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Stress Tolerance of Transgenic Arabidopsis Expressing CkLEA4 Gene during Seed Germination Stage

CkLEA4基因拟南芥种子萌发期的抗逆性分析



全 文 :书!"#$%&
,2016,36(4):0648-0654
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|œ•–‚ƒRD—˜tužŸ l¡¢£

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:(1)D—˜°±NaCl、²³´µABAžŸ ,}犆犽犔犈犃4,|œ•–‚ƒRl¡¢£¶·@J€,¸ ¹
NaCl、²³´µABA°±º»,¼Œ™¡¢£¶½¾,¿J€l¡¢£ ½À±¶·@3”œ•–Œ™,„ÁD
200mmol/LNaClc400mmol/L²³´žŸ ,œ•–Œ™RÂÃO£¶ÄÅ·@J€。(2)D¾°± ABA
žŸ 
,犆犽犔犈犃4œ•–#ŒRÂlÃO£Æ·@J€。HI•Ç,wxyz{犆犽犔犈犃4,|ȷɁ‚ƒ
R¡¢ÊËÌs
、ABAµÍÎtulÏpÐ。
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wxyz{

ÑÒÓkÔÕÖ×,|

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},|‚ƒ
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:Q785;Q786   !KLMN:A
犛狋狉犲狊狊犜狅犾犲狉犪狀犮犲狅犳犜狉犪狀狊犵犲狀犻犮犃狉犪犫犻犱狅狆狊犻狊犈狓狆狉犲狊狊犻狀犵犆犽犔犈犃4犌犲狀犲
犱狌狉犻狀犵犛犲犲犱犌犲狉犿犻狀犪狋犻狅狀犛狋犪犵犲
YUXiumin,ZHANGYanna,YUEWenran,YANGQi,WANYongqing,LIGuojing,WANGRuigang
(ColegeofLifeSciences,InnerMongoliaAgriculturalUniversity,Hohhot010018,China)
犃犫狊狋狉犪犮狋:Basedonourpreviousstudy,犆犽犔犈犃4,a犆犪狉犪犵犪狀犪犽狅狉狊犺犻狀狊犽犻犻LEAgeneinducedbydehydration,
saltandABAtreatments,wastransferredinto犃狉犪犫犻犱狅狆狊犻狊.ThetransgeneexpressionofeightT3trans
genicplantswasdetectedbyquantitativerealtimePCRandthreeindependentoverexpressionlineswith
differenttransgeneexpressionlevelwereselectedforfurtheranalysis.Forthegerminationassays,犆犽犔犈犃4
overexpressionlinesgerminatedmorequicklythanthatofwildtypeonmediumcontainingdifferentcon
centrationsofNaCl,mannitolorABA.Thegerminationrateofwildtypeand犆犽犔犈犃4overexpressionlines
werealdecreasedwiththeincreasedconcentrationsofNaCl,mannitolorABA,whilethegerminationrate
ofwildtypedecreasedmoredramaticalythanthatoftheoverexpressionlines.Thecotyledongreening
ratesof犆犽犔犈犃4overexpressionlineswerealsohigherthanthoseofwildtypeunder200mmol/LNaCl,
400mmol/LmannitolorlowconcentrationsofABA.Altogether,ourresultssuggestedthat犆犽犔犈犃4im
proved犃狉犪犫犻犱狅狆狊犻狊tolerancetosalt,ABAandosmoticstressduringseedgerminationstage.
犓犲狔狑狅狉犱狊:犆犪狉犪犵犪狀犪犽狅狉狊犺犻狀狊犽犻犻;lateembryogenesisabundant(LEA)proteins;abioticstress;transgenic
犃狉犪犫犻犱狅狆狊犻狊
  ÑÒ¢ÙÓkÔÕÖ×(lateembryogenesisa bundantproteins,LEAÖ×)|DÑÒ¢ÙÓk_
‹ÚÛÜÝÞ

ßàáD@·â#$lR


Rãäqrœ?åæçèéêëìípqrîï
lU†

ðDureâ[1]ñòDÑÒ¢ÙóklôõR
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D*+
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çèëì.CâJ$_QRl?Ð

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[89]。
wxyz{
(犆犪狉犪犵犪狀犪犽狅狉狊犺犻狀狊犽犻犻)A@I+
yz{A#$

%&JKlJL¨©ÐcßMÐ

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˜‡wxyz{
lPÂQR

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gghijkMwxyz{5htu icÐ
jklmno
(SSH)‘’Ýæ犆犽犔犈犃4,|p
q
[12],
Sœˆ‰Š‹PCRrstu¢¯犆犽犔犈犃4
pqr

stucABAžŸlv\[13],vuwPw
xyz{©ªtuxybc&z

gHID{,m
n|}É犆犽犔犈犃4,|œ•–~„}OJ€
‚ƒ

QÉ犆犽犔犈犃4,|œ•–},|‚ƒ
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,|D#$§¨©ªtul«¬

ƒ„÷…†~
HIwxyz{LEA,|l«¬µ…†w„‡#
$¨©ÙˆŠ,m

1 efc‰Š
1.1 OPQR
wxyz{
(犆犪狉犪犵犪狀犪犽狅狉狊犺犻狀狊犽犻犻Kom.)
R‹)./0ŒŽ

J€‚ƒ
(犃狉犪犫犻犱狅狆狊犻狊
狋犺犪犾犻犪狀犪L.)Columbia0JL€(Col0)ðgghi
çá

1.2 OPSTUVWX
e/‘$CkLEA4F1(5′GCgtcgacATG
CAGGGAGCAAAGAAAGC3′, ’,ƒ犛犪犾Ⅰ
C “ ” •

c CkLEA4R1(5′GCgagctcCTTA
ACTGTAAGTACCGCCAGTCC3′, ’,ƒ犛犪犮
ⅠC“”•),¤1–—˜wxyz{2c™cD
NAƒš›œº犆犽犔犈犃4,|ž2。…†Ÿ ¡
¢£DNA¤¥¦§¨(©1J$ª«)¤¥¬l™
Ë

­~®¯~p犈犃犛犢BluntSimple("°±²
-J$ª«
),
uph³óÈH´

µ.“C犛犪犾Ⅰc
犛犪犮Ⅰ(Thermoª«)C“ó,­~æðCaMV35S¶
·R¸·l#$•–~pCanGHA,C“h³
¹ºlOéH´}O^»-GV3101,¼½¾Ð®¯
„‡PCR¿Š。PCR‘$Žãµ3$upðnÀJ
>J$ª«Áã

1.3 678YZ4[\]^V_`
‹†ÂõŠoO镖~pCanGCkLEA4
}~J€‚ƒ

†25mg/LÃÄÅ8‘’¾Ð


Ƚ},|#ŒÆRNA,Ç}ÈãcDNA,
Sœg‡ˆ‰Š‹PCRtu},|Œ™l,|
•–‹

ÉÊSYBR犘狉犲犿犻狓犈狓犜犪狇¦§¨(Taka
raJ$ª«)ËÇÌÍU,…†LightCycler480g
‡ˆ‰Š‹PCRÎ(Rocheª«)„‡œº。Çy
™ƒ
:SYBR犘狉犲犿犻狓犈狓犜犪狇10μL,ÏÐlcD
NAš›5μL,n、 Ñ‘$¼0.4μL(10μmol/
L),Ò-r4.2μL。PCRÇy?pƒ:95℃ÓÔÐ
30s,95℃ÔÐ5s,60℃ÕÖ15s,72℃×Ø30s,40
”Ù N

) † ‘ $ ƒ CkLEA4F2(5′ACCCT
GCTGAGGCATTTGGA3′)c CkLEA4R2(5′
CCTATTGGGACGAATAGAGGCAC3′),.ڑ
$ƒAtEF1αF(5′AGAAGGGTGCCAAATGAT
GAG3′)c AtEF1αR(5′GGAGGGAGAGAGA
AAGTCACAGA3′)。,|•–‹¤2-ΔCTÛ,’
½3Œ•–rܛ·lŒ™„‡óݕ€gh。
1.4 abcdef;gghijkSœˆ‰Š‹PCRrstu¢
¯犆犽犔犈犃4 pqr、stuc ABA žŸlv
[13],
vuwPwxyz{©ªtuxybc&
z

D{,mn

tuÉ犆犽犔犈犃4,|œ•–},
|‚ƒRD NaCl、²³´(mannitol)c ABA
žŸ l¡¢€

J€cœ•–‚ƒRµ
75%Þ´Ò-10minó,†ü&0.05% Tween20
l100%Þ´Ò-10min,ߝRà5óQáD
ü&—˜°± NaCl(0、100、150c200mmol/L)、
ABA(0、1、2c3μmol/L)c mannitol(0、200、300
c400mmol/L)l1/2MS^T,n,┌™¼
9464k @AB,â:}犆犽犔犈犃4,|‚ƒR¡¢kl¨©ÐQ
55ãR,4℃˜…OžŸ3d,oܛ½“ä@
22℃,16h‰Ê/8håæxç ^T。¤Ñ1èé
êƒ¡¢ëì

íâ©lR¡¢£

¤RÂî
ï„ð¯Ãñƒëì

íRÂÃO£

tu‚ƒ
œ•–犆犽犔犈犃4ÌtužŸ R¡¢cò—
Jólôx

2 ­®PQ
2.1 狆犆犪狀犌犆犽犔犈犃4OPSTUV=WX
†e/‘$CkLEA4F1cCkLEA4R1œº
õÝó±ƒ510bp犆犽犔犈犃4ž2(ö1,A),÷ø
殯~p犈犃犛犢BluntSimpleup,uph
³ó†犛犪犾Ⅰc犛犪犮ⅠC“„÷øæ,ÐOl•–
~ pCanGHA 。| } ù l O é • – ~ 
pCanGCkLEA4†犛犪犾Ⅰc犛犪犮ⅠúC“¿Š,¬
û““¬l™Ë

ËǕ–~|}ã«

ö1,B)。
2.2 4,5]^VOhij
SœÂõŠoOé#$•–~pCanGCk
LEA4}~J€‚ƒ,ÃÄÅ8‘’ÝæT3 ü
¾Ð#Œ8Œ,Sœˆ‰Š‹PCRÌ},|#Œ„
‡Ž¿Š

„tuɬl,|犆犽犔犈犃4D},
|Œ™l•–‹

ö2),’½•–‹›·l3”
Œ™OE12、OE15cOE19„‡óÝgh。
2.3 4,5678kcdlm@no
ƒtuJ€‚ƒc犆犽犔犈犃4œ•–Œ™
DR¡¢ÊËÌstulÏpÐ

oJ€c3
”œ•–Œ™OE12、OE15cOE19,Dü&—
˜°±NaCll1/2MS^T,,4℃˜…OžŸ
3dó,⩘÷‡û퐝R¡¢£。­®(ö3,
A)Äý,D1/2MS^T,,œ•–Œ™cJ€
l¡¢£þÿ

!&ÇÄ2á

ÜDü&—˜°±
NaCll^T,,3”œ•–Œ™l¡¢£¶·@
J€
;¸
¹NaCl°±º»,¼Œ™¡¢¶pæi
c

¿J€¡¢£ ½À±L·@œ•–Œ™

¤"4©‡R¡¢£ƒë#,$NaCl°±M100
mmol/Lº»æ200mmol/L‡,J€¡¢£ 
½É63.6%,Ü3”œ•–Œ™Ü¶¡¢£% ½
É26.1%。í200mmol/LNaClžŸ ¡¢"
5©‡RÂÃO£,3”œ•–Œ™RÂÃO£ÄÅ
·@J€

ö3,B)。¤n­®ËÇ犆犽犔犈犃4œ•
–óȷɁ‚ƒR¡¢ÊËÌstulÏp
¬&

2.4 4,5678pqcdlm@no
o J€cœ•–Œ™Dü&—˜°±
A.犆犽犔犈犃4;B.pCanGCkLEA4;M.DL5000;
1、2.犆犽犔犈犃4cDNA;3.H´ÌÊ;
4.犛犪犾Ⅰc犛犪犮ⅠC“¿Š
ö1 pCanGCkLEA4•–~l|}P¿Š
A.犆犽犔犈犃4;B.pCanGCkLEA4;M.DL5000;
1,2.犆犽犔犈犃4cDNA;3.Vectorcontrol;
4.Digestedwith犛犪犾Ⅰand犛犪犮Ⅰ
Fig.1 TheconstructionandidentificationofpCanGCkLEA4
recombinantvector
ö2 œ•–犆犽犔犈犃4Œ™•–rÜl
g‡ˆ‰Š‹PCRtu
Fig.2 Theexpressionlevelof犆犽犔犈犃4intransgenic
犃狉犪犫犻犱狅狆狊犻sbyrealtimequantitativePCR
mannitoll1/2MS^T,,퐝R¡¢£。
D0c200mmol/LmannitolžŸ ,œ•–Œ™
cJ€¡¢£þÿ

Ü$ mannitol°±º»
300c400mmol/L‡,œ•–Œ™¡¢£¶ÇÄ
·@J€

ö4,A)。¸¹ mannitol°±º»,¼
Œ™¡¢£¶ ½

¿J€¡¢£ ½À±L·
@œ•–Œ™

¤"3©‡R¡¢£ƒë#,$
mannitol°±M200mmol/Lº»æ400mmol/L
‡

J€¡¢£ ½É66.1%,Ü3”œ•–Œ
™Ü¶¡¢£% ½É23.2%。„Á400mmol/L
mannitolžŸ ¡¢"5©‡,œ•–Œ™RÂÃ
O£ÆÄÅ·@J€

ö4,B)。ËÇ犆犽犔犈犃4œ
•–óºKɁ‚ƒR¡¢ÊËÌÍÎtulÏ


056 ! " # $ % & 36(
A.—˜°±NaClžŸ J€cœ•–Œ™l¡¢£í;
B.200mmol/LNaClžŸ ¼Œ™RÂÃO£í;•ý犘 <0.01
ö3 J€c犆犽犔犈犃4œ•–Œ™DNaClžŸ l¡¢£tu
A.Seedgerminationrateofthewildtypeand犆犽犔犈犃4overexpressionlinesunderdifferent
concentrationsofNaCltreatment;B.Cotyledongreeningrateofwildtypeand犆犽犔犈犃4
overexpressionlinesunder200mmol/LNaCltreatment;indicatesignificantdifferenceamongsamplesat0.01level
Fig.3 Germinationassaysofwildtypeand犆犽犔犈犃4overexpressionlinesunderNaClstress
2.5 4,5678犃犅犃rs@no
q9þ
(Abscisicacid,ABA)ö÷OLl#
$78

ÚPR)*P¡¢

#ŒJó

™q9
µ+,-·âù”#$Jó¢Ùœ?

˜‡ ABA
D#$§¨ØJ$tuœ?Æ¢.¹OLlU
†
[1415]。
jkHI¢¯
,犆犽犔犈犃4 p ABA v
[13]。
ƒHI犆犽犔犈犃4œ•–óö/ôxɁ‚
ƒÌ ABAlB0Ð,gHItuÉü&—˜°±
ABAl1/2MS^T,nœ•–Œ™l¡¢1。
D—üABAl1/2MS^T,,œ•–Œ™l¡
¢£PJ€þÿ

D1、2c3μmol/LABAžŸ
 

œ•–Œ™¡¢£·@J€

ö5,A)。˜‡,
¾°±ABAžŸó¼Œ™lRÂÃO£tu­®
Äý

D0.2c0.3μmol/LABAžŸ ,œ•–Œ
™lRÂÃO£¶·@J€

ö5,B)。¤n­®
•Ç
,犆犽犔犈犃4œ•–ó½¾É‚ƒÌ ABAl
B0Ð

3 ¦ 2
5h

s3

·6c45âNªtu6Oôx¹
#$lJó¢Ù

MÜJ_\½¾U$l3‹

D
ókl„Oœ?

#$7ãÉ÷™qlü8xy
µD9bc:My;yÌ<=ØJ$tu
。LEA
Ö×>öD©ªtu v\3Jl%&çèëìí
pîï

;<#$,gJ`?·l÷øOLÖ×

DghijkHI

?@Aâ
[13]
1BBlast
­®c™í„OQo犆犽犔犈犃4,|Cƒ"4é
LEA,|(DãE。_‹HI•Ç,LEA4(D,
1564k @AB,â:}犆犽犔犈犃4,|‚ƒR¡¢kl¨©ÐQ
A.—˜°±mannitolžŸ J€cœ•–Œ™l¡¢£í;B.400mmol/LmannitolžŸ RÂÃO£í;
cQá•ý犘<0.05c犘<0.01
ö4 J€c犆犽犔犈犃4œ•–Œ™DmannitolžŸ l¡¢£tu
A.Seedgerminationrateofthewildtypeand犆犽犔犈犃4overexpressionlineswithdifferentconcentrationsofmannitol
treatment;B.Cotyledongreeningrateofwildtypeand犆犽犔犈犃4overexpressionlineswith400mmol/Lmannitoltreatment;
andindicatesignificantdifferenceamongsamplesat0.05and0.01level,respectively.
Fig.4 Germinationassaysofwildtypeand犆犽犔犈犃4overexpressionlineswithmannitoltreatment
|D#$§¨ØJ$tuå¹OLlU†

OlveraCarriloâ[16]¢¯œ•–‚ƒ犃狋犔犈犃4
1、犃狋犔犈犃42c犃狋犔犈犃45¬ûºK#$Ì5h
tul§¨&
;Liuâ[17]oFGHI(犅狅犲犪犺狔犵狉狅
犿犲狋狉犻犮犪)l2” LEA4,|犅犺犔犈犃1c犅犺犔犈犃2
DJ;œ•–ó

È·É},|J;l¨hÐ

k
$É™Kr£cL‚HÍM£

„Áº»ÉSOD
c POD l C ? Ð。_ I l 2 ” LEA4 Ö ×
GmPM1c GmPM9¬û­ŽFe3+、Ni2+、Cu2+c
Zn2+ â,kN-ABRÌ#$lîï[18]。Dalal
â
[19]
Qᅆ35Scrd29S¶·RoOP(犅狉犪狊狊犻
犮犪狀犪狆狌狊)l犔犈犃41,|­~恂ƒ,|é,
­®Äý¤<2‰²}~l‚ƒÌsc5ht
u¶&QKlÏpÐ

犆犽犔犈犃4¬ûpæqr、scABAlv\,|
{vuwR¬P#$¨©«¬þz

gHI¢¯

犆犽犔犈犃4œ•–‚ƒDNaCl、mannitolžŸ l
¡¢£¶·@J€

ËÇ犆犽犔犈犃4ºKÉ},|
‚ƒD¡¢ÊËÌscÍÎtulÏpÐ


­2Pœ•–犃狋犔犈犃33,|‚ƒD NaClc
mannitolžŸ lR¡¢£­®÷S[20]。TU
犚犪犫28,|œ•–óD15%PEGžŸ lR¡
¢£Æ·@J€
[21]。
ABAD#$Jó¢Ù¤µ§¨©ªtuœ?
¢.¹OLlU†

wKL«¬ú÷öicR
¡¢
[14]。
gHI

D—˜°±l ABA žŸ ,
犆犽犔犈犃4œ•–‚ƒ¡¢£¶·@J€,D0.2
c0.3μmol/LABAžŸ ,RÂÃO£Æ·@
256 ! " # $ % & 36(
A.—˜°±ABAžŸ J€cœ•–Œ™l¡¢£í;B.0.2c0.3μmol/LABAžŸ RÂÃO£í
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