全 文 :书!"#:10004025(2016)08149908 犱狅犻:10.7606/j.issn.10004025.2016.08.1499
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J K:#klmnoAbc1(p(activityofbc1complex)q#A6rstuvwAxyKz{fV|} E 。 ~34.AtOSA1lm,
F,DEPCR/1 xyAbc1(p,犖狋犗犛犃1,犖狋犗犛犃1F.犃狋犗犛犃1, ¡72.89%|¢£H。犖狋犗犛犃1 ,¤¥¦§¨6©ª2283bp,«¬760,
,¡®¯|ABC1°±²、¢no°±²、³´µ¶·¸¹ º»¼½¾°±² 。 ¿EhÀÁ¶ÃPCR/,犖狋犗犛犃1,qÄÅÆÇtxy、ÈxyÉÊ ËÌ|ÍÎYÏÍÐ :犖狋犗犛犃1,|ÍÎ ¡Æ:ÑH,UVq³ÒKÍÎ;犖狋犗犛犃1,q H2O2 »NaClÊ ËÓÍÎÃÔÕ , ÖqÊËÓ6hÎ×0Ø,YÙªÊËÚ|1.95»2.69Û。ÜÝÞ¶·°ßÍÐ,NtOSA1 lm¶·qÝÞ³´µÔ , Fàá°ß¢£ 。 34ÍÐ ,犖狋犗犛犃1,âFãäxy»Èxy|uv。 ;LM : ; xyAbc1,;,;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oxidativestressrelatedAbc1likeprotein).Theopenreadingframe(ORF)ofthe
犖狋犗犛犃1geneis2283bpwhichencodedadeducedproteinincluding760aminoacidresidues.Theprotein
sequenceofNtOSA1possessesaconservedABC1domain,onekinasedomain,onechloroplastlocalization
signalpeptideandtwotransmembranespans.Therelativeexpressionof犖狋犗犛犃1wasdeterminedbyquan
titativerealtimePCR(qRTPCR).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;oxidativestressrelatedabc1likeprotein;geneclone;expressioncharacter
ization;subcelularlocalization
#qA6rsJKÄåæçèéêëì íbxy , îïð 、 ñð 、 òó 、 f-ô»ÈõÉ 。 ö ÷íbxyèø#ùúûüýþ
,
ÿ£µ!
"#0Ã|H , W%&Axy 。 Ã|
HF(B
、
lmB
、DNAÇt)*A0YZ rA+v , W%,-7µ./0123 [1]。 Aµ!4q5ìâFxyuv|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 , Abc1likelm6Ao QB-|fVCDZ,â FEFG|HZIJ»äJ [38]。 KL 犆犗犙86M¢NO¶|Abc1,,)«¬lm ¡kYZPQH
,
RSTÝÞUVbmRNAW
X|YZÇt[\?@µEFGKbc1]Bµ|
H [910]。 »KL , #| Abc1likelm ¡^5
_|8
[1113]。
.
(犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪),
K¡17,¡®¯| ABC1°±²。M
¢N34|6犃犅犆1犃狋,,~,q.ë
ÆK-¯ÍÎ
,
)«¬|?@µlmFKL
ABC1lm¡45%|`H,a ¡KLABC1l
m`|8
[14]。
bc¢ Abc1lm,AtOSA1
(oxidativestressrelatedAbc1likeprotein)d*e
»KLABC1 ¡45%|`H,f6~lm¶·
;ÝÞ³´µK
,
8ghhiij
,AtOSA1lm
Äk]KL犪犫犮1lmµEFG|H。n¶Ã RTPCR ° ß i j,犃狋犗犛犃1 , | Í Î o CdCl2、H2O2 »ïp©Â|qÿ[11]。YangÉ|3 4ij ,AtACDO1lm(ABC1likekinaserelated tochlorophyldegradationandoxidativestress)¶ ·;³´µK , qÂxyro»³´Vst JKru}E [13]。 .ABC1K1»ABC1K3l me¶·;³´µBµv , CDBµvÑwxy, (ûüJ , q#íbvz»³´µ{|}-K
ru{fV}E
[1516]。AtSIA1(saltinducedABC1
kinase1)FAtOSA1 ¡46%|`H,Å_¶·
;ÝÞ³´µK
,
ͯYÏ~Ð
,AtSIA1» AtO
SA1Ru³´µKFe|Y,CDH |&A»xy|vz [17]。 c , É [18] ã¢
(犜狉犻狋犻犮狌犿犪犲狊狋犻狏狌犿)kABC1, 犜犪犃犅犆1犔,r~,RN、ïÈ、ñðx yÇtABAÊËqÿ。ïÉ[19]34r, (犗狉狔狕犪狊犪狋犻狏犪)犃犅犆1,UVq³ÒKÍÎ, ao5ìwAxyV H2O2、ABA、ñðÇ
tïÈÉ|CD
。
(犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿)6YZA%», IJ34|#
,
6סØ|+3A ¢ 。 äíxyvzJK,| t8|j¢634|g , fNÚ; Abc1lm(p|834ù&。34 WKã犗犛犃1|ÅC,犖狋犗犛犃1, ): 、 ÆÍÎ:H 、 ÜÝÞ¶·tëìw Axyuvã34
,
ªd tÏ犖狋犗犛犃1,
qxy»ÈxyvzK|A%8¡ ¢ãfV⣠。 1 »¤¥ 1.1 45UV ¢¦#ª
‘NC89’,hij§¨
ì#
。
1.2 W X
1.2.1 45犖狋犗犛犃1,<>? E. AtO
SA1lm|,
qSGN ©ª(http://sol
genomics.wur.nl/)(ǻ
SGNmRNA ,Jªlm_¬
)。
®|¼¯EST,SGNE1015449
»SGNE1113121,°±c²³ NtOSA1F1(5′ TTCCCTTGCATGCACATACA3′)» NtOSA1 R1(5′TGATCTCCCAGGGTAACCAT3′);¡ 犛犿犪I»犛犪犾Io´·g|!²³ NtOSA1F2
(5′CCCGGGATGGCGAGTATTTCAGCTAC3′)
» NtOSA1R2(5′GTCGACAGCTGTTCCTGT
0051 ! " # % & 36µ GATCA3′)。¡¶·¸³Ò¹RNA,Ç+ º|cDNAM¢Gª»,NtOSA1F1»Nt OSA1R1ª³,Eï=
PCR&ÁÂ100Û,ÇÁÂÓ| DNA ª», NtOSA1F2» NtOSA1R2 ª³Mx¾
PCR¿À。Mx¾PCR&ÃÄÅÓÆÇ1 pEASYBluntȵ("Éd-A¼½),0
ÊËÌ
(犈狊犮犺犲狉犮犺犻犪犮狅犾犻)DH5α。Ç0ÊËÌÍ
ª»
,NtOSA1F2» NtOSA1R2ª³, ÌÎPCRÏá,^HÐÑ0,+/ÒÓ ¡Ô¼½á 。 1.2.2 45犖狋犗犛犃1,<=YZ[\]HI D EDNAMANv6ÕÖYÏá°ß,׶犖狋犗犛犃1 ,|¤¥¦§¨ (ORF)Øÿv|,
。 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|,
- 、 YZà 、 ÉHgÉËHB ; DEq?ÕÖ TargetP1.1ServeràáNtOSA1lm|ÜÝÞ¶ · 。 ClustalW » MEGA6.0ÕÖ°B32 Abc1lm±}ÙÚ Û , øEBootstraptestÏi(f]ت1000) Û|åÜH 。 1.2.3 45犖狋犗犛犃1,<^_`aG@AbFb cd ÝA¯ìZÞìßà,á;Ââã äjãä 。 ãä¯Öª : ð©25℃,å©60%~ 70%,16hÂâ/8hæç。 °± 犖狋犗犛犃1,qRTPCR ³ NtOSA1
rtF(5′TGTTCTGGTGCCTACTGGTGAC3′)»
NtOSA1rtR (5′AAGTTCTGCTTGTGCCATTTC
3′),Ç犖狋犚犔2(GenBankè¹Z14081)ª!
â ,
,
³ ª NtRL2 rtF (5′GTAAGG GAGCGGGTTCAGTCT3′)» NtRL2rtR(5′ AACGGAGCACCCCTACCTG3′)。éâ Trizol (Thermo¼½)¦êëìÐíî},YÙ¡¶ÝA ¯ 、 ï 、 ³Ò 、 ð»ìZ|¹RNA,ñ+ ºcDNAM¢G。éRealMasterMix(òA +/¡Ô¼½ ) ¦êëìÐí , qCFX96RealTime System(BioRad¼½)qRTPCR¿À,J ª :95℃àmH3min,95℃mH10s,60℃óô 30s,40õa,65℃ö÷5s,*ÓÇøù0.5℃ Õ195℃,61õa。¿E2-ΔΔCt¥±ú。 1.2.4 45=89efB犖狋犗犛犃1,<=@Ab cd ââLee[20]É|¤¥,Þì;1/2MS ûµãä, [21] Ô , A62ü,ý¶6þÿ!,0
¢ £ | · ¸ Y Ù " 10 mmol/L H2O2 » 200mmol/LNaCl|1/2MS#µãä,K,qÊ Ë|0、3、6»12h¶·¸_,#%&Óá;
-80℃(4)E。
ââ HermandÉ[22]|¤¥,Þì;¶
#*+K
,
E1/2Hoagland[23],ä#ãä,A
630dÓ,ý¶6þÿ!,0
¢£|·¸ÆŶ
#*+"YÙ0、30、60»100μmol/LCdCl2
|-.1/2Hoagland,ä#K,qÊË|48h¶
·¸³Ò
,
#%&Óá;-80℃(4)E。
¡¶ÇÔÊË|¹ RNA,ñ+
cDNAM¢G,ÇNtOSA1rtF»NtOSA1rtR,Nt
RL2rtF»NtRL2rtRª³,éâRealMaster Mix(òA+/¡Ô¼½)¦êëìÐí,q CFX96RealTimeSystem(BioRad¼½)qRT PCR,YÏ犖狋犗犛犃1,|ÍÎÃ。¿ÀJ ª :95℃àmH3min,95℃mH10s,60℃óô 30s,40õa,65℃ö÷5s,*ÓÇøù0.5℃ Õ195℃,61õa。¿E2-ΔΔCt¥±ú, DPSv7.05i/HYÏ。 1.2.5 gh@Aij=kl、45mYnjo7B pqrstuv úABµÈµpTEXGFP ¡012V3ý45 、CaMV35S68Z、5 ·g»犌犉犘,«¬7。 E犛犿犪I»犛犪犾I(Thermo¼½)ÅÀ8o´á 9×| 犖狋犗犛犃1,Ò:»pTEXGFPȵ, ;<|犖狋犗犛犃1,«¬7="pTEXGFP ȵ|5·g , ø犖狋犗犛犃1»犌犉犘 ,>B ÍÎ , ÆÇ&0ÊËÌDH5α。Ç0ÊË
Ìͪ»
,NtOSA1F2» NtOSA1R2ª³
, ÌÎPCRÏá,^HÐÑ0,+ /ÒÓ¡Ô¼½á 。 ÇA (犖犻犮狅狋犻犪狀犪犫犲狀狋犺犪犿犻犪狀犪)ªhi , ââYooÉ[24]|¤¥T)úABµ,o t¯ÖGªæçot4h。±}!|>BÍÎÈ µpTEXNtOSA1GFPPEG?ÿ¥ AúABµK , jðÌ (20~25)℃æçqÿ24 ~48h。DEnÂ@ABCDÁÂiEF(GHI ½¼½ ,¯ ¹LSM710),JKNtOSA1GFP>Bl m|ÜÝÞ¶·LM 。 °¶â :GFPlm|nr 10518N OPQ,É:xy,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|cDNAt)Øÿ|,
Chloroplasttargetingpresequencesarepresentedinbold;UnderlinerepresentstheABC1conserveddomain;Putative transmembranespansarepresentedinbolditalic;Stopcodon Fig.1 cDNAanddeducedaminoacidsequencesof犖狋犗犛犃1 VWU¨ûÍABC1°±²;XWU¨ûÍno°±²; mU¨ûͽ¾°±² U2 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 á°ßij , º|Ò:F.犃狋犗犛犃1 ,_¬
¡ï©|¢£H (72.89%), [Zª 犖狋犗犛犃1(GenBank è¹ KU565475)。 犖狋犗犛犃1,|¤¥¦§¨6©ª2283bp,«¬ 760,
。 ªãtNtOSA1lm|A%Ht[q8 , DEq?ÕÖYÙNtOSA1lm|ËHB »°±²YÏ»àá , °ßij :NtOSA1lm YZê86.07kD,Ë\ÉHgª8.92;q,
|277~397 ¡ ABC1°±²,q364~502 ¡no°±² ,NtOSA1lmC] ¡2½¾ °± , YÙ·;,
|703~721»726~ 743(U1、2)。.、、KL»^Þ_4 ì|®¯Abc1lmFNtOSA1lm
,
r NtOSA1 ¡®¯| ABC1=>°±²
(
U3),ÍÐNtOSA1ô;Abc1(p。
2051 ! " # % & 36µ AtOSA1(At5g64940)»AtABC1(At4g01660)` ).;OsABC1(Os02g0575500)` );SynABC1(P73627)` )^Þ_; ScCOQ8(EWH18611.1)` )KL U3 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]1000|ḩ
U4 NtOSA1F)bAbc1lm|ÙÚÛ
ValuesatnodesshowtheconfidencelevelofBootstrapreplication1000
Fig.4 PhylogenetictreeofthealignmentofNtOSA1deducedaminoacidsequencewithotherAbc1proteins
30518N OPQ,É:xy,犖狋犗犛犃1|ÍÎtÜÝÞ¶·YÏ
2.2 45犖狋犗犛犃1,
、
.
、
、
cd
、^
Þ_É12
ì|32Abc1lm5f,±} Û , °ß ( U4)ij,NtOSA1lmF. AtOSA1lm|efÙ×g,¢£Hª76.04%; Fcd ADB13188lm|¢£Hª74.15%;F Os02g0575500|¢£Hª73.45%。ÍÐ,Nt OSA1F.、cd»|efÙhg, %FM¢NO¶| Abc1lm、KLScCOQ8、Ç t. AtABC1»ik HsCABC1|efÙ hj ( U4)。 2.3 45犖狋犗犛犃1,<=~@A DEqRTPCRÏá犖狋犗犛犃1,qÄÅ ÆK|ÍÎLM 。 °ßij ( U5),犖狋犗犛犃1, UVq³ÒKÍÎ ( ÍÎÃ6K| 137.83Û),qðK¡kÃÍÎ(ÍÎÃ6K |10.75Û),%q、ï»ìZK|ÍÎÃlñ,m nÏáÄ 。 ÍÐ ,犖狋犗犛犃1, ¡Æ:ÑÍ Î|: 。 2.4 _`8945犖狋犗犛犃1,<=@A ¿ E qRTPCR Ï á 犖狋犗犛犃1 , q 10mmol/LH2O2 »200mmol/LNaClÊËÓÄÅ Àog|·¸K|ÍÎÃm , °ßij ( U 6,Ⅰ),犖狋犗犛犃1,q H2O2 » NaClÊËÚNÍ ÎÃÔÕ , ÖqÊËÓ6hÎ×0Ø,YÙªÊË Ú|1.95»2.69Û。q H2O2 ÊËÓ12h,犖狋犗 犛犃1,ÍÎÃopYST,ªÊËÚ|0.6Û, qNaClÊËÓ12h,犖狋犗犛犃1,ÍÎÃk] ÊËÚq 。 EÄÅBÃr©| CdCl2 ÊËã¸, qRTPCR°ßij(U6,Ⅱ),犖狋犗犛犃1,|Í ÎÃqÄÅBÃr©CdCl2 ÊËÌs¡i/Ht Ñ , çj~,åÄâF#uµ!CdCl2 |vz 。 2.5 45犖狋犗犛犃1=CDEFG TargetP1.1ServerÕÖàá°ßij,NtO SA1lmN]¡¢46,
|³´µ¶· ¸¹º ( U1),àá~lm¶·;ÝÞ³´µK,å ÜHÉvª1,ª×ïÉv。EnÂ@ABCDÁ ÂiEFÏáNtOSA1lm|ÜÝÞ¶·,°ßi j ,NtOSA1¶·;ÝÞ³´µK(U7),FÕÖà á°ßwB 。 U5 犖狋犗犛犃1,qÄÅÆK|ÍÎ Fig.5 Expressionprofilesof犖狋犗犛犃1genein differenttissuesoftobacco ÄÅ0xyLÍjÅ¢ÊËÄÅÀog ( r© ) |i/HtÑ (犘<0.01,LSDÏi) U6 犖狋犗犛犃1, H2O2,NaCl(Ⅰ)»CdCl2(Ⅱ)|uv Differentcapitallettersrefertosignificantdifferencesamongdifferenttimepoints(cencentration)ofthesametreatment (犘<0.01,LSDtest) Fig.6 Expressionanalysisof犖狋犗犛犃1geneinresponsetoH2O2,NaCl(Ⅰ)andCdCl2(Ⅱ)treatment 4051 ! " # % & 36µ
a.³´µ)rÁÂ;b.NtOSA1GFPlmÁÂ;c.BU
U7 NtOSA1GFPlmqúABµK|ÁÂiEJK
a.Thefluorescenceofchlorophyl;b.ThefluorescenceofNtOSA1GFP;c.Mergedimage
Fig.7 FluorescencemicroscopeobservationofNtOSA1GFPproteinsintobaccomesophylprotoplasts
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