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Cloning and Functional Analysis of CiNAC1 from Caragana intermedia

中间锦鸡儿转录因子基因 CiNAC1的克隆及功能分析



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bP犆犻犖犃犆1R,ëy„…†‡cDNA ”I J , K,-¦LéPrimeSTARù!PCRHn, ‚犆犻犖犃犆1ˆgcDNA‰uŠ‹(‚1)。犆犻犖犃犆1 ˆgcDNA Š‹‰u1066bp,Wy‘’H“ (ORF)921bp,ؕ–306—˜ˆ™(‚2)。 2.2 犆犻犖犃犆1€Y‚aƒ犖犃犆K€„…† ‡@ˆ‰Št‹CD   p,DNAMAN5.2£«#CiNAC1˜ˆ™Š ‹ÄÇÈÉ ANAC019、ƒ„ MtNAC1、` B Gm NAC35、ÒK`BGaNAC21/22l˜ˆ™Š‹ù! ‚1 犆犻犖犃犆1ˆgcDNAHn M.DL5000 1.犆犻犖犃犆1 Fig.1 Amplificationof犆犻犖犃犆1cDNA ÐzŠ‹Ö0 ,¨ ™ ( ‚3)¶ÕCiNAC1›œÄW ^NAC4›œo*,N¤l˜ˆ™Š‹-獵, NMA~E.5—¾¨©ª。 K, MAGE6£«#y„…†‡CiNAC1Ä ƒ„ NAC1(犕犲犱犻犮犪犵狅狋狉狌狀犮犪狋狌犾犪)、¹…†(犃犿 犿狅狆犻狆狋犪狀狋犺狌狊犿狅狀犵狅犾犻犮狌狊)、` B NAC35(犌犾狔犮犻狀犲 犿犪狓)、ÒK`BNAC21/22(犌犾狔犮犻狀犲狊狅犼犪)、‡"ˆ ‰ (犕犪犾狌狊犺狌狆犲犺犲狀狊犻狊)、ØØL NAC21/22(犜犺犲狅 犫狉狅犿犪犮犪犮犪狅)、pŠNAC40(犕犪狀犻犺狅狋犲狊犮狌犾犲狀狋犪)、‹ ™KNAC1(犘狅狆狌犾狌狊狋狉犻犮犺狅犮犪狉狆犪)、ŒŽK׏ œKNAC1(犘狅狆狌犾狌狊狋狉犲犿狌犾犪×犘狅狆狌犾狌狊犪犾犫犪)、 L (犆犪犿犲犾犾犻犪狊犻狀犲狀狊犻狊)、ÇÈÉ NAC1(犃狉犪犫犻犱狅狆狊犻狊 狋犺犪犾犻犪狀犪)ù!Ë\ù¬LP,¨ ™(‚4)¶Õy „…†‡CiNAC1ă„MtNAC1‘Go’,œ l-”£·‚89%,¡•ÞËm2。 2.3 45678犆犻犖犃犆1€=UtŒwCD@ ABŽ 2.3.1 犆犻犖犃犆1€=UtŒwCD K,Ex PasyGXA¥ProParam,PCiNAC1›œ•– 306—˜ˆ™,-0PQq34.57kD,.žŸpI ”8.35,B–i—Δ68.50,_›œj혳› œ 。 ™¢£MµPvÁ€—КFo›k76— œž–QATG,‹ŸX¶, Gž–QTGA,¡b‹ŸX¶ ‚2 犆犻犖犃犆1ˆgcDNA‰uŠ‹;•–˜ˆ™Š‹ TheATGinitiationcodonisunderlined;Theterminalcodonisunderlinedwithasterisk Fig.2 ThecDNAandthededucedaminoacidsequenceof犆犻犖犃犆1 8821 ! " # $ % &                   363 ‹ŸXrP¶NAC¨©ªl5—¾¨©ªA~E;û¢£¤¶‰o*l˜ˆ™Š‹;¥¢£¤¶oÆ£l˜ˆ™Š‹;ANAC019.ÇÈÉ (NP_175697.1);CiNAC1.y„…†‡;MtNAC1.ƒ„(XP_003595973.1);GmNAC35.`B(NP_001235901.1);GsNAC21/22.ÒK`B(KHN26800.1) ‚3 CiNAC1›œÄW^NAC4›œ¦§¨©ªlÖ0 TheunderlinedsectionsarefiveconservativesubdomainsAtoEinNAC.Aminoacidresiduesconservedinalsequencesareboxed inblack,whilesimilaraminoacidsareboxedingray.AlabbreviationandGenBankAccessionNo.areshownas:ANAC019. 犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪(NP_175697.1);CiNAC1.犆犪狉犪犵犪狀犪犻狀狋犲狉犿犲犱犻犪;MtNAC1.犕犲犱犻犮犪犵狅狋狉狌狀犮犪狋狌犾犪(XP_003595973.1); GmNAC35.犌犾狔犮犻狀犲犿犪狓 (NP_001235901.1);GsNAC21/22.犌犾狔犮犻狀犲狊狅犼犪(KHN26800.1) Fig.3 AlignmentofNACdomainbetweenCiNAC1andotherNACs ¦”˜ˆ™§f ; P¨2lΩ¶BootstrapêyˆÑ1000kze}«ŸlØSïl¬PÖ;­®¶¯¬°± ‚4 CiNAC1ÄW^$ NACslË\ù¬P TheaccessionNo.wasgiveninbracket;Thenumbersonthebranchesrepresentthereliabilitypercentofbootstrapvaluesbased on1000replications;Scaleplaterepresentstheevolutiondistanceoftheseplants Fig.4 PhylogeneicanalysisofCiNAC1andNACsfromotherspecies 98217ø           CDE,.:y„…†‡efgQˆg犆犻犖犃犆1l€;{|P ™¢£˜ˆ™b230—¡¢˜ˆ™,¶ÕCiNAC1 ›œj¡¢›œ 。 2.3.2 犆犻犖犃犆1€=ABjŽYCD  K, NCBICDD(ConvervedDomainDatabase)Î ÇÈPCiNAC1›œl¦§¨©ªvÁ,GN¤ <11~135—˜ˆ™n„ko—S125—˜ˆ™V ûlNAM¦§¨©ª。g_,Ø뚲by„… †‡犆犻犖犃犆1ˆg³ÑNAChi。 K,NetPhosbNetGlycateGX£«MµCi NAC1›œl«™¬­Ÿb®ˆ¬­Ÿ°vÁ, 犆犻犖犃犆1ˆg•–l›NM7—®ˆ¬­Ÿ(10、 32、48、121、124、226、269)b26—«™¬­Ÿ,W y26—«™¬­Ÿä”g˜™(Thr)«™¬­Ÿ (50、82、112、161、199、236、267、268、334、341、379、 395、429、453、455、466、485、559、584、670、686、736、 737、791、877、890)。 2.3.3 犆犻犖犃犆1€=‘’j=Ž K, SOPMAGX£«MµCiNAC1›œlZ[¨©, vÁCiNAC1›œlZ[¨©´µ3¶Ð·Ö¸ mÐ , Wy145—˜ˆ™úû´µ3¶,зָ” 47.39%;70 — ˜ ˆ ™ ú û α¹ º,Ð · Ö ¸ ” 22.88%;66—˜ˆ™úû¡PF,зָ” 21.57%;25—˜ˆ™úûβe˜,зָ”8.17%。 2.4 犆犻犖犃犆1,;u]^_`a“”bcd=e fCD   ”ƒùoË12犆犻犖犃犆1ˆgl{|,¯à ¨, qRTPCR 5~´µƒy„…†‡G÷x NaCl、¼¢、»,¹º、-pH"%ëì犆犻犖犃犆1ˆ g¶·lP¬ 。 ¯Ã¨™Ž ( ‚5),¼¢"% ëìy„…†‡犆犻犖犃犆1ˆg¶·»Á¼2½ 싑l¾ý , [G6h°m-,·‚6¿;NaCl" %ì犆犻犖犃犆1ˆg¶·ÀÁ½-,‚12h°·‚ Â"%l6¿ÃÄ,Z[o~‚48hŦº-¢z; -pH"%ì,犆犻犖犃犆1ˆg¶·ÀÁ½-,[G 48h°·‚m-¢z,T”Â"%l13¿;»,¹ º"%ì犆犻犖犃犆1ˆg¶·»Á¼2½ì‹‘ l¾ý , G16d°·‚m-¢z,T”Â"%l4 ¿ 。 ÆÕy„…†‡犆犻犖犃犆1ˆgØ|jÄ0¹ º 、 Xçb»stlÙp 。 2.5 9,;opq4犆犻犖犃犆1,;=efCD îïT3 hiO}eˆgÇÈÉþÿl~‚, åï<RNAZ&efûcDNA,K,dQ£
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tonewmedium(n=50)
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rootformationin犃狉犪犫犻犱狅狆狊犻狊
e1 –`—n˜ef™š~e—›Œ†œ
Table1 Comparisonoftherootphenotypicpropertiesbetweenwildtypeandoverexpressionlines

Plant
¶ÓdÑPhenotypiccharacteristic
ÌÍÎ
Numbersoflateralroot/number
Íu
Rootlength/cm
ÌÍÎ

Íu
Lateralrootdensity(N/L)
WT 10.28±4.40 4.443±1.19 2.565±1.33
OE5 12.42±4.39 4.564±1.45 3.235±1.93
OE42 15.58±6.05 5.293±1.71 3.265±1.57
OE60 14.36±4.81 5.095±1.49 3.131±1.45
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