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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



全 文 :书!"#$%&
,2016,36(7):1285-1293
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vÁCiNAC1³­‚¿ÀÂy,ÃÄÅT”efgQl{|joÆl;e犆犻犖犃犆1ˆgÇÈÉÊËÌÍÎ4ŽÏ
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犆犾狅狀犻狀犵犪狀犱犉狌狀犮狋犻狅狀犪犾犃狀犪犾狔狊犻狊狅犳犆犻犖犃犆1犳狉狅犿犆犪狉犪犵犪狀犪犻狀狋犲狉犿犲犱犻犪
YUEJunyan,YUEWenran,YANGQi,YUXiumin,YANGFeiyun,
WANGGuangxia,LIGuojing,WANGRuigang,CONGJingyu
(InnerMongoliaAgriculturalUniversity,Hohhot010018,China)
犃犫狊狋狉犪犮狋:NACsareoneofthelargestplantspecifictranscriptionfactorfamilies.Membersinthisfamily
playimportantrolesingrowthandabioticstressresponses.Inthisstudy,thefullengthcDNAsequence
of犆犻犖犃犆1from犆犪狉犪犵犪狀犪犻狀狋犲狉犿犲犱犻犪wasclonedbyPCRtechnique.Itwas1066bpinlength.Bioinfor
maticsanalysisshowedthat犆犻犖犃犆1hadanopenreadingframeof921bp,encoding306aminoacids.The
calculatedmolecularweightofCiNAC1was34.57kDandtheisoionicpointwas8.35.TheCiNAC1pro
teinwasahydrophilicproteinwhichhadaconservedNAMdomainintheNterminus.Itcontained26
phosphorylationsitesand7glycosylationsites.RealtimequantitativePCRanalysisshowedthat犆犻犖犃犆1
wasinducedbydrought,NaCl,dehydration,andhighpH.LocalizationassaysrevealedthatCiNAC1lo
calizedinthenuclei,consistentwithitsroleastranscriptionfactor.Overexpressionof犆犻犖犃犆1promoted
thelateralrootformationandlengthoftheprimaryroot.Theseresultsindicatedthat犆犻犖犃犆1mightbe
relatedtoresponsetoenvironmentalstressin犆.犻狀狋犲狉犿犲犱犻犪.
犓犲狔狑狅狉犱狊:犆犪狉犪犵犪狀犪犻狀狋犲狉犿犲犱犻犪;NACtranscriptionfactors;abioticstress;functionalanalysis
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犖犃犆1ˆg¸¹º、¼¢、-»、-pHl½]。
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çèébêÝ£ëé.Ö*Thermoìí。
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1.2.1 PQTU îïðñòóly„…†‡ 
Q #Ñôõbö÷»
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ˆÂoÆly„…†‡þÿù!"%
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þÿlÍr‰r%&G200mLï”300mmol·
L-1NaCll(} MSú÷ˆy,P)%&0、1、3、6、
12、24b48hìï*。
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‡þÿlÍr‰r%&G 200 mL ï” 200
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þÿ@+N»,yïb

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012

P)G0、1、3、6、12、24b48hìï*。
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ìok4¢24hì,5ÿ630d°”0ù,nì7
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84dï1k*,¹º16dìe¢,e¢48
hì9ï*,ï*°„P)”0、4、8、12、16b18d。
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Ku30dly„…†‡þÿ,/,Trizol:;ù!
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lRNA, MMLV&eféOûcDNA1.2.3 45678犆犻犖犃犆1,;犮犇犖犃[\=>
? ÍǯÃĹºefVÎÇÈy NAC1Š
‹G@Hn犆犻犖犃犆1ˆgcDNA‰u$$P犆犻
犖犃犆1F(5′GCGTCGACATGAGCAACATAAG
CATGGTAG3′)bP犆犻犖犃犆1R(5′GCGAGCT
CAAGAAAGCTTAAAGAGGAGTGAA3′),ë&
efy„…†‡cDNA ”IJ,K,-¦Lé
PrimerSTAR(TaKaRaìí)ù!PCRHn&p。
&pBŠ”98℃MP£3min,98℃P£30s,
55℃NO10s,72℃¡P1.5min,72℃QR¡P
5min,30—Sö。
1.2.4 ]^_`abcTUd犆犻犖犃犆1,;ef
CD y„…†‡þÿP), NaCl、-pH、¼¢、
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犖犃犆1 ± ² ³  ¶ · $ $ q 犆犻犖犃犆1F(5′
TCAAAACCAAACCAACCCAATC3′)b q犆犻
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3′),qRTPCR¨,SYBRGreenⅠ±²T¾Ï´
µ

&p}˔10μLSYBRPrimixExTaq
TM,
$
$þ0.4μL(10μmol·L
-1),
UVlcDNAIJ
5μL,DEPC¢4.2μL,<}Ë20μL。PCR&pBŠ
”95℃MP£1min,95℃P£5s,60℃NO30s,
72℃¡P10s,95℃QR¡P1min,40—Sö。
y„ … † ‡ l j $ $ ” qCiEF1αF(5′
CAAAAAGTCCCCTCGTTGTCTC3′)b qCiEF1αR
(5′AGCAATCGTTCTTCCTAATGATCTAA3′),Ç
ÈÉ j $ $ ” qAtEF1αF(5′AGAAGGGTGC
CAAATGATGAG3′)b qAtEF1αR (5′GGAGG
GAGAGAGAAAGTCACAGA3′),¯ è™,2-ΔΔCt
Ï@W

1.2.5 45678犆犻犖犃犆1,;=`aghiC
D K,DNAMAN £«ù!˜ˆ™Š‹ÐzÖ
0

K,ExPasyGXA¥ProParam b ProtScale
P›œlþ ˜ˆ™M

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;NCBICDDÎÇÈ(http://www.nc
bi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)Mµ›
œl{|¨©ª
;NetPhos(http://www.cbs.dtu.
dk/services/NetPhos/)b NetGlycate (http://
www.cbs.dtu.dk/services/NetGlycate/)Mµ›
œl«™¬­Ÿb®ˆ¬­Ÿ
;SOPMA(http://
npsapbil.ibcp.fr/cgibin/npsa_automat.pl?page
=npsa_opma.html)Mµ›œlZ[¨©;MEGA6
£«ù!Ë\ù¬LP

1.2.6 jkeflm狆犆犪狀犌犆犻犖犃犆1n狆犆犃犕
犅犐犃1302犆犻犖犃犆1 ,犛犪犮Ⅰb犛犪犾ⅠêÝ£ë
é0犆犻犖犃犆1bpCanGHAù!{éë,NCD
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/, T4DNA çèéçè,©Å
pCanG犆犻犖犃犆1x¶·Õ};,犅犵犾Ⅱb犛狆犲犾Ⅰ
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©ÅpCAMBIA1302犆犻犖犃犆1x¶·Õ}。
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ïG23℃、16h²ù/8hûülæÄyKu3~4
j6lÇÈÉþÿ~‚

,PEGk]ÏÝlÇÈÉ
~m ? K q }

å ï pCAMBIA1302犆犻犖犃犆1
GFPbnÕ}pCAMBIA130235SGFPqñþT
30μg,oì#WP)e¬‚ÇÈÉ~m?Kq}
y

GpqJyÄærw16h,,±²Ž>stu
GFP±²S。
1.2.9 9犆犻犖犃犆1,;opq}~CD ÒKÓ
bx¶·ÇÈÉ Q
(OE5、OE42bOE60)÷x
75%†vwá10min,,Mk0.05% Tween20l
100%†vwá10min,x Qy¹ì G1/2MS
ú÷ˆ2
,4℃îˬ"%3d,#zJïb/Ñ
23℃,16h²ù/8hûüÊ«‹ú÷36~40h,î
ïuýoÆl50{þÿ,Z|‚7l1/2MSú÷
ˆ2}~ú÷12d,\@ÌÍÎ,ZµÍu。
2 ¨™ÄP
2.1 45678犆犻犖犃犆1,;=>?YCD
N¹ºefV‚犆犻犖犃犆1ˆglo_Š‹,
78217ø           CDE,.:y„…†‡efgQˆg犆犻犖犃犆1l€;{|P
RxNCBIBlastù!Ö0,vÁ¥k€l‘’

(ORF)。ÍÇÃ_HŠ‹G@P犆犻犖犃犆1F
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
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K, NCBICDD(ConvervedDomainDatabase)Î
ÇÈPCiNAC1›œl¦§¨©ªvÁ,GN¤
<11~135—˜ˆ™n„ko—S125—˜ˆ™V
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NAC1›œl«™¬­Ÿb®ˆ¬­Ÿ°vÁ,
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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¸


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47.39%;70 — ˜ ˆ ™ ú û α¹ º,Ð · Ö ¸ ”
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fCD
  ”ƒùoË12犆犻犖犃犆1ˆgl{|,¯Ã
¨, qRTPCR 5~´µƒy„…†‡G÷x
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(WT)bx¶·ÊË(OE5、
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Fig.5 Theexpressionof犆犻犖犃犆1underdifferentstressesbyqRTPCR
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W^h”eˆgÊË
;B.eˆgÊË犆犻犖犃犆1¶·¢zl¯°³PCRdz
‚6 犆犻犖犃犆1eˆgÊËdz;¶·¢zdz
A.RTPCRidentificationof犆犻犖犃犆1transgenic犃狉犪犫犻犱狅狆狊犻狊lines:C-.犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪cDNA(Negativecontrol);C+.犆犪狉犪犵犪狀犪
犻狀狋犲狉犿犲犱犻犪cDNA(Positivecontrol);Othersaretransgeniclines;B.QuantitativerealtimePCRanalysisof犆犻犖犃犆1expressionin
犆犻犖犃犆1transgeniclines
Fig.6 Theexpressionlevelof犆犻犖犃犆1intransgenic犃狉犪犫犻犱狅狆狊犻狊
‚7 ÇÈÉ~m?Kq}yGFP犆犻犖犃犆1l¾¿À³­
Fig.7 ThenuclearlocalizationofGFP犆犻犖犃犆1
in犃狉犪犫犻犱狅狆狊犻狊mesophylprotoplast
ÇÈÉ QG1/2MSú÷ˆy­$Ku40hì,e|‚7l
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The40hoursseedlingson1/2MSmediumweretransferred
tonewmedium(n=50)
Fig.8 Overexpressionof犆犻犖犃犆1promotedthelateral
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
Ò

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[31]。
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…ùàã-Þl NAC1‘áüâKuãS;
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QlmRNAü‹*KuãS,N.*+ÇÈÉÌ
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;Wang.[34]vÁKuãbãbã½]
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He .[3536] v Á Ç È É 犃狋犖犃犆2(犃犖犃犆092、
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. 犃犚犉7、犃犚犉19、犔犅犇12、犃犐犚1
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K$st&pylT,
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ùô
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