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Overexpression of NtMTP1 Gene from Nicotiana tabacum Improved Zinc Tolerance of Tobacco

NtMTP1基因过表达对增强烟草Zn胁迫耐受性的研究



全 文 :书!"#$%&
,2016,36(7):1308-1314
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ZnSO4 Ž,«¬犖狋犕犜犘1,„ž‘’|}~Zn­®€H¯°。±²‘³:犖狋犕犜犘1,„R}~K´µ‡
ˆ:¶H‘’

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;犖狋犕犜犘1,„‘’¨Zn2+¹›,R400μmol/LZnSO4 Žº,‘’z
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犗狏犲狉犲狓狆狉犲狊狊犻狅狀狅犳犖狋犕犜犘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
犐犿狆狉狅狏犲犱犣犻狀犮犜狅犾犲狉犪狀犮犲狅犳犜狅犫犪犮犮狅
RANQiao1,WUChanjuan1,2,GAOYongfeng1,TANGYunlai1,2,
HUANGCaizhi1,LIUJikai1,2
(1SchoolofLifeScienceandEngineering,SouthwestUniversityofScienceandTechnology,Mianyang,Sichuan621010,China;
2FundamentalScienceonNuclearWastesandEnvironmentalSafetyLaboratory,SouthwestUniversityofScienceandTechnolo
gy,Mianyang,Sichuan621010,China)
犃犫狊狋狉犪犮狋:Inthisstudy,therelativeexpressionof犖狋犕犜犘1wasdeterminedbyquantitativerealtimePCR
(qRTPCR).Theresultsshowedthat犖狋犕犜犘1genewashighlyexpressedinflowersandleaves.After
treatmentof400μmol/LZnSO4for48h,theexpressionof犖狋犕犜犘1increasedtothemaximum,which
was3.81foldsofthecontrol.PlantexpressionvectorpBI12135S∶∶MTP1wasconstructed,andthere
combinantplasmidwasintroducedinto犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿L.cvNC89bymeansof犃犵狉狅犫犪犮狋犲狉犻狌犿狋狌犿犲
犳犪犮犻犲狀狊mediatedtransformation.TheqRTPCRrevealedadistinctincreaseofendogenous犖狋犕犜犘1ex
pressionlevelintheoverexpressingtransgeniclines,whichwere10.42,7.61and11.84foldsofthatof
wildplants,respectively.Seedsofwildtypeandtransgenicplantsweretreatedwithdifferentconcentra
tionsofZnSO4.Theresultsshowedthatoverexpressionof犖狋犕犜犘1conferredenhancedzinctolerance.
Theseresultsprovideanimportantbasistoiluminatethefunctionof犖狋犕犜犘1geneinZn2+transportin
tobacco.
犓犲狔狑狅狉犱狊:犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿;metaltoleranceprotein1;zinc;tolerance
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MnCDF[67]。
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altoleranceprotein,MTP),ÌD ] E 8 Zn2+、
Fe2+、Co2+ &-/Z¡Ê,EY½ 7 áF
‡
[89]。
R±G!

H1F‡ MTP‚ƒR6æI
Ⅰ、Ⅱ、Ⅴ、ⅥKÿÛáOÿ‡PQÝBjR

STU½F¡Ê:
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VÌ]½ÜÝBÉ Zn2+ WXYZ
[
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Ê

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µ
,犃狋犕犜犘1,„UVR_`¸aK‘’,Rb
cZndYef Δ狕狉犮1犮狅狋1K¶C‘’犃狋犕犜犘1
ógef bc‘µ0|žzZn2+€H[13]。
Nh

Þß#$犕犜犘1,„iTjk。Rl+m
n#$opqr
(犕犲犱犻犮犪犵狅狋狉狌狀犮犪狋狌犾犪)Kst
Zn2+º,犕狋犕犜犘1,„RuK‘’z$%,R
vK‘’z"¼

R¸Kw<³Äf¢
[14]。
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-Ox#$y.z{
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(犜犺犾犪狊狆犻犮犪犲狉狌犾犲狊犮犲狀狊)KBY/0•犕犜犘1,„,
34êµ犕犜犘1,„R2ßOx#$K€RÞ
၂

ƒ„ MTP1‚ƒR#$o-…†Á‡õ
žJKoV]E
[15]。

(犗狉狔狕犪狊犪狋犻狏犪)犗狊
犕犜犘1,„Ò‰Šêµ犕犜犘1,„,t,„‹8
WˆH5ŒŽ !,UVR=¸ÁvK¼
Œ‘’

S‘’z‘¨o-÷ Zn2+、Cd2+、
Cu2+ÁFe2+¹›[1617]。
}~
(犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿)ÒÛßoV’“
]$

”•jk–—˜¢

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

:ÀÒ|o-€H34i=½34?
>Ÿp
。Shingu&W}~Kjk¨ 犖狋
犕犜犘1,„,t,„¶C‘’ÌD ¡bcZn
dYef Δ狕狉犮1|Zn›H,ªêµ NtMTP1
‚ƒy‹8bc¢£æ!

¤±²ƒ„ Nt
MTP1‚ƒÌDZn2+ ¥/R¢£K¦-§õ
H
[18]。
ïÒ

¤±²¨©„• 犖狋犕犜犘1,„R
bcKËÌ

SR}~ ÉA$%Ë̪*«
¬

T34“Û­Y–•犖狋犕犜犘1,„‘’m
n

†vG®•犖狋犕犜犘1,„ž‘’Ÿ ªS¡
¢¨£A¤}~#©K

[žº¯qrY–•犖狋
犕犜犘1ž‘’¡,„#©|Zn­®€Ì,
½34}~|o-Zn2+¡ÊÁY°±²ÍÎ
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1 ´µÁ¶œ
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η#$´µ½}~
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#

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狉犻狌犿狋狌犿犲犳犪犮犻犲狀狊)EHA105 Á # $ ‘ ’ Ÿ  
pBI121»¼Tqrs½€。
1.2 T U
1.2.1 V犚犖犃WXY犮犇犖犃Z[ ¾¾Trizol·
¿
(InvitrogenÀÁ)³ÃÍÄ}~Å RNA,E
PrimeScriptTM RTreagentKitwithgDNAEraser
Æ¡â·¿Ç
(TakaraÀÁ)“”ơ⧨cD
NA,È8-20℃Éʽ€ËE。
1.2.2 ;<犖狋犕犜犘1,4B\]67^_ DE
qRTPCR¶œY–}~ 犖狋犕犜犘1,„(GenBank
ÌâŒAB20124)R…†‡ˆ[ÍK‘’WX,
‰}~ 犖狋犚犔2(ribosomicproteinL2,GenBankÌ
âŒZ14081)]½ÉÎ,„,ÏÐø$ NtRL2rtF
(5′GTAAGGGAGCGGGTTCAGTCT3′) Á
NtRL2rtR(5′AACGGAGCACCCCTACCTG3′);
NtMTP1rtF(5′TCGAGGTTGTTGGTGGTATTAAA
3′)Á NtMTP1rtR(5′CGATTCTGAAAAACCCAT
AGGAC3′)。uE RealMasterMix(SYBR Green
90317¯            O P,&:犖狋犕犜犘1,„ž‘’|ÆÇ}~Zn­®€H34
I)·¿Ç({uA¢+/("Ñ)<ÒÀÁ),RBio
RadÀÁ CFX96RealTimeSystem “” qRT
PCR2Æ。JÓ½:95℃ÔfH3min,95℃fH
10s,60℃ÕÖ30s,40á×j,65℃ØÙ5s,*º
‰ÚÛ0.5℃"Ü95℃,61á×j。ÝÐuE
2-ΔΔCtœÐÞ。
1.2.3 ;<犖狋犕犜犘1,4`犣狀=>aBbc6
7 E1/2HoaglandÖ×¢[19]ß×£A¤}~_
à30dº,¦Ä6áâã,0FÛä_à¡åY
Àÿ0、50、100、200Á400μmol/LZnSO4 ‰æ
1/2HoaglandÖ×¢K,Ž48hºÄ_àòç,
¢èéêºÈ8-80℃ë€ËE。ÍÄ_àòç
ÅRNA,’ơ⧨HÛìcDNA,DEqRT
PCRY–犖狋犕犜犘1,„Â|‘’z。
1.2.4 ;<犖狋犕犜犘1,4de567fgBhi
 uÐ 犖狋犕犜犘1,„Óí,DEPrimerv5.0î
ï

ÏÐðn PCR ø$ NtMTP1F1(5′ATTT
GATGATTTGGGTCGG3′)Á NtMTP1R1(5′
TGAAACAGATACTGGA ACCG3′),‰ŠK<
犡犫犪IÁ犛犪犮Iñàí‹p:¶Hø$ NtMTP1
F2 (5′TCTAGAATGGAGACGCAGAACCTGG
3′)Á NtMTP1R2(5′GAGCTCTTACTCTCTTTC
TATTTGAATGG3′)。‰£A¤}~cDNA½m
ò“”PCR2Æ,§¨犖狋犕犜犘1,„aó,ÆZJ
Ó½94℃ fH15s,55℃ÕÖ15s,72℃ØÙ10
s,30á×j,72℃tYØÙ5min。PCRô$F
狆犈犃犛犢BluntŸ ("Ñmn-A$ÀÁ)ôõ,¡
¢0¹˜™DH5αºÍÄo‡Bö,÷ø0,„+
/ùú<ÒÀÁ¬Ó

¬Óûüº

E 犡犫犪IÁ
犛犪犮I(ThermoÀÁ)†vñàí¬Óûü 犖狋
犕犜犘1,„aóÁ#$‘’Ÿ pBI121, 犖狋
犕犜犘1,„ýå¨CaMV35Sþ8Zºÿ,gH
jk÷ø0,„+/ùú<ÒÀÁ¬Ó

o‡B
öEê!œ
[20]
¡å¨—˜™ EHA105Y"Ü
ÝK

1.2.5 ;A¤}~

Eÿ<pBI12135S∶∶MTP1o‡Bö
—˜™EHA105$£A¤}~¸a,”º¸
aÈ8 MS[22]+3%%&+0.8%ú+200μmol/
L()*+,Eß×,!,8(24±1)℃、-ƒ.ï
ß×2dº¡/¨ÿ50mg/L0Q123Ù(ka
namycinsulfate,Kan)Á 500 mg/L 45678
(ceftriaxonesodium,Cef) MS+3.0%%&+0.
8%ú+3.0mg/L6BA+0.2mg/LNAAY¢
ß×,!

Ú7~15d9:1;ß×,,8!<.ï
¥¦ß×1~2á=。>Y¢ß×,!?@‡ˆ
601.5cmAB‰Cv,íÄFC¡/Üÿ50
mg/LKan1/2MS+3.0%%&+0.5%ú
Auß×,!“”Auß×Á¥¦

DÄçè
Kan›H#©[23]。Eu+꒺¡/ÜFK,RG

(24±1)℃、x¾16h/d.ïß×。
1.2.6 ;<犖狋犕犜犘1,4567dnBopqr
8犣狀=>?@ABst ξ_`a&Hy¡,
„gH#©¶œ
[24],
|‰3ÙIQ¡/àⅡ(ne
omycinphosphotransferaseⅡ,犖犘犜Ⅱ),„aó
“”2Æ

Ӽ

ÍÄ¥¦¨¡,„gH#©
RNA,Æ¡âA=cDNA,DEJyzPCR«¬£
A¤Á¡,„#©犖狋犕犜犘1,„‘’z,W¥
¦§¨RRNAWXž‘’¡,„}~#©,“
Û­ß륦§¨ 犖狋犕犜犘1,„ž‘’K* 


¦Ä3á犖狋犕犜犘1,„ž‘’K*Z©+F
£A¤}~©+“”Zn­®Ž。4á©+
ßZE15% NaClOLõº,YÀMß8ÿ<‹Œ
½0、200、500、1000Á2000μmol/LZnSO4 1/2
MSN ß×,!OPß×,EßZQêº,RS_
àA6êëTU

RH45{¬y_àu6。
1.2.7 uevwxYyzvTU ‰!qr»“
”3;VWA$%o¡,ÚáòTÿ3©Š‰!


ÏÈ3;/{o¡“”qRTPCR。Zn­®
€HY–K

YÀ¬yÚዌŽ10©‰!
_àu6“”,ÐY–

DEDPSv7.05îï
“”X„Ù·r,ÐY–ÁLSDÞoÃY«r
ŽNZ¶JŒ

uEExcel2013îï][。
2 ±²FY–
2.1 ;<犖狋犕犜犘1,4B\]67^_
YÀÍÄ£A¤}~u

v
、¸ 、
·ÁßZÅ
RNA,ơ⧨HÛìcDNA,DEqRTPCR«
¬犖狋犕犜犘1,„R}~…†‡ˆK‘’TU。
±²

[1)Ą,犖狋犕犜犘1,„UVR}~·F
¸K‘’

YÀ½uK16.81Á7.26¿,Ru、v
ÁßZK‘’z\%

¤±²‘³

}~ 犖狋
犕犜犘1,„;<‡ˆ:¶‘’:]。
2.2 犣狀2+{bc犖狋犕犜犘1,4`;E…†Bz‹ŒZnSO4 Ž£A¤}~48h,
DEqRTPCR| 犖狋犕犜犘1,„‘’z“”Y
–

±²÷[2^„,FE0μmol/LZnSO4 Ž
0131 ! " # $ % &                   36_
|¾‡}~ÂÃ
,犖狋犕犜犘1,„‘’zR50
μmol/LZnSO4 Ž}~K`³Äf¢,>‹
Œ’¨100μmol/Lº,S‘’zab"¼,RE
400μmol/LZnSO4 Ž}~K’¨»¼,½|¾
‡3.81¿。¤ÝБ³,}~犖狋犕犜犘1,„
‘’¨Zn2+¹›。
2.3 ;<犖狋犕犜犘1,4567dnB|}
gE—˜™š›¸#œ¡¢£A¤}~

ED
[1 }~犖狋犕犜犘1,„R…†‡ˆK‘’
Fig.1 Expressionprofileof犖狋犕犜犘1gene
indifferenttissuesoftobacco
…†0cdcä‘eŽNÄÅHZ¶
(犘<0.01,LSD«r)
[2 }~犖狋犕犜犘1,„|ZnSO4 °Z
Differentlettersrefertosignificantdifferencesamong
thetreatments(犘<0.01,LSDtest)
Fig.2 Expressionanalysisof犖狋犕犜犘1genein
responsetoZnSO4treatment
§16©‡ß#©,‰ÍÄD§‡ß#©DNA
½mò
,NPTIIF/R½ø$“”PCRrfgH#
©

Ô¯2Æ6ŒZ½857bp。±²‘³([3),£
A¤}~«Ì2Æ0.K

›H#©ÌD2Æ0
Ô¯0F.K

E«¬012©›H#©,gHg
½75%。”º,DEJyzPCR|›H#©K犖狋
犕犜犘1,„‘’WX“”h­Hy,¥¦0犖狋
犕犜犘1,„‘’z»¼3©#©,YÀ½OE6、
OE9ÁOE10。DEqRTPCR|犖狋犕犜犘1,„
R£A¤Á¡,„#©K‘’TU“”yzY
–

±²Ä„

[4,A),犖狋犕犜犘1,„R3©¡,
„#©K‘’zÄż8£A¤#©

YÀ½£
A¤10.42、7.61Á11.84¿。|犖狋犕犜犘1,„
ž‘’#©“”‘¤RS

êµRû¸A6TU

¡,„#©F£A¤ÂÃ

ªw<‘µ0³ÄZ


[4,B)。
2.4 ;<犖狋犕犜犘1,4567dn8犣狀=>B
?@AL~
  ijKžzo-ó|#$uA6ôAk
7]E

„l

#$u6m¸]½Æn#$|o-
€HÛáoVop

£A¤Á3á 犖狋
犕犜犘1,„ž‘’K* ©+ßZq™º,YÀÈ
8ÿ<…†Bz‹ŒZnSO4 1/2MSß×,!A
6

RS_àA6TU

±²Ä„

[5),A6R
ÿ<0μmol/LZnSO4 1/2MSß×,!£A
¤Á¡,„#©w<³ÄZ¶

R200μmol/LZn
SO4 Ž,£A¤}~uA6‘µ0rYk7,
ž‘’#©u+ÂãA¤V60s3cm;R
500Á1000μmol/LZnSO4 Ž,t*£A¤Á
ž‘’#©A6u¨•ÛyJŒk7

ïž
‘’#©u+ª*ãA¤6

R2000μmol/L
ZnSO4 Ž,£A¤Á¡,„}~#©ui…Ì
û¸A6

uA6¨vok7

¼lVw

”•
ß×,KZnSO4 ‹Œ"¼,£A¤Á¡,„}~
M.DL2000;1~16.¡,„#©;WT1、WT2.£A¤#©
[3 ¡,„#©犖犜犘犜犐犐,„PCR«¬
M.DL2000;1~16.Transgenicplants;WT1andWT2.Wildtype
Fig.3 PCRanalysisfor犖犘犜犐犐geneintransgenicplants
11317¯            O P,&:犖狋犕犜犘1,„ž‘’|ÆÇ}~Zn­®€H34
_àA6u¨ÛyJŒk7

ïR200、500
Á1000μmol/LZnSO4 Ž,犖狋犕犜犘1,„ž
‘’#©_àubxuãA¤}~_à6

‘³犖狋犕犜犘1,„ž‘’ÆǕ}~|Zn­
®€H

A.¡,„#©犖狋犕犜犘1‘’qRTPCR«¬;B.犖狋犕犜犘1,„ž‘’#©‘¤;WT.£A¤#©;OE6、OE9、OE10.
¡,„}~©+

†
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