘绿黄花’梨HHT基因克隆与表达分析



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,2016,36(6):1105-1109
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HHT),ˆcDNAq‰Š,‹Œr 犘狆狔犎犎犜(Žr KX131155)。‘’“”S•–,[,ˆ—˜™=š
(ORF)r1335bp,›œ444ž,。B$HŸ%’“ ¡,¢£qPpyHHT¤¥¦§¨’©¦ªr49.91kD,
«¬­®4.75,¯ ¥`§°±²³98%,´ µ¶·¸¹。º»¼½£ªPCR(qRTPCR)•³’“ ¡,2b`S
e¾犘狆狔犎犎犜,ˆ
lcd
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犆犾狅狀犻狀犵犪狀犱犈狓狆狉犲狊狊犻狅狀狅犳犎犎犜犌犲狀犲犻狀‘犎狌犪狀犵犺狌犪’犘犲犪狉犪狀犱
犐狋狊犅狌犱犕狌狋犪狀狋‘犔ü犺狌犪狀犵犺狌犪’犘犲犪狉(犘狔狉狌狊狆狔狉犻犳狅犾犻犪犖犪犽犪犻)
LZhaoqing,RENDandan,ZHOUHe,QIAOYushan
(ColegeofHorticulture,NanjingAgriculturalUniversity,Nanjing210095,China)
犃犫狊狋狉犪犮狋:Thestudyusedfruitpeelofsandpear(犘狔狉狌狊狆狔狉犻犳狅犾犻犪Nakai)cultivars‘Huanghua’pear(rus
setfruit)anditsbudmutant‘Lühuanghua’pear(greenfruit)at8weeksafterfulbloom(WAFB)asex
perimentmaterials.thecDNAfullengthof犎犎犜gene,whichwasnamed犘狆狔犎犎犜 (GenBankaccession
No.KX131155),wasclonedbyconventionalandnestPCRtechniques.Sequenceanalysisshowedthatthe
fullengthofthe犘狆狔犎犎犜orthologconsistedofa1335bpopenreadingframe(ORF)encodingapoly
peptidecontaining444aminoacidresidues.Themolecularweightofdeducedaminoacidswas49.91kD,
withanisoelectricpoint(狆犐)of4.75,whichhadthehighestsimilarity(98%)andclosestrelationship
withthatin犘狔狉狌狊犫狉犲狋狊犮犺狀犲犻犱犲狉犻.RealtimefluorescencequantitativePCR (qRTPCR)analysisdemon
stratedthattheexpressionof犘狆狔犎犎犜genein‘Huanghua’and‘Lühuanghua’pearpeelwerechanging
constantlyduringthekeyperiod(6-9weeks),andsignificantlyhigherin‘Huanghua’pearpeelthanthat
in‘Lühuanghua’.犘狆狔犎犎犜geneinvolvedintheformationofsandpearrusset/greentraits,itsexpres
sionleveldifferencesmayplayaroleintheformationofsandpearskincolor.
犓犲狔狑狅狉犱狊:犘狔狉狌狊狆狔狉犻犳狅犾犻犪Nakai;犎犎犜gene;cloning;bioinformatics;geneexpression
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çíî”ëï
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PD]xæçíð”ëï[suberinpoly(aliphatic)do
main,SPAD]ìËqW’©”ë。SPPD4C®~
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CYP86A()ω~,Ç«[912]。234{|q`
æç¦0˧¶,ˆü犘犃犔、犆4犎x犆犆犚«[1315]。
ω~,€ O‚ƒ„…†‡(ωhydroxyp
almitiateOferuloyltransferase,HHT)®÷øù
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DÙDESPADxSPPDW’©q”ëìË。
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CTABXS]SegRNA[18],1%h(ijk¬l
mÅino±

] 1μg g RNA,tu Prime
scriptTM reagent Kit with gDNA Eraser(¾
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CATTCGTCA3’)x 犎犎犜R(5’AGCATCAG
CAATCTCAGTG3’)。PCR r€;·g;èr
25μL,‚ƒ„1μL,|$…0.5μL,dNTPMix
ture2.0μL,10×Buffer2.5μL,Ex犜犪狇DNAí0
‡0.2μL,uddH2O†‡。PCR~ˆ‰r94℃
Šk±5min;94℃ k±20s,58℃‹Œ20s,72℃
Ž1min,35;72℃Ž10min。PCRÍ
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Ÿ

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¤¥¦B$3Fü§pqŐ

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(5’GGTGGTAGAGCTCGCAGGACTGCAGCT
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‘’“

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dÀno6Èo9pq`SºgRNA,r…Ž¸É
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|q犘狆狔犎犎犜,ˆ‘yz1¨{M|$COF
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Ñ1 犘狆狔犎犎犜,ˆqÒӁ‘hž,‘
ATG.Startcodon;TAG.Stopcodon;Shadow.Conserveddomainsequence
Fig.1 Thenucleotideacidsequenceandaminosequenceof犘狆狔犎犎犜gene
(5’TCTTGTTCCTTTCTAATGGG3’)x COR
(5’GGGCTCTTTCACCCACTT3’)。 ^ _ `
犝犫犻狇狌狋犻狀,ˆrÔÈ,ˆ,|$r CO138(5’
GCTCGCAGTGCTCCAGTTCTAC3’)x CO139
(5’AACATAGGTCAACCCGCACTT3’)。È9
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¾ TaKaRapq“BÍ)ՖAuqRT
PCRr€。r€;·r10μLSYBRPremixEx
TaqTMⅡ,0.3μLx、¯ °|$,1μLcDNA,ddH2O
†‡È20μL。r€´r95℃Šk±4min,
95℃k±20s,60℃‹Œ20s,72℃Ž40s,40


º»¼½£ªPCR\]’uABI7300cy
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,ˆqߒê}

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Žr KX131155。wI DNAMAN6.0·‰¢
Å[¤¥’©ª®49.91kD,«¬­®4.75。
2.2 犘狆狔犎犎犜,<[\]^_BC
tuNCBI¤¥bƔëïHIè(conserved
domaindatabase,CDD)¨ 犘狆狔犎犎犜 ,ˆ›œ¤
¥qbÆæAuŠÅ

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[¤¥<11~443
ž,  é , ê C ü v  HHT ( ë ” ë ï
(PLN02481,Eì0e+00)(Ñ2)。í犘狆狔犎犎犜,
70116À          89:,«:‘cd’` hijk‘lcd’` 犎犎犜,ˆ{|¯•³’“
Ñ2 犘狆狔犎犎犜,ˆŠÅ¤¥q”ë
Fig.2 Conserveddomainanalysisofprodicted
proteinof犘狆狔犎犎犜gene
Ñ3 ‘cd’` ¯i¹$b 犎犎犜,ˆž,‘
q·ÀAÃT
Fig.3 Phylogenetictreeoftheaminoacidresidues
encodedby犎犎犜genein‘Huanghua’pear
andotherspecies
ˆ›œqž,‘Au¾¨’“

ÝÞPpyHHT
¯½b#$ HHTq§°±<76.6%x98%êC,
i¾ § ° ± < 92% ^ x q ü ¥ ` (犘狔狉狌狊
犫狉犲狋狊犮犺狀犲犻犱犲狉犻)、î S (犕犪犾狌狊 犱狅犿犲狊狋犻犮犪)、ï
(犘狉狌狀狌狊犿狌犿犲)、ðñòó(犉狉犪犵犪狉犻犪狏犲狊犮犪)«,Ò
¥`¸²³98%。’“”S"ÝÞ,犘狆狔犎犎犜 ,
ˆ›œqž,‘ôü HHTq2bƔëæ
ï

õ HXXXD(o175~179öž,é,)÷±ö
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犎犎犜 qܺ,ˆ。
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<“]Ûq4»À,‘cd’` 犘狆狔犎犎犜 ,
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Fig.4 Therelativeexpressionof犘狆狔犎犎犜inthe
peelof‘Huanghua’and‘Lhuanghua’pearat
differentstages
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[1] DUPREEZMG,REESDJG.Investigatlingthemolecular
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犮狅犿犿狌狀犻狊L.[J].犃犮狋犪犎狅狉狋犻犮狌犾狋狌狉犪犲,2008,800:383390.
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狅犳犉狉狌犻狋犛犮犻犲狀犮犲,2011,28(3):485492.
[3] aZb,dL¥,p3e._`SefËˆhýg¨«[J].
¾UWST
,2005,34(3):5254.
[4] SUGARD,BASILSR.Colorandrussetvariationamongse
lectionsof‘Bosc’pear[J].犑狅狌狉狀犪犾狅犳狋犺犲犃犿犲狉犻犮犪狀犘狅犿狅
犾狅犵犻犮犪犾犛狅犮犻犲狉狔,2008,62(2):7781.
[5] GANGDR,WANGJ,DUDAREVAN,犲狋犪犾.Aninvestiga
tionofthestorageandbiosynthesisofphenylpropenesinsweet
basil[J].犘犾犪狀狋犘犺狔狊犻狅犾狅犵狔,2001,125(2):539555.
[6] BERNARDSMA.Demystifyingsuberin[J].犆犪狀犪犱犻犪狀犑狅狌狉
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[7] GRACAJ,SANTOSS.Suberin:abiopolyesterofplants,
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