全 文 :!23" !3#
Vol.23 No.3
! " # $
ACTA AGRESTIA SINICA
2015$ 5%
May. 2015
犱狅犻:10.11733/j.issn.10070435.2015.03.017
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犛狋狌犱狔狅狀犇犻犳犳犲狉犲狀狋犻犪犾犘狉狅狋犲狅犿犻犮狊狅犳犔犲狔犿狌狊犮犺犻狀犲狀狊犻狊犝狀犱犲狉
犛犪犾犻狀犲犪犾犽犪犾犻狀犲犛狋狉犲狊狊
XIELinan,ZHANGYang,NIEYuzhe
(ColegeofLifeScience,NortheastForestryUniversity,Harbin,HeilongjiangProvince150040,China)
犃犫狊狋狉犪犮狋:Torevealthemolecularmechanismofsalinealkalinetolerancefrom犔犲狔犿狌狊犮犺犻狀犲狀狊犻狊,thepro
teinexpressionprofilewasconstructedby2DDIGEandMALDITOF/TOF.Therewere74proteinspots
presentingtoexpressdifferentialyafterthetotalproteinswereseparatedby2DDIGE.The44proteinsof
alpresentingspotswereupregulated,and30proteinsweredownregulated.Amongthe74proteinspots,
theannotationsofthe33differentialexpressedproteinswereknown.Theexpressedproteinswereana
lyzedandcomparedbymassspectrum.Identifieddifferentialproteinstookpartintheenergyexchange,
metabolicprocessesofbasalmetabolism,photosynthesis,plantdefense,andDNAsynthesis,respectively.
Theseworkswilbefundamentaldatatoiluminatethemolecularmechanismofplantdefensetosalineal
kalinestress.
犓犲狔狑狅狉犱狊:犔犲狔犿狌狊犮犺犻狀犲狀狊犻狊;Salinealkalinestress;2DDIGE;Differentialproteomics
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<à«__:ÉGôI©ZIËÌY]
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(isoelectricfocusing,IEF),!2_
SDSPAGE。ëÒÀÂJíî5W3~ñ&.
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W¤ô=ðâ3HÏÐÑaðâCy2,Cy3j
Cy5{ÁIëÒF»§¢£¤24cm¶ñÁN
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(Vh)G}Ø55000Vhcv8lJ。
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75mmol·L-1TrisHClpH8.8,29.3%Æ,2%
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(sodiumdodecylsulfate,SDS),
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mol·L-1E¥,75mmol·L-1 TrisHClpH8.8,
29.3%Æ,2% SDS,0.002%Ô[q)8)u)
15min¢5W12.5% SDSPAGEJ,kÁ@
óæ3W,Â45min¢@óæ17W Â3.
5h。J¢IÀÁ¤40%ûêj10%û½fÞy
+¢¤®Ä{OIÀÁðn5Wð
。
ô=árâ
(Typhoon9400)árJ¢IÀ
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ð2JGß
:
ár:|Cy2D[·488nm
Þ=._520BP40,;ár:|Cy3D[·530
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,“+Gð”+ÅëÒÀ+}"
DYG
,“-Gð”+ÅëÒÀ+}"DYG。
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PlusMALDITOF/TOFAnalyzer)JK。WôK
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sciece.com)f NCBInrGHe85Wö。ëÒ
KJv6eÑÞ90%IëÒÀuæfGeneontolo
gyGHe8(http://amigo.geneontology.org/
cgibin/amigo/blast.cgi)85WóôJa。
2 mnU,c
2.1 v*ãäÐÄÂqÑÔo}Õ
í2DDIGEJ¢ôKÝ3~11LM
IãRëÒÀ+}Ç
(
1),ëÒÀÂJí3
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uv~öØIëÒÀÝGÉ_
1800A,ôKIëÒÀÝÅqëÒÀdÝ、{
獆
、
J>a2
。
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!3# ݺÄ:ÕD4#NOY]ãR+}ëÒÀ«.IÌÍ
<1 xt2犇犇犐犌犈}Õov*ãäÐÄqÑÔ
Fig.1 Differentialexpressionprofilingoftotalproteins
in犔犲狔犿狌狊犮犺犻狀犲狀狊犻狊by2DDIGE
:
ñ_Cy3{ÁëÒÝ(<à),_Cy5{ÁëÒÝ
(Na2CO3Ë572h),c_Cy3jCy5{ÁëÒÝI`ú
Note:Proteinsextractedfromtheseedlingsgrewonthecontrol
conditionweredyedbyCy3showningreen;proteinsextracted
fromtheseedlingsgrewonthe200mmol·L-1Na2CO3
conditionweredyedbyCy5showninred;theoverlapped
spotswereshowninyelow
2.2 ÁÃßU犖犪2犆犗3 klßv*ãäÐÄÂo
Ö))
íDeCyderÃ_×ãRJKÉOJK,ôK
<૶Na2CO3 Ë5«ëÒÀ+}FG,UJí
DeCyderÃ_×ãRJKÉOJK¢ôKI<à«
¶Na2CO3 Ë5«UJãR+}IëÒÀÝ,6ä
nz_íî8IUJãR+}IëÒÀÝ
(
2)。WëÒ+}7e\ãR1.5YPx_p×
{%
,
KØ74AãR+}IëÒÝ,,|8
Na2CO3 Ë5¢ëÒÀ7eDÝ_44A(+1),
Na2CO3 Ë5¢7eDÝâ30A(+2)。
2.3 ÁÃßU犖犪2犆犗3 klßv*ãäqÑÐÄ
Âo×Æ-
WãR+}IëÒÀIÀÇJKa5WGHe
ö
,
f74AãR+}IëÒÀ8,67éÀIëÒ
À33A,67dÀIëÒÀ41A。67éÀIëÒÀ
mVYZ¶Ú¬·IëÒ18A,¶=x¤¬·I
ëÒ9A,¶&^ñ¬·IëÒ2A,¶ô"}ä¬
·IëÒ3A,¶}¬·IëÒ1A,ß3nÅ,æ
ç1aëÒÀIãR+}FG|¬。
q1 ÁÃßU犖犪2犆犗3 klßv*+ð\,ð^qÑÐÄÂoÖ))|[
Table1 UpregulationinproteinsexpressionaccumulationfromtheseedlingsofbothcontrolandNa2CO3treatedgroups
U
No.
ëÒU
SpotNo.
7eChangesin
accumulation
U
No.
ëÒU
SpotNo.
7eChangesin
accumulation
U
No.
ëÒU
SpotNo.
7eChangesin
accumulation
1 900 +1.73 16 1716 +4.41 31 2246 +1.72
2 1156 +1.60 17 1724 +1.64 32 2258 +1.67
3 1258 +1.62 18 1726 +7.57 33 2264 +1.51
4 1369 +1.93 19 1750 +3.14 34 2336 +1.98
5 1504 +1.79 20 1864 +1.81 35 2337 +1.64
6 1510 +1.64 21 1901 +2.67 36 2389 +2.13
7 1512 +1.75 22 1960 +2.82 37 2425 +2.32
8 1516 +2.28 23 1993 +1.59 38 2426 +1.97
9 1539 +2.36 24 2018 +1.59 39 2457 +1.79
10 1580 +1.52 25 2160 +2.38 40 2466 +1.83
11 1595 +1.89 26 2188 +1.66 41 2528 +2.19
12 1639 +2.23 27 2194 +3.65 42 2531 +3.57
13 1687 +3.46 28 2218 +1.57 43 2682 +1.55
14 1700 +2.00 29 2220 +1.63 44 2727 +2.13
15 1704 +4.00 30 2221 +1.77
:“+Gð”+ÅëÒÀ+}"DYG
Note:“+number”indicatestheupregulatedfoldofproteinexpression
155
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Table2 DownregulationinproteinsexpressionaccumulationfromtheseedlingsofbothcontrolandNa2CO3treatedgroups
U
No.
ëÒU
SpotNo.
7eChangesin
accumulation
U
No.
ëÒU
SpotNo.
7eChangesin
accumulation
U
No.
ëÒU
SpotNo.
7eChangesin
accumulation
1 1200 -2.16 11 2241 -2.15 21 2308 -18.80
2 1357 -2.16 12 2249 -2.14 22 2360 -1.61
3 1429 -1.82 13 1769 -1.78 23 2372 -2.16
4 1439 -1.94 14 1819 -3.14 24 2453 -1.74
5 1443 -1.72 15 1822 -2.68 25 2594 -2.69
6 1526 -1.53 16 2052 -1.51 26 2604 -1.98
7 1557 -1.63 17 2091 -2.44 27 2612 -4.52
8 1614 -2.58 18 2182 -3.82 28 2650 -4.17
9 1623 -1.52 19 2195 -2.30 29 2651 -2.73
10 1629 -1.94 20 2251 -1.63 30 2655 -3.99
:“-Gð”+ÅëÒÀ+}"DYG
Note:“-number”indicatesthedownregulatedfoldofproteinexpression
<2 ÁÃßU犖犪2犆犗3 klß\,ãäqÑoÐÄÂù
Fig.2 DifferentialexpressedproteinspotsofcontrolandNa2CO3treatedgroups
:A:1726UëÒÝ,+}7eD7.57Y;B:2194UëÒÝ+}7eD3.65Y;C:2612UëÒÝ+}7eD4.52Y;
D:2594UëÒÝ,+}7eD2.69Y。6änz_íî8IUJãR+}IëÒÀÝ
Note:A,proteinofNo.1726upregulated7.57fold;B,proteinofNo.2194upregulated3.65fold;C,proteinofNo.2612downregulated
4.52fold;D,proteinofNo.2594downregulated2.69fold.Thespotsindicatedwitharrowsweresomedifferentialyexpressedproteinspots
<3 v*ãäqÑÐÄÂØÙ|[
Fig.3 Annotationsofthedifferential
expressedproteins
2.3.1 ÊAHÈ Na2CO3 CÚHp>qrsb
ctuÈ+vwxKL@bc ãR+}ëÒÀí
óôÞ¢Q8`¶Ú¬·IëÒÀ(+
3),óôéÀëÒ8nätuzàI¥a。ëÒ
Ý1156ëÒÀflNOQÕË5NOY]I
ãRëÒÀtS8
,
7e\_D
,
De_
1.60 Y,í ë Ò À Þ _ Ý ê £ (Enolase2,
ENO)。
tS<૶Na2CO3 Ë5«Y]IãRëÒ
ÀÇ,
,
ëÒÀÝ1357,1526j1629J*D
2.16,1.53,1.94Y,æ1504,1510J*D
1.79,1.64Y。ëÒÝ1357,1504,1510,1526,
1629_aüÃŽ£(fructosebisphosphate
255
!3# ݺÄ:ÕD4#NOY]ãR+}ëÒÀ«.IÌÍ
aldolase,FBA),|81504UëÒÝ_aüÃŽ
£+ÍÀR£
。
flNOQÕË5NOY]IãRëÒÀ
tS8
,
ëÒÀÝ1512,1516,1595ëÒÀ7ev
_D
,
eJ*_1.75,2.28,1.89Y,æ1439
j1614J*D1.94,2.58Y。<ëÒÀÝ
Þ_Æ3Ž1þ£(glyceraldehyde3phos
phatedehydrogenase,GAPDH)。
ãRëÒÀÝ1700j1750_+}7eD,
eJ*_2.00,3.14Y,æ1769D1.78Y。
|8ëÒÀÝ1700j1750Þ_ia½1þ£2
(malatedehydrogenase2,MDH),1769_SÞû
½2åIia½1þ£á2
。
ëÒÀÝ2336
De_1.98Y,ëÒÀÞ_?U½£2(fu
maratehydratase2,FH)。ëÒÝ21957e_D
,
De_2.30Y,ëÒÀÞ_ĽÞ
£
(carbonicanhydrase,CA)。ëÒÝ2220D
e_1.63Y,ëÒÀÞ_ßëÒ3(ferritin3,
FER)。ëÒSÝ2425De2.32Y,ëÒÀ
Þ_Sàáâ¼ü½1®£û(sadenosylmethi
oninedecarboxylaseproenzyme,SAMDC)。
2.3.2 ÊAHÈ Na2CO3 CÚHp>qrsb
ctuÈÙIyäKL@bc flNOQÕË
5NOY]IãRëÒÀtS8
,
ëÒÀÝ
1539,1557,1623,1639,2604,2612,2650,2655
j2682|87AÝëÒÀ+}7eD,J*_-
2.36,-1.63,-1.52,-1.98,-4.52,-4.17,
-3.99Y,2AëÒÝ7eDJ*_2.23,
1.55Y,ëÒÀÞ_ÃŽëZü®\£/úp£
(ribulosebisphosphatecarboxylase/oxygenaseac
tivase,RuBisCO),|8¢5AÝ_ÃŽëZü
®\£HJ
(
+4)。
2.3.3 ÊAHÈ Na2CO3 CÚHp>qrsb
ctuÈJrzMKL@bc ëÒÀÝ1716,
17267e\v_D,eJ*_4.41,7.57
Y
,
_;p\&£2(Peroxidase2,POD)(+5)。
2.3.4 ÊAHÈ Na2CO3 CÚp>qrsbc
tÈs{|KL@bc ëÒÝ1200,1580j
2246+}7e\J*_D2.16,D1.52j
D1.72Y。íëÒÀÞJ*_ ATP£
(ATPsynthase)Iβ、γjα&2(+6)。
2.3.5 ÊAHÈ Na2CO3 CÚHp>qrsb
ctuÈ{}KL@bc ëÒÀÝ17047e
\_D
,
e_4.00Y,_DNAzIRNAl
£2(DNAdirectedRNApolymerase2)(+7)。
q3 ãäqÑÐÄÂ;UÚÛd8oÐÄÂ|[
Table3 Annotationsofdifferentialyexpressedproteinsrelatedtometabolization
ëÒU
Spot
No
7eChange
fold
ÏU
AccessionNo.
ëÒÀé9
Proteinname
:?&`
Sources
Jr"
/
Ý
MW/pI
Jð
Protein
score
ã{ÚùG
Peptide
count
ã{æ
Intensity
matched
1156 1.60 P42895|ENO2_MAIZE Ýê£ENO [h犣犲犪犿犪狔狊 48417.6/5.7 308 14 38.028
1504 1.79 P08440|ALF_MAIZE aüÃŽ£FBA [h犣犲犪犿犪狔狊 39036.2/7.52 77 4 20.398
1510 1.64 P08440|ALF_MAIZE aüÃŽ£FBA [h犣犲犪犿犪狔狊 39036.2/7.52 65 4 14.425
1512 1.75 P26517|G3PX_HORVU Æ3Ž1þ£GAPDH àE犎狅狉犱犲狌犿狏狌犾犵犪狉犲 36604.8/6.67 546 9 65.522
1516 2.28 P26517|G3PX_HORVU Æ3Ž1þ£GAPDH àE犎狅狉犱犲狌犿狏狌犾犵犪狉犲 36604.8/6.67 383 6 71.578
1595 1.89 P08477|G3PC_HORVU Æ3Ž1þ£GAPDH àE犎狅狉犱犲狌犿狏狌犾犵犪狉犲 33442.9/6.2 610 11 51.992
1700 2.00 Q43744|MDHM_BRANA ia½1þ£2MDH2 e犅狉犪狊狊犻犮犪狀犪狆狌狊 35859.9/8.81 135 3 7.523
1750 3.14 Q9LKA3|MDHM2_ARATH ia½1þ£2MDH2 vTä犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪36024/8.3 177 2 24.046
2220 1.63 Q9LYN2|FRI3_ARATH ßëÒ3FER3 vTä犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪28875.7/5.54 77 2 16.906
2336 1.98 Q9FI53|FUM2_ARATH ?U½É£2FH2 vTä犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪54505.5/5.93 34 5 47.213
2425 2.32 Q9M6K1|DCAM_IPOBA
Sàáâ¼ü½1®£û
SAMDC
Æå犐狆狅犿狅犲犪犫犪狋犪狋犪狊 40215.4/4.88 32 2 64.481
1357 -2.16 P46256|ALF1_PEA aüÃŽ£FBA a;犘犻狊狌犿狊犪狋犻狏狌犿 38707/6.38 73 1 19.407
1526 -1.53 O65735|ALF_CICAR aüÃŽ£FBA æç;犆犻犮犲狉犪狉犻犲狋犻狀狌犿 19783.8/8.52 100 6 13.703
1629 -1.94 P16096|ALFC_SPIOL aüÃŽ£FBA èe犛狆犻狀犪犮犻犪狅犾犲狉犪犮犲犪 42726.8/6.85 74 2 30.349
1439 -1.94 P25857|G3PB_ARATH Æ3Ž1þ£GAPDH vTä犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪48085.7/6.33 154 3 29.12
1614 -2.58 P09043|G3PA_TOBAC Æ3Ž1þ£GAPDH é]犖犻犮狅狋犻犪狀犪狋犪犫犪犮狌犿 42121.8/6.6 143 2 13.625
1769 -1.78 P37228|MDHG_SOYBN ia½1þ£á2 MDH c;犌犾狔犮犻狀犲犿犪狓 37826.9/7.55 51 1 20.573
2195 -2.30 P40880|CAHC_HORVU ĽޣCA àE犎狅狉犱犲狌犿狏狌犾犵犪狉犲 35736.1/8.93 177 3 58.938
(Note):ENO,Ýê£(Enolase);FBA,aüÃŽ£(Fructosebisphosphatealdolase);GAPDH,Æ3Ž1þ£(Glyceralde
hyde3phosphatedehydrogenase);MDH,ia½1þ£(Malatedehydrogenase);FER,ßëÒ3(Ferritin3);FH,?U½É£2(Fumarate
hydratase2);SAMDC,Sàáâ¼ü½1®£û(Sadenosylmethioninedecarboxylaseproenzyme);CA,Ľޣ(Carbonicanhydrase)
355
! " # $ !23"
q4 ãäqÑÐÄÂ;U~Otd8oÐÄÂ|[
Table4 Annotationsofdifferentialyexpressedproteinsrelatedtophotosynthesis
ëÒU
Spot
No.
7eChange
fold
ÏU
AccessionNo.
ëÒÀé9
Proteinname
:?&`
Sources
Jr"
/
Ý
MW/pI
Jð
Protein
score
ã{ÚùG
Peptide
count
ã{æ
Intensity
matched
1539 2.36 Q40073|RCAA_HORVU
ÃŽëZü®\£
/
úp£
RuBisCO
àE犎狅狉犱犲狌犿狏狌犾犵犪狉犲 51383.5/8.04 867 15 68.732
1639 2.23 P05698|RBL_HORVU
ÃŽëZü®\£
/
úp£
RuBisCO
àE犎狅狉犱犲狌犿狏狌犾犵犪狉犲 53671.8/6.22 608 11 62.037
2682 1.55 P07398|RBS3_WHEAT
ÃŽëZü®\£
/
úp£
RuBisCO
4E犜狉犻狋犻犮狌犿犪犲狊狋犻狏狌犿 13274.5/5.84 373 9 26.809
1557 -1.63 Q40073|RCAA_HORVU
ÃŽëZü®\£
/
úp£
RuBisCO
àE犎狅狉犱犲狌犿狏狌犾犵犪狉犲 51383.5/8.04 733 14 80.048
1623 -1.52 Q05800|RBL_NELLU
ÃŽëZü®\£
/
úp£
RuBisCO
ËSÖ犖犲犾狌犿犫狅犾狌狋犲犪 44231.4/6.4 239 4 61.025
2604 -1.98 P26667|RBS2_WHEAT
ÃŽëZü®\£
/
úp£
RuBisCO
4E犜狉犻狋犻犮狌犿犪犲狊狋犻狏狌犿 19783.8/8.52 252 6 24.308
2612 -4.52 P10795|RBS1A_ARATH
ÃŽëZü®\£
/
úp£
RuBisCO
vTä犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪1351.5/6.62 109 2 100
2650 -4.17 P10795|RBS1A_ARATH
ÃŽëZü®\£
/
úp£
RuBisCO
vTä犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪20488.1/7.59 147 2 11.642
2655 -3.99 P26667|RBS2_WHEAT
ÃŽëZü®\£
/
úp£
RuBisCO
4E犜狉犻狋犻犮狌犿犪犲狊狋犻狏狌犿 19783.8/8.52 320 6 27.704
(Note):RuBisCO,ÃŽëZü®\£/úp£,Ribulosebisphosphatecarboxylase/oxygenaseactivase
q5 ãäqÑÐÄÂ;UºlÐd8oÐÄÂ|[
Table5 Annotationsofdiferentialyexpressedproteinsrelatedtoresistance
ëÒU
Spot
No.
7eChange
fold
ÏU
AccessionNo.
ëÒÀé9
Proteinname
:?&`
Sources
Jr"
MW
(kDa)
Jð
Protein
score
ã{ÚùG
Peptide
count
ã{æ
Intensity
matched
1716 4.41 Q01548|PER2_HORVU ;p\&£2POD àE犎狅狉犱犲狌犿狏狌犾犵犪狉犲 1661.807 96 2 100
1726 7.57 Q01548|PER2_HORVU ;p\&£2POD àE犎狅狉犱犲狌犿狏狌犾犵犪狉犲 1661.807 75 2 99.942
(Note):POD,;p\&£2,Peroxidase2
q6 ãäqÑÐÄÂ;UåÜd8oÐÄÂ|[
Table6 Annotationsofdiferentialyexpressedproteinsrelatedtoenergy
ëÒU
Spot
No.
7eChange
fold
ÏU
AccessionNo.
ëÒÀé9
Proteinname
:?&`
Sources
Jr"
/
Ý
MW(kDa)/pI
Jð
Protein
score
ã{ÚùG
Peptide
count
ã{æ
Intensity
matched
1580 1.52 P0C1M0|ATPG_MAIZE
ATP£γ&2
ATPsynthase,subunitγ
[h犣犲犪犿犪狔狊 40107.1/8.44 264 5 31.341
2246 1.72 P22778|ATPO_IPOBA
ATP£α&2
ATPsynthase,subunitα
Æå犐狆狅犿狅犲犪犫犪狋犪狋犪狊 27298.3/9.56 40 1 10.73
1200 -2.16 P0C2Z7|ATPB_ORYSA
ATP£β&2
ATPsynthase,subunitβ
É¿犗狉狔狕犪狊犪狋犻狏犪 53978.1/5.381320 21 66.604
q7 ãäqÑÐÄÂ;UÝd8oÐÄÂ|[
Table7 Annotationsofdiferentialyexpressedproteinsrelatedtotranscription
ëÒU
Spot
No.
7eChange
fold
ÏU
AccessionNo.
ëÒÀé9
Proteinname
:?&`
Sources
Jr"
/
Ý
MW(kDa)/pI
Jð
Protein
score
ã{ÚùG
Peptide
count
ã{æ
Intensity
matched
1704 4.00 Q8VWF8|RPOT2_NICSY
RNAl£2
RNApolymerase2
ËLé]
犖犻犮狅狋犻犪狀犪狊狔犾狏犲狊狋狉犻狊
116958.4/9.24 37 3 16.299
455
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@qIã
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,
8`,
18AëÒÀÝ。|8YZütݾ¿8IÝê£
ENO(ëÒÀÝ1156)jÆ3Ž1þ£GAPDH
(
ëÒÀÝ1512,1516,1595,1439,1614)。ütݾ¿
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fý2¡pNO()ñí!×
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fñ\ÖI©
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Ýê£
(
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a¶¬·IÝê£fë
p
、
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y4#Ôq,2ü+}"
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Æ3Ž1þ£(GAPDH)îfD+}j
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\YGJ*_1.75,2.28,1.89Yj
-1.94,-2.58Y,-²2ÞD+},e&ý
ØÕD4#¢
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Æ3Ž1þ£+}"ùú,!
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>?
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(
%&( , -)
655