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Cloning, Expression Characterization and Subcellular Localization of NtOSA1 Gene from Nicotiana tabacum

烟草氧化胁迫相关基因NtOSA1的克隆表达及亚细胞定位分析



全 文 :书!"#$%&
,2016,36(8):1499-1506
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
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0123

OPQ
(1983-),R,12,ST,UVWX#$YZA$%34。Email:kateryan@163.com
456789:;,<犖狋犗犛犃1=>?@A
BCDEFGHI
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1,2,
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1,
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1,2,
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(1!.+/0% A[+%FIJ%\,>?]^621010;2!.+/0% _`$Fabcdefghij,>?]^621010)
J K:#$klmnoAbc1(p(activityofbc1complex)q#$A6rstuvwA$xyKz{fV|}
E

~34€‚.ƒAtOSA1lm„,…†‡Fˆ‰Š‹ŒŽ‘’,DE“”PCR/•–—˜™1
šˆ‰›œxyžAbc1(p,Ÿ犖狋犗犛犃1,犖狋犗犛犃1F‚.ƒ犃狋犗犛犃1,Ÿ ¡72.89%|¢£H。犖狋犗犛犃1
,Ÿ¤¥¦§¨6©ª2283bp,«¬760š„,…,­¡®¯|ABC1°±²、¢šno°±²、³´µ¶·¸¹
º»¼š½¾°±²

¿EhÀÁ¶ÃPCR/•,’犖狋犗犛犃1,Ÿqˆ‰ÄŌÆÇt›œxy、ÈxyÉÊ
ËÌ|ÍÎYÏÍÐ
:犖狋犗犛犃1,Ÿ|ÍΠ¡ŒÆ:ÑH,UVq³ÒKÍÎ;犖狋犗犛犃1,Ÿq H2O2 »NaClÊ
ËÓÍÎÃÔÕ

ÖqÊËÓ6hΙ×0Ø,YÙªÊËÚ|1.95»2.69Û。ÜÝÞ¶·°ßÍÐ,NtOSA1
lm¶·qÝÞ³´µÔ

Fàá°ß¢£

34ÍÐ
,犖狋犗犛犃1,ŸâF㈉䛜xy»Èxy|uv。
;LM

ˆ‰

›œxyžAbc1,Ÿ;,Ÿ–—;ÍÎ:H;ÜÝÞ¶·
NOHP$
:Q785;Q786 !QRST:A
犆犾狅狀犻狀犵,犈狓狆狉犲狊狊犻狅狀犆犺犪狉犪犮狋犲狉犻狕犪狋犻狅狀犪狀犱犛狌犫犮犲犾狌犾犪狉犔狅犮犪犾犻狕犪狋犻狅狀
狅犳犖狋犗犛犃1犌犲狀犲犳狉狅犿犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿
LIUJikai1,2,GAOYongfeng1,WUChanjuan1,2,ZHANGLin1,CHENCaixia1
(1SchoolofLifeScienceandEngineering,SouthwestUniversityofScienceandTechnology,Mianyang,Sichuan621010,China;
2FundamentalScienceon Nuclear Wastesand EnvironmentalSafety Laboratory,Southwest UniversityofScienceand
Technology,Mianyang,Sichuan621010,China)
犃犫狊狋狉犪犮狋:Membersoftheactivityofbc1complex(Abc1)familyareproteinkinasesthatarefunctionalydi
verseproteinswithmultiplerolesintheregulationofplantgrowthanddevelopmentandabioticstresstol
erance.AccordingtotheaminoacidsequenceofAtOSA1andthetranscriptomedataof犖犻犮狅狋犻犪狀犪狋犪犫犪
犮狌犿,aAbc1likegenewasisolatedanddesignatedas犖狋犗犛犃1,asforthehighidentitywiththe犃狋犗犛犃1
(犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪oxidativestressrelatedAbc1likeprotein).Theopenreadingframe(ORF)ofthe
犖狋犗犛犃1geneis2283bpwhichencodedadeducedproteinincluding760aminoacidresidues.Theprotein
sequenceofNtOSA1possessesaconservedABC1domain,onekinasedomain,onechloroplastlocalization
signalpeptideandtwotransmembranespans.Therelativeexpressionof犖狋犗犛犃1wasdeterminedbyquan
titativerealtimePCR(qRTPCR).Theresultsshowedthat犖狋犗犛犃1genewasexpressedparticularlyin
leaves,butalsoinflowers.AftertreatmentofH2O2andNaClfor6h,theexpressionof犖狋犗犛犃1in
creasedtothemaximum,whichwas1.95and2.69foldsofthecontrol,respectively.Whenfusedtogreen
fluorescentprotein(GFP),NtOSA1localizedtothechloroplastintobaccoprotoplasts,whichwasconsist
entwiththepredictionresultsofthesoftwareTargetP1.1Server.Theseresultssuggestedthat犖狋犗犛犃1
maybeinvolvedinresponsetooxidativeandsaltstressintobacco.
犓犲狔狑狅狉犱狊:犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿;oxidativestressrelatedabc1likeprotein;geneclone;expressioncharacter
ization;subcelularlocalization
  #$qA6rs€JKÄåæçèéê™ëì
íbxy

îïð

ñð

òó

f-ô»ÈõÉ

ö
÷íbxyèø#$›œùúûüýþ

ÿ£µ!
"#0Ã|$H›

W%&A›œxy

€Ã|$
H›F(B

lmB
、DNAÇt)*A$0YZ
rA+v

W%,-7µ./0123
[1]。
A$µ!4q5ìâF›œxyuv|lm(
p

‘îAbc1(p,~(p6¢k70| ¡5ì
A$%8|lm(p

9:4q;ú_»<_A
$K

öklm ¡ï©=>| ABC1°±²»l
mno°±²
[2]。
qú_»<_A$?@µK

Abc1likelm6Ao QB-|fVCDŸZ,â
F™EFG|HZIJ»ä›œ€J
[38]。
KL
犆犗犙86M¢šNO¶|Abc1,Ÿ,)«¬lm 
¡kYZPQ$H

RSTÝÞUVbmRNAW
X|YZÇt[\?@µEFGKbc1]Bµ|
$H
[910]。
»KL‘

#$| Abc1likelm ¡^5
_|8
[1113]。
‚.ƒ
(犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪),
ŸŒK¡17š,Ÿ­¡®¯| ABC1°±²。M
¢šN34|6犃犅犆1犃狋,Ÿ,~,Ÿq‚.ƒë
šŒÆKŒ-¯ÍÎ

)«¬|?@µlmFKL
ABC1lm¡45%|`H,a ¡KLABC1l
m`|8
[14]。
bc¢š Abc1lm,AtOSA1
(oxidativestressrelatedAbc1likeprotein)d*e
»KLABC1 ¡45%|`H,f6~lm¶·
;ÝÞ³´µK

8ghhiij
,AtOSA1lm
Äk]KL犪犫犮1lmµEFG|$H。n¶Ã
RTPCR ° ß i j,犃狋犗犛犃1 , Ÿ | Í Î o ™
CdCl2、H2O2 »ïp©Â|qÿ[11]。YangÉ|3
4ij
,AtACDO1lm(ABC1likekinaserelated
tochlorophyldegradationandoxidativestress)¶
·;³´µK

q›œxyro»³´Vst€
JKru}E
[13]。
‚.ƒABC1K1»ABC1K3l
me¶·;³´µBµv

CDBµvÑwxy,
(ûü€J

q#$íbvz»³´µ{|}-K
ru{fV}E
[1516]。AtSIA1(saltinducedABC1
kinase1)FAtOSA1 ¡46%|`H,Å_¶·
;ÝÞ³´µK

ͯYÏ~Ð
,AtSIA1» AtO
SA1Ru³´µKFe|Y€,CD’$H›
|&A»›œxy|vz
[17]。
c

‚ƒ„É
[18]
–—㢚…†
(犜狉犻狋犻犮狌犿犪犲狊狋犻狏狌犿)kABC1,Ÿ
犜犪犃犅犆1犔,‡rˆ~,ŸRN‰Š、ïÈ、ñðx
yÇtABAÊËqÿ。É[19]34rˆ,
Ž
(犗狉狔狕犪狊犪狋犻狏犪)犃犅犆1,ŸUVq³ÒKÍÎ,‡
ao5ìwA$xyŸV H2O2、ABA、ñðÇ
tïÈÉ|CD

ˆ‰
(犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿)6YZA$%»,
ŸIJ34|‘”#$

6ס’Ø|+3A$“
”•¢

ˆ‰äíxyvz€JKž,Ÿ|–—
t8|–j¢—634|˜g

fNڞ;ˆ‰
Abc1lm(p|834ù™š&›。œ34
žWˆ‰K–—™ã犗犛犃1|ÅC,Ÿ犖狋犗犛犃1,
’)†‡:Ÿ

ŒÆÍÎ:H

ÜÝÞ¶·tëìw
A$xyuvã34

ªd tÏ犖狋犗犛犃1,
Ÿqˆ‰›œxy»ÈxyvzK|A$%8¡
¢ãfVâ£

1 “”»¤¥
1.1 45UV
¢¦#$“”ªˆ‰
‘NC89’,hij§¨
ì#

1.2 W X
1.2.1 45犖狋犗犛犃1,<>? E‚.ƒ AtO
SA1lm|„,…†‡qSGN ©ª(http://sol
genomics.wur.nl/)†‡‘’(Ž«ªˆ‰
SGNmRNA †‡,J†ªlm†‡’_¬…†
‡
)。
­Ž‘’®|¼¯EST†‡,SGNE1015449
»SGNE1113121,°±c²³$ NtOSA1F1(5′
TTCCCTTGCATGCACATACA3′)» NtOSA1
R1(5′TGATCTCCCAGGGTAACCAT3′);­¡
犛犿犪I»犛犪犾Io´·g|!²³$ NtOSA1F2
(5′CCCGGGATGGCGAGTATTTCAGCTAC3′)
» NtOSA1R2(5′GTCGACAGCTGTTCCTGT
0051 ! " # $ % &                   36µ
GATCA3′)。¡¶ˆ‰·¸³Ò¹RNA,Ç+Š
‹º˜|cDNAM¢Gª‘»,NtOSA1F1»Nt
OSA1R1ª³$,Eï=(TaKaRa¼½)M¢¾PCR¿À。M¢¾
PCR&$ÁÂ100Û,ÇÁÂÓ| DNA ª‘»,
NtOSA1F2» NtOSA1R2 ª³$Mx¾
PCR¿À。Mx¾PCR&$ÃÄŜÓÆÇ1
pEASYBluntȵ("Éd”-A$¼½),Šœ0
ÊËÌ
(犈狊犮犺犲狉犮犺犻犪犮狅犾犻)DH5α。Ç0ÊËÌ͖
—ª‘»
,NtOSA1F2» NtOSA1R2ª³$,
ÌÎPCRÏá,^H–—ÐÑ0,Ÿ+/ÒÓ
¡Ô¼½á†

1.2.2 45犖狋犗犛犃1,<=YZ[\]HI D
EDNAMANv6ÕÖYÏᆰß,׶犖狋犗犛犃1
,Ÿ|¤¥¦§¨
(ORF)†‡‡Øÿv|„,
…†‡

DEq?ÕÖInterPro(http://www.
ebi.ac.uk/interpro/)»TMpred(http://www.ch.
embnet.org/software/TMPRED_form.html)YÙ
àá NtOSA1lm|8=>°±²»½¾°±
²

DEq?ÕÖProtParamYÏNtOSA1|„,
…Œ-

YZÃ

ÉHgɘHB

DEq?ÕÖ
TargetP1.1ServeràáNtOSA1lm|ÜÝÞ¶
·

€ClustalW » MEGA6.0Õ֝°B’32
šAbc1lm†‡†‡‘’‡±}Ùڏœ
Û

øEBootstraptestÏi(f]ت1000)œ
Û|åÜH

1.2.3 45犖狋犗犛犃1,<^_`aG@AbFb
cd ÝA¯ˆ‰ìZÞì™ßà,á;Ââã
äjãä

ãä¯Öª

ð©25℃,å©60%~
70%,16hÂâ/8hæç。
°± 犖狋犗犛犃1,ŸqRTPCR ³$ NtOSA1
rtF(5′TGTTCTGGTGCCTACTGGTGAC3′)»
NtOSA1rtR (5′AAGTTCTGCTTGTGCCATTTC
3′),Lj‰犖狋犚犔2(GenBank苹Z14081)ª!
â , Ÿ

³ $ ª NtRL2 rtF (5′GTAAGG
GAGCGGGTTCAGTCT3′)» NtRL2rtR(5′
AACGGAGCACCCCTACCTG3′)。éâ Trizol
(Thermo¼½)¦êëìÐíî},YÙ¡¶ÝA
¯ˆ‰­

ï

³Ò

ð»ìZ|¹RNA,ñ+Š‹
º˜cDNAM¢G。éRealMasterMix(ò­Aœ
+/¡Ô¼½

¦êëìÐí

qCFX96RealTime
System(BioRad¼½)qRTPCR¿À,J†
ª
:95℃àmH3min,95℃mH10s,60℃óô
30s,40šõa,65℃ö÷5s,*ÓÇøù0.5℃
Õ195℃,61šõa。Ž¿E2-ΔΔCt¥±ú。
1.2.4 45=89efB犖狋犗犛犃1,<=@Ab
cd ââLee[20]É|¤¥,ˆ‰Þì;1/2MS
ûµãä,
[21]
Ô

A62ü,ý¶6þÿ!,0…¢
£ | · ¸ Y Ù Š " ­ 10 mmol/L H2O2 »
200mmol/LNaCl|1/2MS#µãä,K,qÊ
Ë|0、3、6»12h¶·¸_$,#%&Óá;
-80℃(4)E。
ââ HermandÉ[22]|¤¥,ˆ‰Þì;¶
#*+K

E1/2Hoagland[23],ä#ã䈉,A
630dÓ,ý¶6þÿ!,0…¢£|·¸ÆŶ
#*+Š"YÙ­0、30、60»100μmol/LCdCl2
|-.1/2Hoagland,ä#K,qÊË|48h¶
·¸³Ò

#%&Óá;-80℃(4)E。
¡¶ÇÔÊ˓”|¹ RNA,ñ+Š‹˜™
cDNAM¢G,ÇNtOSA1rtF»NtOSA1rtR,Nt
RL2rtF»NtRL2rtRª³$,éâRealMaster
Mix(ò­Aœ+/¡Ô¼½)¦êëìÐí,q
CFX96RealTimeSystem(BioRad¼½)qRT
PCR,YÏ犖狋犗犛犃1,Ÿ|’ÍÎÃ。¿ÀJ†
ª
:95℃àmH3min,95℃mH10s,60℃óô
30s,40šõa,65℃ö÷5s,*ÓÇøù0.5℃
Õ195℃,61šõa。Ž¿E2-ΔΔCt¥±ú,
DPSv7.05i/HYÏ。
1.2.5 gh@Aij=kl、45mYnjo7B
pqrstuv úABµŠœÈµpTEXGFP
­¡„012V3ý45
、CaMV35S68Z、5–
—·g»犌犉犘,Ÿ«¬7†‡。
E犛犿犪I»犛犪犾I(Thermo¼½)ÅÀ8o´á
†9×| 犖狋犗犛犃1,ŸÒ:»pTEXGFPȵ,
;<|犖狋犗犛犃1,Ÿ«¬7="™pTEXGFP
ȵ|5–—·g

ø犖狋犗犛犃1»犌犉犘 ,Ÿ>B
ÍÎ

ÆÇ&$Šœ0ÊËÌDH5α。Ç0ÊË
Ì͖—ª‘»
,NtOSA1F2» NtOSA1R2ª³
$

ÌÎPCRÏá,^H–—ÐÑ0,Ÿ+
/ÒÓ¡Ô¼½á†

ǜAˆ‰
(犖犻犮狅狋犻犪狀犪犫犲狀狋犺犪犿犻犪狀犪)ªhi“
”

ââYooÉ[24]|¤¥T)ˆ‰úABµ,o
t¯ÖGªæçot4h。±}!|>BÍÎÈ
µpTEXNtOSA1GFP€PEG?ÿ¥Šœ™œ
Aˆ‰úABµK

jðÌ
(20~25)℃æçqÿ24
~48h。DEnÂ@ABCDÁÂiEF(GHI
½¼½
,¯
¹LSM710),JKNtOSA1GFP>Bl
m|ÜÝÞ¶·LM

°¶â
:GFPlm|nr
10518N         OPQ,É:ˆ‰›œxyž,Ÿ犖狋犗犛犃1|–—ÍÎtÜÝÞ¶·YÏ
OµÍj³´µ¶·¸¹º

ÌP?ÍjABC1°±²;OQµÍj½¾°±²;ÍjRST¬Z
U1 犖狋犗犛犃1|cDNA†‡t)Øÿ|„,…†‡
Chloroplasttargetingpresequencesarepresentedinbold;UnderlinerepresentstheABC1conserveddomain;Putative
transmembranespansarepresentedinbolditalic;Stopcodon
Fig.1 cDNAanddeducedaminoacidsequencesof犖狋犗犛犃1
VWU¨ûÍABC1°±²;XWU¨ûÍno°±²;
mU¨ûͽ¾°±²
U2 ˆ‰NtOSA1lmB°±jYU
Darkgraybox,ABCdomain;Lightgraybox,kinasedomain;
Whitebarrels,predictedtransmembranedomains
Fig.2 SchematicrepresentationoftheNtOSA1protein
ª488nm,FĪ500~525nm;³´µ|n
rª633nm,FĪ650~720nm。
2 °ßFYÏ
2.1 45犖狋犗犛犃1,<>?BwxHI
ᆰßij

º˜|Ò:F‚.ƒ犃狋犗犛犃1
,Ÿ_¬…†‡ ¡ï©|¢£H
(72.89%),Ÿ
[Zª 犖狋犗犛犃1(GenBank 苹 KU565475)。
犖狋犗犛犃1,Ÿ|¤¥¦§¨6©ª2283bp,«¬
760š„,…。
ªãtNtOSA1lm|A$%$Ht[q8


DEq?ÕÖYْNtOSA1lm|˜HB
»°±²YÏ»àá

°ßij
:NtOSA1lm
YZê86.07kD,Ë\ÉHgª8.92;q„,…
†‡|277~397 ¡ ABC1°±²,q364~502
 ¡no°±²
,NtOSA1lmC] ¡2š½¾
°±

YÙ·;„,…†‡|703~721»726~
743(U1、2)。‚.ƒ、Ž、KL»^Þ_4š
$ì|®¯Abc1lmFNtOSA1lm†‡‘
’

rˆ NtOSA1 ¡®¯| ABC1=>°±²

U3),ÍÐNtOSA1ô;Abc1(p。
2051 ! " # $ % &                   36µ
AtOSA1(At5g64940)»AtABC1(At4g01660)` )‚.ƒ;OsABC1(Os02g0575500)` )Ž;SynABC1(P73627)` )^Þ_;
ScCOQ8(EWH18611.1)` )KL
U3 ˆ‰NtOSA1F)*$ìABC1°±²‘’
AtOSA1(At5g64940)andAtABC1(At4g01660)arefrom犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪;OsABC1(Os02g0575500)isfrom犗狉狔狕犪狊犪狋犻狏犪;
SynABC1(P73627)isfrom犛狔狀犲犮犺狅犮狔狊狋犻狊;ScCOQ8(EWH18611.1)isfrom犛犪犮犮犺犪狉狅犿狔犮犲狊犮犲狉犲狏犻狊犻犪犲
Fig.3 ComparisonofABC1domainalignmentsamongNtOSA1andotherspecies
agÔ|ØÍjBootstrapf]1000ž|ḩ
U4 ˆ‰NtOSA1F)bAbc1lm|ÙڏœÛ
ValuesatnodesshowtheconfidencelevelofBootstrapreplication1000
Fig.4 PhylogenetictreeofthealignmentofNtOSA1deducedaminoacidsequencewithotherAbc1proteins
30518N         OPQ,É:ˆ‰›œxyž,Ÿ犖狋犗犛犃1|–—ÍÎtÜÝÞ¶·YÏ
2.2 45犖狋犗犛犃1,ý¶KL

‚.ƒ

Ž

cd
、^
Þ_É12š
$ì|32šAbc1lm†‡5f‘’,±}
œÛ

°ß

U4)ij,NtOSA1lmF‚.ƒ
AtOSA1lm|efžÙ×g,¢£Hª76.04%;
Fcd ADB13188lm|¢£Hª74.15%;F
ŽOs02g0575500|¢£Hª73.45%。ÍÐ,Nt
OSA1F‚.ƒ、cd»Ž|efžÙ’hg,
%FM¢šNO¶| Abc1lm、KLScCOQ8、Ç
t‚.ƒ AtABC1»ik HsCABC1|efžÙ
hj

U4)。
2.3 45犖狋犗犛犃1,<=~@A
DEqRTPCRÏá犖狋犗犛犃1,Ÿqˆ‰ÄÅ
ŒÆK|ÍÎLM

°ßij

U5),犖狋犗犛犃1,
ŸUVqˆ‰³ÒKÍÎ

’ÍÎÃ6­K|
137.83Û),qðK¡kÃÍÎ(’ÍÎÃ6­K
|10.75Û),%q­、ï»ìZK|ÍÎÃlñ,m
nÏáę

ÍÐ
,犖狋犗犛犃1,Ÿ ¡ŒÆ:ÑÍ
Î|:Ÿ

2.4 _`89€‚45犖狋犗犛犃1,<=@A
¿ E qRTPCR Ï á 犖狋犗犛犃1 , Ÿ q
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ab+%%&
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