全 文 :2016
y
3
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41
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Vol41,No.6 March,2016
Þß
DNA
àÔáâÓ
(GlMET1)
ÔÕãä
ÐåÜÝæç
l]i
1,2,
;mn
2,3,
!o
2,
Lpq
2,
r2
2,
.UV
2
(1
&ÁVWz?Z zZY
,
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611137;
2
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,`
a
100700;
3
£bÄÅXZVv
,`
a
100190)
[
lm
] DNA
àÔáâÓé
DNA
àÔ_òû$7ôSÓ
,
°8ÔÕÜÝêëZGÖühi
。
î.Þßá=.
,
@òwÔÕ=!]ãä=rÞß
DNA
àÔáâÓ
GlMET1,Genbank
÷<ò
KU239998,`
ÔÕ.¨ös
<^nw÷°v
。
ÐåÜÝæçÜ{
pET28a(+)GlMET1
Ö´½²
BL21(DE3)
$ÒÜÝ$í
Ñ
,
æçn8_
,
=$Ñ 7=¬ÜÝò
:
æç-ò
16℃,
Ö1°±
25h(A600ò08),IPTGË
ò
02mmol·L-1,
æçOsò
12h。
Oæ¢vö
PCR
<ÄÜ{£¤³?Þß
GlMET1
ÔÕÜݪÝ:B{ã±
,
9
GlMET1
²h}ÜݪÝ:H°¼y}
,
ù{
GlMET1
ÜÝöFGÞß°±m¾ÁÛî&>
。
ñ
<Äò!
DNA
àÔáâÓÑ hifù÷vúÔä
。
[
nop
]
Þß
;
àÔ_
;
àÔáâÓ
;
ÔÕãä
;
ÐåÜÝ
[
qr)(
] 20151209
[
stuT
]
£b¨ÆÛÍXZEfij
(81325023);
£bPk«]^efgh
(863)
ij
(SS2014AA022201)
[
vwAx
]
.UV
,Tel:(010)84044340,Email:huangluqi01@126com;
r2
,y_yuan0732@163com
[
Ax
]
l]i
,
rA]^7
,Email:chaliangliang520@126com
CloningandprokaryoticexpressionanalysisofDNAmethyltransferas1
(MET1)inGanodermalucidum
ZHALiangping1,2,WANGYajun2,3,LIUShuang2,ZHAOYuyang2,YUANYuan2,HUANGLuqi2
(1ColegeofPharmacy,ChengduUniversityofTraditionalChineseMedicine,Chengdu611137,China;
2StateKeyLaboratoryBreedingBaseofDaodiHerbs,NationalResourceCenterforChineseMateriaMedica,
ChinaAcademyofChineseMedicalSciences,Beijing100700,China;
3NationalCenterforNanoscienceandTechnology,Beijing100190,China)
[Abstract] DNAmethyltransferaseisthekeyenzymeintheprocessofDNAmethylation,playinganimportantroleinregulationof
geneexpressioninvivoAccordingtotheGanodermalucidumtranscriptomedata,afullengthcDNAsequenceofMET1fromGlucid
umwasclonedforthefirsttime,theGenBankregistrationnumberisKU239998,andweconductedacomprehensivebioinformaticsa
nalysisofthegeneticcharacteristicsandspatialstructureTheprokaryoticexpressionanalysisshowedthatEcoli[pET28a(+)
GlMET1]inBL21(DE3)couldinduceobjectiveprotein,shakingthecultureat16℃ untilthehostbacterium(A600)wasapproximate
ly08,andaddedIPTGtofinalyconcentrationof02mmol·L-1,andthentheoptimalexpressionofGlMET1recombinantprotein
wasaccumulatedfortheinductiontimeof20hTherealtimePCRresultsshowedthattheexpressionlevelsofGlMET1hadobvious
diferencesamongvarietiesofGlucidumDuringthematuritystage,theexpressionlevelsofGlMET1werelowerthanthatinjuvenile
stage,theresultsshowedthatwiththegrowthofGlucidum,theexpressionlevelsofGlMET1wereonthedeclineTheresearchpro
videdanimportantbasisforstudyingthemechanismofDNAmethyltransferasethoroughly
[Keywords] Ganodermalucidum;methylation;methyltransferas;genecloning;prokaryoticexpression
doi:10.4268/cjcmm20160608
·1201·
2016
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Vol41,No.6 March,2016
Ü,©W$øéA£Ð¯
DNA
¹T©W
ºÔ¨Ñ _$ø
,
ÖüàB
DNA
àÔ
_
、
Ñ $ø
、
hK´ÖÖì/Ë
RNA
ö
[1]。
DNA
àÔ_hò[?ÖüÜ,©W$ø
,
²°
8ÔÕÜÝêë÷ZGÖühi
,
*á
T3ùú
,
ÔÕh4¨
X
hKÜö°ò
û
。DNA
àÔ_é²
DNA
àÔáâÓå_Û
,
àÔáâÓ/
SAM
àÔÔ}áârŵ¶
5
XûÐ3bêàÔ_òû
[2]。DNA
à
Ô_Òóå_1
DNA
àÔáâÓ
(DNAmethyl
transferase,Dnmt)
²hK´ÖÖ¨ÔÕÜÝêø$
ZGôShi
。
íî²$luw?më
3
?
DNA
àÔáâÓ
,
oàÔáâÓ
l(methyltransferas,
METl)、
<^NÖSàÔáâÓ
(domainsrearanged
methyltransferas,DRM)¨
hK´àÔ_Ó
(chro
momethylase3,CMT3)[35]。MET1
uPòéDBâ
FX
DNA
àÔáâÓ
,¯
DBXå]¹T
$
CG
ºÔŵ¶àÔ_hi
,
¤O
METl
Ò
µ¶0Ø./
、
êøØÌOsö
[6]。
$,Þß
Glucidum
éÍ,Ïi[?X1
,
sB>Å
、
kd_=
、
°º
、
å
、
Ô]>
ü¨Xö~Oö>?
,
$Þß>ŨL
kd_=éÞßÖü
,
Ü{¯Ð
DBe/0
、
et_
、
eÎϨî¡Åö>?°
[79]。
Õ
20
Ƕ
80
yPìì
,
Þß3°
、
]°P¶Æ¨,ö:nú!
,
E
éPÐÞßLk]°=²ôÔÕ§¨
、
ã
ä¨ÒwBök.
,
á\¨ÔÕv
ò
ÞßÔÕ>÷vúÔä
,
ÞßuPò
é
X1]°P¶d5£?
[1011]。
íî
w>?$ãä$
METl
ÓÔÕ
,
x9
¤
[12]、
¬Q
[13]、
aý
[14]、
sþ
[15]
ö
,
6,i
MET1
ÓÔÕk.Ãu
。
N
!]Þßá$§¨=rÞß
DNA
àÔáâÓÔÕ
GlMET1,
µiwÔÕ=ãä=r
cDNA
¹T
,
ÔÕ
.¨ös<^nw÷°v
,`
^
¿
pET28a(+)GlMET1
Ö´½
,
á©lÜÝ1
BL21(DE3)
$
,
µ¶Ñ ÜÝæç-
、
æç
Os
、IPTG
ËÄÖ1¿¢ö
4
ÕOnú
8_
,
¤OµiOæ¢vö
PCR
qu£¤³?¨
£¤°±O}
GlMET1
ÜݪÝ
,
ò[H
DNA
àÔáâÓÔÕÒ²Þßm¾òû
$êøÒ÷vÔä
。
1
ʸú
11
,Ê
ÿß
1
<µÕ°Ñ!åÑ4ÿÙßÿ#D
E
,
$ß
10
<µÕ°Ñ!åÑ4ÿÙ[³#D
E
,
×ß
1
<µÕ°Ñ!åÑ4×%Î,F
,
_°ÞßµÕ°Ñ!×%&*
。
?Ñ!$A
,,%+¹»Vv
,
³?Vv~ý
°
-80℃
ÝZ
。
12
1°
、
e¨¥1
Ò1
BL21(DE3)
òN(¸ý
。
Ðå
ÜÝe
pET28a(+)
òN(¸ý
;DNA
u¹
723(°&(DE
,
)å]27b
H°g°gûDE=
。
Cù8%Ó´Õ
NEB
BCDE
。
13
u
RNA
pq
cDNA
=
¦q
100mg
¢sÞß&3
,
¢©Ø
°Yèfbè
,
µi
CTAB
ú&3$
pqu
RNA,1%
VWÅEFUi°TU
RNA
´ö
。ND2000
uvu
RNA
A260,A280,¨ O
A260/A280ò18~20uRNAÒá\cDNA。
14 GlMET1
wÔÕ=ÐåÜÝe^¿
Þß
DNA
àÔáâÓ
1(GlMET1)
ÔÕì>
°Þßá\=.4
。
¨O+¹T
3554
º
Ôhòãä,
,
²2`ux:;ú
BamHI
¨
SalI
Ó%X診Ô
,
¹Tuw±ò
3554
bp,
wÔÕ=72ddx*°&(BCDE
。
¿BÓ%Xè
GlMET1
¨
pET28a(+)
e_nbÓ%
,
Ái
T4DNA
ðèÓnð
è
,25℃
ðè
3h。
/ðèÆá_l
DH5αD
ØÄÅ
,
²sB+,ÌO
LB
Æ¥6b
37℃。
Â
ãä1¢
PCR,
bÓ%¨u¹Vv
。
15 GlMET1
°v
/u¹÷=¹T<ÄXi
ORFFinder(ht
tp://wwwncbinlmnihgov/gorf/gorfhtml)
5Ø
--Ò.
(ORF)。
Ái²ÈgD
Protparam(ht
tp://wwwexpasych/tools/protparamhtml)
:uÔ
Õ/ËÑ ²3ö
、
Ô]=í
、
öUè
、
£
/võ=
、
W¨A=
、
ª
/
0ª¨/ËJw±
ö _ ´
;
µ i
CDD (htp://
wwwncbimlmnihgov/Structure/cdd/wrpsbcgi)
nÑ ´ < ^ N
;
µ i
CFSSP(htp://
·2201·
5#Ýö
:
Þß
DNA
àÔáâÓ
(GlMET1)
ÔÕãäÐåÜÝæç
wwwbiogemorg/tool/choufasman/)
nÑ ´8
^ < ^
;
Á i
Swiss Model(htp://
swissmodelexpasyorg/)
û¹
,
î.ÔÕÔ]¹T
n¿5
,
:uÑ ´L^<^
。
i
DNAMAN
é
¹Tn>ÖÂìCùÓ%Xè:
u
,
i
ClustalW
é|Ô]¹Tn
ÂÃ
,
i
MEGA606
é^¿
Neighborjoining
õâ
_¹
,bootstrap
Ö]=ò
1000
]
。
16 pET28a(+)GlMET1
ÐåÜÝ8_
/^¿$
pET28a(+)GlMET1
´½á_r
BL21(DE3)
ÜÝDØ$
,
ná_ÜÝÄÖ1
。
qá_ÜÝ1¢[1∶100
rs+,ÌOe
LB
Æ¥¢$
,37℃ 250r·min-1
tÃÆ¥+
25h
l
A60004~06,IPTG°16℃H-æç12h,À
À1
,
ö¦
2×loadingbufer
12
,
b
,
i
10%
®à¯3EFn
SDSPAGE
。
~æç-
、
æçOs
、IPTG
Ë
,
IPTG
Os
(
oæçZl1¢Ë
A600)ö4ÕOÐ
åÜÝƦmB[vµ¶
,
ú
4
ÕO
Þß
DNMT1
ÖÑ Üݵ¶
。
17 RealtimePCR
á\$=.$÷qÞß8*GlGPD
¨
GlMET1
å]¹T
,
Ái
PrimerPremier50
:
;Oæ¢vö2
,
¾Æ±²
100~250
bp,
47°g°gû
(
bH
)
DE=
,
$
GPD
bO2ò
5′CGCTCAACAAGAACTTCGTCAA3′,
GPD
ÛO2ò
5′CGTAGACAAGGAGGTCACA
GA3′,GlMET1
bO2ò
5′ATCGATCCAACG
GCAAAG3′,GlMET1
Û O 2 ò
5′CACA
GAAGACGAACGAATCC3。
Òóõ
:5μL2×
SYBRgreen,02μLROXReferenceDye,02μL2
1,02μL2 2,10μLcDNA,34μLH2O。
PCR
Òóò
:95℃30s;95℃5s,60℃34s,
40
¾NM
;dissociationstage。
²
ABI7500
æ¢
vö
PCR
Ábnæ¢vö
,
。
xÔÕÜÝöì8*ÔÕhòB|n²v
ö
,
|ÒóÖ
3
]
,
²vöúµi
2-ΔCT
ú
<Ä
。
2
<Ä
21 GlMET1
ÔÕwÔÕ=°v
.Þßá\=.÷=Þß
GlMET1
cDNA,
Ái
DNAMAN
é<=
ORFFinder
²Èé
GlMET1
ÔÕw±
cDNA
¹Tn
,
:u
s
3554bp
+Ø--Ò.
,
@òwÔÕ=
()
GlMET1
ãä
,
u¹<Ä:u<Ä
²[÷
。NCBIBLASTX
ãÿ
GlMET1
µ5Ò?
1
Dichomitussqualens65%
²õ
,
ß
Trametesversi
color46%
²õ
,
§Ú¨1
Coniophoraputeana42%
²
õ
。
ÂÜ{³÷ÔÕ°
MET1
üÓ
,
/ñÔÕ
Wò
GlMET1,GenBank
÷<ò
KU239998。
Ái
NCBI
Conserveddomains
²ÈgD
GlMET1
Ñ Ò<^N
,
<ÄÜ{
GlMET1
DB
4
ÒN
:39~166aa
üu
DNMT1RFDsu
perfamily
Bä>
,365~496aa
39~637
aa
:ò9
BAH
<^N
,700~1160aa
Dcm
üÓBä>
。
Ái²È°gD
TM
HMM20Server
nÑ 6`<^N:u
,
<Äãÿx6`J
。
Xi
ExPASy
²È¾Rj
SWISSMODELHomologyModeling
GlMET1
Ñ
n¤>¿5
,
=rúÑ L\ös5
,Gl
MET1
Ñ 5
4yoc1A
=ò
062,
Ñ ¹T
²õò
2599%,
q
1。
22 GlMET1
¹TüÓ¤>ÔÕõâ_
/
GlMET1
GenBank
$
20
?|¤
>Ñ nÂ
,
²é
MEGA606
Ýbµi
²çðèú^¿_¹
,
ndôõ
,
q
2。
_¹$
,
ì=¶£¤d²_¹$
_£¤[
,
b3{
GlMET1
ò[
[
,
ê3{
MET1
ò[
[
,
GlMET1
ôõ7EòX1
Ter
mitornycessp
¨
Moniliophthoraroreri,
Kd
MET1
³Bôõ7b
。
23 pET28a(+)GlMET1
ÐåæçÜÝ
/^¿
pET28a(+)GlMET1
´½á_lÜ
Ý1
BL21(DE3)
$
,
IPTG
æçÜÝ
,
³=1
?S12~n
SDSPAGE
Ñ U
,
q
3。
<ÄÜ{²
149kDa
I$ëíÑ ¿
,
GlMET1
ÔÕ
cDNA
7
3545
ºÔ
,
/Ë
133kDa
Ñ
,
ÐåÜÝe
pET28a
b
HisTag
B7:ò
16kDa,
³ìÖÜÝe/ËÑ
+ò
149kDa;
êÕEcoli[pET28a]
æçÜ
Ý~²
149kDa
IB¿$ë
,
Ü{sB
GlMET1
ÔÕÖ´½
,
²
Ò1
BL21(DE3)
$
ÜÝ
。
·3201·
2016
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41
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}
Vol41,No.6 March,2016
AGlMET1
Ñ Ò<^N
;BGlMET1
6`<^N:u
;CGlMET1
Ñ L^<^
。
q
1 GlMET1
Ñ ös<^:u
Fig1 PredictedstructureofthededucedproteinGlMET1
At
x9¤
Arabidopsisthaliana(NP_1997271);At
ëë®
Aegilops
tauschi(EMT234551);Ca
8É
Cicerarietinum;Ci
_Ì
Chrys
anthemumindicum;Cs
F9
Camelinasativa;Gs
É
Glycinesoja;
Mn
v:
Morusnotabilis;Mt
;<ÚÛ
Medicagotruncatula;MrMo
niliophthoraroreri;Nt
ña
Nicotianatabacum;Os
ªð
Oryzasativa
JaponicaGroup;Ot
ÓK
Ostreococcustauri;Pt
ßÄ
Populustricho
carpa;Pp
|=ã
Prunuspersica;Pp
:R>?
Physcomitrelapatens;
Sl
«
Solanumlycopersicum;Si
:Q
Setariaitalica;Tc
Theo
bromacacao;Vv
MN
Vitisvinifera;TsTermitornycessp。
q
2 GlMET1
|
MET1
Ô]¹T_¹
Fig2 PhylogeneticanalysisofGlMET1andotherMET1amino
acidsequencesfromplants
24 pET28a(+)GlMET1
ÜÝ8_
241
æç-
pET28a(+)GlMET1
Ñ ÜÝ
µ¶
ËæçOs
12h,IPTG
Ëò
04
mmol·L-1,A600ò 06£¯,,&£¤æç-
16,20,25,30,37℃
pET28a(+)GlMET1
Ñ Ü
ݵ¶
,
q
4。
-ò
16,20℃
O
,pET28a
(+)GlMET1
BÜÝ
,
9æç-ò
16℃
OÜ
Ýöý
,
6FG-0Ìbø
,pET28a(+)
M
Ñ ´
Marker;C:Escherichiacoli[pET28a];1
bæç
Ecoli
[pET28a(+)GlMET1];2
æç~
Ecoli[pET28a(+)
GlMET1]。
q
3 pET28a(+)GlMET1
ÐåÜÝ
Fig3 AnalysisofthepET28a(+)GlMET1fusionproteinex
pressionbySDSPAGE
GlMET1
²
25,30,37℃
:bÆ°ÜÝ
,
Ü{ñÑ
¬=H-æç
,
7=¬-ò
16℃。
M.
Ñ ´
Marker;C.Escherichiacoli[pET28a];15Ecoli[pET28a
(+)GlMET1],
æç-_ò
16,20,25,30,37℃。
q
4 pET28a(+)GlMET1
²£¤æç-ÛÜÝ
Fig4 ExpressionofpET28a(+)GlMET1proteinatdiferent
temperture
·4201·
5#Ýö
:
Þß
DNA
àÔáâÓ
(GlMET1)
ÔÕãäÐåÜÝæç
242
æçOs
pET28a(+)GlMET1
Ñ ÜÝ
µ¶
Ëæç-
16℃,IPTG
Ëò
04
mmol·L-1,A600ò06£¯,,&£¤æçOs2,
4,8,12,24h
pET28a(+)GlMET1
Ñ Üݵ
¶
,
q
5。
FGæçOs
,pET28a(+)Gl
MET1
ÜÝÁ&>
,
²æçOsò
12h
OÜ
Ýö7
,
ù{æçOsBÁ°íÑ ÜÝ
。
M.
Ñ
Marker;C.Escherichiacoli[pET28a];1~5Ecoli[pET28a
(+)GlMET1],
æçOs_ò
2,4,8,12,24h。
q
5 pET28a(+)GlMET1
²£¤æçOsÛÜÝ
Fig5 ExpressionofpET28a(+)GlMET1proteinatdiferent
inductiontime
243 IPTG
Ë
pET28a(+)GlMET1
Ñ Ü
ݵ¶
Ëæç-
16℃,
æçOs
12h,A600
ò
06
£¯
,
,&£¤
IPTG
Ë
0,01,02,04,
06,08,10mmol·L-1
pET28a(+)GlMET1
Ñ
Üݵ¶
,
q
6。
FG
IPTG
Ë
,
pET28a(+)GlMET1
ÜÝB{ã¯_
,
ù{Ë
IPTG
£ÒíÑ ÜÝ
,
HË
IPTG
ÒêBÁ°íÑ ÜÝ
。
M
Ñ
Marker;C.Escherichiacoli[pET28a];1~7Ecoli[pET28a
(+)GlMET1],IPTG
Ë_ò
0,01,02,04,06,08,10
mmol·L-1。
q
6 pET28a(+)GlMET1
²£¤
IPTG
ËIÛÜÝ
Fig6 ExpressionofpET28a(+)GlMET1proteinatdiferent
IPTGconcentration
244
ÄÖ1¿¢
A600 pET28a(+)GlMET1
Ñ Üݵ¶
Ëæç-
16℃,
æçOs
12h,IPTG
Ëò
04mmol·L-1
£¯
,¨
OæçO
ÄÖ1¿¢
(A600)_ò02,04,06,08¨
1
O
,
,&
pET28a(+)GlMET1
Ñ ÜÝ
,
q
7。
FGÄÖ1¿¢
(A600),pET28a(+)
GlMET1
ÜÝBbø~Ûî
,
²
A600ò08OZí
Ñ ÜÝ7
。
M
Ñ
Marker;C.Escherichiacoli[pET28a];1~5Ecoli[pET28a
(+)GlMET1],
ÄÖ1¿¢
(A600)_ò02,04,06,08,1。
q
7 pET28a(+)GlMET1
²£¤ÄÖ1§ÛÜÝ
Fig7 ExpressionofpET28a(+)GlMET1proteinindiferent
culturedensitiesbeforeIPTGinduction
25
Þß
GlMET1
ÔÕÜݪÝ
ò[Hú×ÞßàÔáâÓÔÕÜÝ5
£
,
N
@ò
RealtimePCR
()Ñ!_ÙJ
Þß
3
ÖƳ?¨
1
_°³?núÜÝ
±
,
q
8。
<Äãÿ
GlMET1
²£¤³?Þß
$ÜݪÝB{ã±
,
9²£¤°±O}B
±
。
[÷
,GlMET1
²£¤³?sÜÝö
]ò_°Þß
>
ÿß
1
<
>
×ß
1
<
>
$ß
10
<
;
r[÷
,
£¤m¾O}
GlMET1
Üݪݲ
4
³?$:Üë$h}H°¼y}
,
ù{
GlMET1
ÜÝöFGÞß°±m¾ÁÛî&>
。
3
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