,2018,38(1):0035-0040 犃犮狋犪犅狅狋. 犅狅狉犲犪犾. 犗犮犮犻犱犲狀狋. 犛犻狀. :1000 4025(2018)01 0035 06 犱狅犻 :10.7606/j.issn.1000 4025.2018.01.0035 "# 犘狅犛犃犇 $%&',,,,, ( F,G H, 401331) :IJKLK( 犘犪犲狅狀犻犪狅狊狋犻 )MNO, RACE P RT PCR QR,STUVJKLKWXY ACP P 犛犃犇 cdna Z,G[O 犘狅犛犃犇 (GenBank\]^O KY038819) _` abc, cdna _` Z1559bp, 9 1197bp, 398,3 Z 172bp,5 Z 123 bp _` bc,jklk 犘狅犛犃犇 _` 2 a,jklk, TMHMM P TargetP au,posad, M / a b c, 犘狅犛犃犇 8 J K L K M b, 8 b,,8 b ;,60db, 80d,10d b ()*:JKLK;WXY ACP ;b a;race;rt PCR +, -:Q785;Q786./01:A 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犛狋犲犪狉狅狔犾 犃犆犘犇犲狊犪狋狌狉犪狊犲犌犲狀犲犘狅犛犃犇犳狉狅犿犘犪犲狅狀犻犪狅狊狋犻 LIAOBingnan,LUJunxing,HUANG Xinglin,GUAN Li,BAIHuiyang,ZHANG Tao (ColegeofLifeSciences,ChongqingNormalUniversity,Chongqing401331,China) 犃犫狊狋狉犪犮狋 :Thestearoyl ACPdesaturase ( 犛犃犇 )genefrom 犘犪犲狅狀犻犪狅狊狋犻 wasclonedusing RT PCR and RACE PCRstrategies,namedas 犘狅犛犃犇 (GenBankaccessionnumber:KY038819).Thefullengthof 犉犃犇 3cDNAis1559bp,containsa1197bpopenreadingframe(ORF)encodingaputativeproteinof398 aminoacids.thenon codingdistrictsof3 and5 endare172bpand123bp.multiplesequencealignment resultsshowedthatitcontainstwoconserveddomainsof 犘狅犛犃犇 gene.phylogenetictreeanalysisshowed thatthe 犘. 狅狊狋犻 hadtheclosestevolutionaryrelationshipwith 犚犻犮犻狀狌狊犮狅犿犿狌狀犻狊.Subcelularlocalization analysisoftmhmmandtargetpindicatedthatitmightbetargetedtotheendoplasmicreticulum without transmembraneregion. 犘狅犛犃犇 genewasexpressedintheroot,stem,leaf,petal,pistil,stamenandseed of 犘. 狅狊狋犻 bythetissue specificityexpression.theexpressivecontentof 犘狅犛犃犇 geneindiferenttissues of 犘. 狅狊狋犻 wasdiferent:thehighestwaspetal,thenextwaspistil,andthelowestlevelwasinroot;in diferentperiodsofseeds,thehighestexpressionwasinseedsof60d,thenextwas80d.theexpressionlevel of10dwasthelowest. 犓犲狔狑狅狉犱狊 : 犘犪犲狅狀犻犪狅狊狋犻 ;stearoyl ACPdesaturasegene( 犛犃犇 );expressionanalysis;race;rt PCR :2017 07 10; :2017 11 28 : (31171588);!"#(CXTDX201601018); $%& ' ( % (2015 3); $)*+,!- (cstc2016shmszx80051);,./01 ( 23(AS201613)!:456(1991-),7,89:;(,, <=( E mail:569960239@qq.com >?@A:B C,D;,E, <=,( E mail:zht2188@126.com
36 38 JK L K( 犘犪犲狅狀犻犪狅狊狋犻 ) (Paeoni aceae) ( 犘犪犲狅狀犻犪 )L K (Sect.Moutan DC.), [ P LK@ O,,8 3000 8 500, U =,!! "#$ @ [1 3] %&', ()F *+,, -. /0 1 2345 6789:; [4 6] L K $ <X= α, ω 3 ` PX=, DHA P EPA >,?@FA BCD P AE EX P F : @ [7 9] 2011 G 9 ^ H,IJKJ,IL,!MNO LK $ ; P Q,X= R ( OS' [10] W X Y ACP (stearoyl ACP desatu rase,sad) :X= TUVW X,Y WXY ACP 8 C9 C10 X $Y ACP,8 PX= O PX = @ > ( Z, 犛犃犇 8 [ \ ] [11] ^ _ [12] [13] [14] F_ [15] P ` a [16] : b c S T,! ( 8 " # $ [17] 2 [18] [19] [20] P. [21] b c @ 犛犃犇 ST ap JKLK, >8 (,JKLK 犛犃犇 ( 8X= P Q c I ( [5], JKLK P X= I R b P X= R R #( J K L K W X Y ACP 犘狅犛 犃犇 @ RACE STI,? a, * PCR 犘狅犛 犃犇 @ b a, a PX=,O 犘狅犛犃犇 8 PX=, @,O 犘狅犛犃犇 I 0 b P 1 PQR 1.1 JKLK( 犘犪犲狅狀犻犪狅狊狋犻 ) F LK ; 2016 3~6 8 F < = * L K M P, -80, 1 犈狊犮犺犲狉犻犮犺犻犪犮狅犾犻 DH5α O < = * 犘狉犲犿犻狓犜犪狇 T4DNA DL2000 Marker T/A S T pgem T ] TaKaRa J EASYspin RNA (DNaseI) D J b J 1.2 1.2.1 234 $%& NCBI U 1 L K EST _ `?,GenBank \] ^ O JI454401.1 5 P 3 RACE, U _`> U Z cdna _ ` PrimerPremier5.0 /,5 P 3 RACE 2, RACE G P : :PsSAD GSP1 PsSAD GSP2; :PsSAD NGSP1 PsSAD NGSP2(b1) c RNA ] cdna, 1 犘犆犚 Table1 TheprimersusedforPCR [ Primername PsSAD GSP1 PsSAD GSP2 PsSAD NGSP1 PsSAD NGSP2 犘犝犉 1639Forward 犘犝犉 1639Reverse 犘狅犛犃犇 Forward 犘狅犛犃犇 Reverse _` Sequence(5 3 ) 5 GCTGAATGGAACAAGCGATGACGGT 3 5 CACAGAAGAAGCCCTCCCAACATACCAG 3 5 CGCCCTTCACCAGACAGACCTTCC 3 5 CTGGACAAGGGCATGGACGGCTG 3 5 AAACGAGTCGGTTGAAGATGAG 3 5 TATGCGGTGGATTTCGGAG 3 5 GCACCTAAAGCCTGTTGAGAAATGTTGG 3 5 GGTATGTTGGGAGGGCTTCTTCTGTGA 3
1 456,::LK 犘狅犛犃犇 ST b a 37,I cdna O,@ RT PCR, S T pgem T, DH5α,@ /,PCR ST_ _ J 1.2.2 犘狅犛犃犇 $ 犮犇犖犃 567 89: VectorNTIAdvance11 L @ _ ` 9 P,SignalP4.1server a? ^,TMHMM Serverv.2.0 a : Phyre2 8 a 0 P MEGA 7.0 R N @ 1.2.3 犘狅犛犃犇 $; c ST JK 犘狅犛犃犇 cdna a,, I M P cdna O,I 犘犝犉 1639 O! [22], : 犘犝犉 1639 Forward 犘犝犉 1639 Reverse 犘狅犛犃犇 Forward 犘狅犛犃犇 Reverse(b1) * PCR " 犘狅犛犃犇 8 #$ b RT PCR SYBR Premix Ex TaqTM I(TaKaRaJ ) c,8%# CFX96 Real TimePCR DetectionSystem(Bio Rad J ) @ & 3, ',3 ', U Excel@,I SPASS @ Q a, RT PCR a 2 a 2.1 犘狅犛犃犇 $ 犮犇犖犃 <%& > RACE R,I J K L K M N cdna O @ PCR TA STP_ a, U 5 _`Z 1200bp, U 3 _ ` Z 900bp( 1) 1% (X)*+,", BF-. _ U / N 0 > NCBI Blast a,1 ATG, 犘狅犛犃犇 5, 8c poly(a) Z2 @, JKLK ST V Z 犘狅犛犃犇 (GenBank \ ] ^ O KY038819), Blastn _ ` b c,s T 犘狅犛犃犇 ( 犚犻犮犻狀狌狊犮狅犿犿狌狀犻狊 ) 犛犃犇 (NM 001323709.1) Q 34 ATG;5^3467 TGA 1 犘狅犛犃犇 8 9 _` : TheinitiationcodonATGismarkedwithbox;ThestopcodonTGAismarkedwithasterisk Fig.1 Thesequencemapof 犘狅犛犃犇 nucleotideandputativeaminoacid
38 38 2 PoSAD B Fig.2 PredictionofPoSADconserveddomainsof 犘. 狅狊狋犻 3 JKLK SAD ; SAD @ Fig.3 PhylogenetictreeanalysisofSADof 犘. 狅狊狋犻 andsadsofotherplants 82% R / VectorNTIAdvance11 a Z Z _ ` 1559bp, 5 123bp,3 172bp, 1197bp 9 2.2 犘狅犛犃犇 $ 犮犇犖犃 56<7 89: SignaIPserver B? ^,TM HMM Serverv.2.0 a WoLFPSORT B PoSAD 0 NetPhos2.0B 25 8 < ', =14 > (Serine) 8? (Threonine)P 3 @ (Tyrosine) A 9 PoSAD./ < NCBI8 a1 CDD a (CD), bc,posad Ferritin like B( 2) 2.3 犘狅犛犃犇 =>?<@A NCBI V 11 SAD _ `, MEGA7.0 R JKLK SAD _` C @ JKLK ( 犚犻犮犻狀狌狊犮狅犿犿狌狀犻狊 ) `a( 犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿 ) [ \]( 犎犲犾犻犪狀狋犺狌狊犪狀狀狌狌狊 ):11 : SAD _`,11 SAD _ ` D EO/F, 2 `aeo ;J - Tb I /( 犘 <0.05) 4 JKLK RT PCR a Diferentnormalletersshowsignificantdiferencesin diferenttissues( 犘 <0.05) Fig.4 RT PCRanalysisof 犘狅犛犃犇 expression indiferenttissuesof 犘. 狅狊狋犻 KLK,, _ `./O 81.0% ; $. : ; SAD 8@! ( 3), 9JKL K 犛犃犇 犛犃犇 R 2.4 犘狅犛犃犇 $; * abc, /b LF, 犘狅犛犃犇 8 #$ b,8 b, O 8 G, P b,
1 456,::LK 犘狅犛犃犇 ST b a 39 - Tb /( 犘 <0.05) 5 JKLK RT PCR a Diferentnormalletersshowsignificantdiferencesin diferentgrowthstages( 犘 <0.05) Fig.5 RT PCRanalysisof 犘狅犛犃犇 expressionin diferentgrowthstagesof 犘. 狅狊狋犻 seeds b ( 4), b M b / ;b M / ; b b P / 8,b HI JKLM NO, J 60d b,80d, 10db ( 5) 60d P 10d b 4 b / 3 #( IJKLKO( 2,8c LK ] _, RACE S T U 犘狅犛犃犇 ZcDNA _ `,> N! Z U N0 R / _` abc,pos AD [\] $. : 11 : SAD _`R / 70%I, b SAD 1, Ferritin like B,b c 8 a@ P /!,Q 8,ZTU R [23] _ ` @ a b c,j K L K PoSAD _` EO, $,Q c_ `R 8@ R,8 X= R [24] ( B P au,posad, M,B?^,Q ( PoSAD R!R ( Z,/ S ) $ ^ W X Y ACP 犘狅犛犃犇 8 M T U V WX b, 8 b, MN [25] #(,Y 犘狅犛犃犇 8JKLK #$ "Vb, #$ 8 /, 犘狅犛犃犇 8 b, JK 犘狅犛犃犇 8 b,, b AZ2bc ]. / 8 b b, 60 d b,b c 犘狅犛犃犇 @ ;H I K L J 犘狅犛犃犇 b M NO, 犘狅犛犃犇./ WX R, c#( STUV 犘狅犛犃犇 8LK X= VW @ [26] ( 犘狅犛犃犇 8 J K L K / b, a PX=,@ O( X =, & TUVW, 1 Z0L X= &,O [\ b BC.: [1] BERUTO M,CURIRP. 犐狀狏犻狋狉狅 CultureofTreePeonythrough AxilaryBudding [M].ProtocolsforMicropropagationofWoody TreesandFruits,Berlin:SpringerNetherlands,2007:477 497. [2] LISS,WANGLS,SHU Q Y, 犲狋犪犾.Fatyacidcomposition ofdevelopingtreepeony( 犘犪犲狅狀犻犪狊犲犮狋犻狅狀 MoutanDC.)seeds andtranscriptomeanalysisduringseeddevelopment[j]. 犅犕犆犌犲狀狅犿犻犮狊,2015,16(1):1 14. [3] ] ^,_ `.a$ LK Zb & c [J]. %&(,2014,27(1):68 71. ZHOU L,WANG Y.Developmentandutilizationofoilseed peonyanditsindustrialdevelopmentstrategyin China [J]. 犠狅狉犾犱犉狅狉犲狊狋狉狔犚犲狊犲犪狉犮犺,2014,27(1):68 71. [4] B C,,,:.$ LK ( @c[j]. F( ),2015,(2):143 149. ZHANG T,GAO T S,BAIR Y, 犲狋犪犾.Utilizationandre searchprogressofoiltreepeony [J]. 犑狅狌狉狀犪犾狅犳犆犺狅狀犵狇犻狀犵犖狅狉犿犪犾犝狀犻狏犲狉狊犻狋狔 (NaturalScience),2015,(2):143 149. [5] %, -,,:.$ LK JK */b X = a[j]. %(,2015,(1):67 72. LINP,YAO X H,CAO Y Q, 犲狋犪犾.Variationanalysisof fruitcharacteristicsandfatyacidcomponentsin 犘犪犲狅狀犻犪狅狊狋犻 Phoenix White [J]. 犖狅狀狑狅狅犱犉狅狉犲狊狋犚犲狊犲犪狉犮犺,2015,(1):
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