免费文献传递   相关文献

Cloning and High Prokaryotic Expression of the Light Harvesting Chlorophyll a/bBinding Protein Gene from Bamboo(Phyllostachys edulis)

毛竹捕光叶绿素a/b结合蛋白基因 cab PhE1 的克隆与表达分析


Abstract: The light harvesting chlorophyll a/bbinding protein is one of key proteins in the transformation from light energy to chemical energy. An open reading frame coding precursor protein of cab gene was cloned from the first strand of bamboo cDNA through RTPCR methods, and named as cabPhE1 (cab gene 1 from Phyllostachys edulis EF207229). The sequence analysis showed that the deduced polypeptide was highly homologous to some other CAB proteins from monocotyledon, and the gene belonged to lhcb2 family. Tissue specific expression showed that cabPhE1expressed higher in leaf than sheath and stem. The prokaryotic expression vector of cabPhE1gene encoding the mature protein was constructed by subcloning the fragment into pET23 a and was expressed in Escherichia coli induced by IPTG. The molecular weight of the induced protein was about 28 ku,approximate to that of the mature protein. This work is a key to the further research on in vitro reconstitution of lightharvesting Chl a/b complexes.


全 文 :! !"" ! ##
$ % % &$ # %
& ’ ( )
’()*+,)- ’)./-* ’)+)(-*
/012!"*+02#
3456*
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
$ % % &
34r_OPQ 4 7 88Wjk(g !"#$%&’( %stg5u)@"
;~(9 * >$ *)%9 Y*##
992 ;‘4q9d:g ;@&’A4q()G$OEFZoGHI JK 9%%9%$L
$2 :;()>1f<=? JK 9%%%&#L #2 :;&’()<=>&’<=? JK 9%%%&9Q
$%&k 94LLbrrI 4 7 853n5|HLk37)4ibL‘pib
’()*+#’:9;2!<1=&!#2$ ,-./0#- ,12+#9%%9 > :!;;3$%%&4%# > %9!" > %"
wxy#k$%%; > %# > 9#g
|}z{k;@&’A &!;z{9$%%! > ! > ";QL;@&’AEFz{9$%%: > 9Qg
"V"ds,-./g
!"#$%$& ’$( )%&* +,#-’,.#/%0 123,455%#$ #6 /*4 7%&*/ )’,845/%$& !*"#,#3*.""
’ 7 9?:%$(%$& +,#/4%$ ;4$4 6,#< :’<9##9!"#$$%&’()"#& *+,$-&Q
@40 ABCDCE9 .CF (BGEH$ .CF ICEH1C9 JGEH ABGEBF4#
992 )*+ ,"#-."/-.+ -0 1"2#-- "34 5"//"3 6!7*3!* "34 8*!&3-9-:+ -0 6/"/* ;-.*3/*.3"/7-3"9 ?*3/*. 0-. 1"2#-- "34 5"//"3 1*7@73: 9%%9%$L
$2 >33=95/,’0/k ,BG 1CHBK B45LGMKCEH NB1050OBP11 4 7 8?8CEQCEH O50KGCE CM 0EG 0R SGP O50KGCEM CE KBG K54EMR05D4KC0E R50D 1CHBK GEG5HP K0
NBGDCN41 GEG5HP 6 -E 0OGE 5G4QCEH R54DG N0QCEH O5GNF5M05 O50KGCE 0R !"# HGEG T4M N10EGQ R50D KBG RC5MK MK54EQ 0R 84D800 NU+-
KB50FHB V,?J(V DGKB0QM*4EQ E4DGQ 4M !"#$%&’(9 !"# HGEG 9 R50D %&+99-MB0TGQ KB4K KBG QGQFNGQ O01POGOKCQG T4M BCHB1P B0D010H0FM K0 M0DG 0KBG5 (-Y O50KGCEM R50D D0E0N0KP1GQ0E*4EQ KBG HGEG
8G10EHGQ K0 9&!#B R4DC1P 6 ,CMMFG MOGNCRCN GZO5GMMC0E MB0TGQ KB4K !"#$%&’( GZO5GMMGQ BCHBG5 CE 1G4R KB4E MBG4KB 4EQ MKGD6 ,BG
O50S45P0KCN GZO5GMMC0E LGNK05 0R !"#$%&’( HGEG GEN0QCEH KBG D4KF5G O50KGCE T4M N0EMK5FNKGQ 8P MF8N10ECEH KBG R54HDGEK CEK0 O*,?
$# 4 4EQ T4M GZO5GMMGQ CE ’4OO50ZCD4KG K0 KB4K 0R KBG D4KF5G O50KGCE6 ,BCM T05S CM 4 SGP K0 KBG RF5KBG5 5GMG45NB 0E 73 C7/.- 5GN0EMKCKFKC0E 0R 1CHBK?B45LGMKCEH
(B1 4[8 N0DO1GZGM 6
>4. ?#,(5k 84D8009 %&+99-GZO5GMMC0ELO50S45P0KCN GZO5GMMC0E
b3.oV{#|&*(wU|^*‘q1f
b3.ob+UL)G=OrGV,9Lb@In
5$3D9.\(!*.\("Q.iFU*f: .\("V
r@1frrDSxD:;e_MNUs[1dn
59y>Kq*$%%%4Qg.\("Uo4rGVj
9&!#(*9&!#B*9&!#D*9&!#E*9&!#F Z 9&!#G ][hX4
0‘Up|H?T*5;XEG@I‘?I538
F .\(Y9*.\(Y$*.\(Y#*.\(Y!*.\(Y" Z .\(Y<
@In5$3D.=Kb+=O‘F{‘Xvqm+
9]1CDDGS */ "9 6*$%%/D)<=7KU@A*u .\("$3DU/D5
6UXeym$T9uHq*9&&;L.CF */ "9 6*
$%%!Q*ufm+U<=5iL^g
.\("VsREGUhn5*f1fb3.o
9^:{lLgU.og,|HUX]‘bTbi
b‘f?Tn5RDM%jrrDU9^‘ua@+
f $RbMCa4UXv3SxDh[N56UT
b*h3n5dUl+{Z@IGn5UEkq!
o7j*R.smTMC*u .\("<=2jmU<
=9(4RR455C */ "9 6*$%%!Q*isZA4bU^L<=
a|2>?U}~gEAU<=ij*,9fct
6JFibU@In5G@IfD5mh<=E
k*Rbr74|6G12U .\("‘B^j*RDU56G12U .\(Y9 U56/|xst
9J4F1MGE */ "9 6*9&&%Ly>Kq*$%%%8QgH@*,
9fct6J:ib .\(Yn5V<=f56Zm
+Us[AIlWg
4bSA&sp‘ie‘q7F*4Cmhv
o*45mh,o*VB,UA[ob9M9:*
$%%$Qgx<=hEGUqZ4[949%&+99-*4A97!"#$% S|H #&$’(")**R3 #&$’(")* |H?TF
pn5U:6&XF*6AF‘pibVD !"#$(’)$*+"%$,’-./0*fc
t6J 12&394"%Q:-#ib*h#s<=4bL
brrI ’ 5 )53n5G@IUD5Ek74S:
|^*,orSAIZ@Z_f7o4ba|4Ak’g
! A2gn+
363 ^#<= o."M7OZZ{UU94[
bJ[*CZFV|G&K.U;@&’A4q(
)G$OEFZoGHIUCZ4*+f/Erf
oU^Hg
36& " 789Z[\ (489bi .o 2:;<-/=>0:F
$U #/0:-a+94G&Ko:/ErfU
789 A’= +, &! B*&CCDQ*o */=,0>’F$U_F9^
#p3F (489g
36% X8Ncd]]^\49 h’r4Lbrr
I ’ 5 )53n5|H@a:UhX|H:69‘(
{q*&CCEQ#k *F7 Pf*Pf 3kGH$9#AAFFAF
A#FAAFAF#AF$%H ZPf &k GH$#F9F##FFF#AAA
9FA99A$%HLj|O&>&f>^$Ow*A$3Fgh94 (489 smj*<==_ *F7gP
f 3ZPf & _JHIskIJ K*G ,<:LIJ K 3
,<:*GG L DG K 3 ,<:*E& K 36G ,<:*%G RGbLE&
K%0 3C ,<:g*F7 if|rj !A"M$#0’NO VD
M|!*4JK}-K(($A489P#97‘PM97#Z F@.N-@ Qq&fDIXe|:
U (489:63f?Tn5dU567F*RG;
‘p+Zn5d\’%q9<= )@’N-d0Xeg
36J #&$’(")* X8NYcef~/gh] h9
4 9(-<:|H9RSDC3I3TQ.F"*7o 7#$*F7K:
: #&$’(")* |Hfghd4:Uibeg*F7_
JHIkIJ K*G ,<:LIJ K 3 ,<:*GG K 3 ,<:*E& K
3 ,<:*&TRGbLE& K%0 3C ,<:g
36G #&$’(")* X8iY/gj2Nk: h’
#&$’(")* |H?TFpU:6Z‘pibVD
9!"#$&%’QUo|riF*.oVw72^WiFU
7K#k2Pf EkGH$ (’-’->AFA9FF9FF9FF9#A9 $
%HZPf TkGH$ (-(>’>F##AFFAAAF9FA99A $%H*Pf
Tg6*fJTb*h[oG UU4B6wg
so2 -"+!Z ."/!F^W3{U|HfYj !"#$&%’Uo|riF*8FEkibVDg
36D #&$’(")* X8N7l/g 3 #&$’(")* |H
UEkdzJo94"%Q*s+K|r*qJR *F7M|!*CZ9qg
{3QJ 3CC5wQU;;JydIU V1CZ|
:*OJ5&st 9DCC s C6D L C6T i*2T 2*#A
<=-#CZ!*wpJD<=n5" 89g)@
&63 X8Ncd\49 h94 (489smj*P
f 3ZPf &U *F7iff 3WUjmkIJ:<
K*"1o@5hBT*f TCC )!NOAsH|69V
kQ*GU|U|HfY|x^x*q&::s{U
|HfYgo|O&+-r&f($Apyw *F7if*K] */=,0>’U !A"M$# 0’NOVD
pngw^#p:.%^*3ywU 489fY2 9
!gwjVD|*F{ct6J9 )0#"+12#"2& #/!2Q
4UG"Jt*qS5@As*a+04|rdzR^
WVrXe!*4JK}-K(($A|:5hij*U*?T &D% R;|+9A0:01’:X 8=B "R&CE&&IQg
&%Ci?;LTULbrrI ’ 5 ) 53n5m+
>*! %J Y %D*%T Y JC*&%& Y &%Jisn5a^ FU
l+{iF*! &3J Y &3EisXk|{iF*&C% Y
&CDis6;+a^#l+{iF*%C Y %G*3CD Y
333*3&C Y 3&G*3ED Y 3T3 is 8 Y k7G{iF*
%J Y %D*%T Y JC*&%& Y &%Jisn5a^ FUl+{
iFg #&$’(")* |H?TUn5UqdeXEs G6JJ Z &T JDC6%J .gq|H*6s
#&$’(")*9 #&$ >0:0 3 Z/=, (" B +34!20QgJo 1@’N-
fdDI3 #&$’(")* U:6GEv-U|H<=
hX4d0*5hBT #&$’(")* |HIU !"#$% S
|H@a*?TLbrrI ’[) 53n5gqfd
DI9+--!k[[\\\B ()N B ]-. B ]X[N0/;<(0N[(+@=/=*[QU|*
f?TU;|+! 3 Y %Js#4*a!sFpn5
9V 3Qg
&6& #&$’(")* X8mPQQnN/g a+94
rf‘r8‘oFZhU 789*_F9F (489!s
G*94 9(-<:|H.F"*TG (489oeZGb
\*KF"|HUibM_stghTG!U (489
mje<= #&$’(")* |HUVWg5h9V &Qi
jk#&$’(")* |Hfrf‘r8‘oF’Aib*f
:frf:UibM__‘*fr8ZoF:Ui
bd0w}Lofh:3oM|gjibg
&6% #&$’(")* X8iY/gj2Nh] .o‘
[^ 9^!O/=)0N- !=@O,0/’N0Q<= *F7gVWifU
IJDJ3 & ’ ( ) JG"
V ! !"#$%&’( ?TUp+:63fp#LU;|+:6
"#$% ! &’()*+,#-* .*/’*0(* 10- -*-’(*- 12#0+ 1(#-. .*/’*0(* +3 !"#$%&’(
#4 45*(’5.+5 !!L Fpn5 61,’5* 75+,*#0%
V 8 !"#$%&’( |HU 9:;4<9 Xe
"#$% 8 9:;4<9 101)=.#. +3 !"#$%&’( $*0*
!krf >*13L8kr8 ?@*1,@LAkoF ?,*2LBkh 9++, %
V A ‘pibVD 7C:;(1D;4@C!;21,’5*
"#$% A 45+E15=+,#( *F75*..#+0 G*(,+5 7C:;(1D;4@C!;21,’5*
OAsHNtU|69VOkQ*GU|U|HfY
^xg3ywU H&IfY2 I!gwj :;*1.=V
D!|:*5hijF )&*!Z +&,!ZFpn5U?TUgh 7C:8A1
sVD*o )&*!Z +&,!F^W*3 !"#$%&’( |H
?TFpU:6&X9 !"#$%&’($21,’5*QVw7C:8A1VDUo|riF*Y6A2;A :J de
b‘!"#$%&’(;21,’5*‘k;+:6"n9M#.I:1$QU‘
pibVD 7C:;(1D;4@C!;21,’5*9V AQguEkd
z<=^WM|9Qg N>8!9HCAQ*oUn5U-#ibg
8OB !"#$%&’( X8N7l/gh] h !"#$%&’(
|HEkibVDUK|rJRsmj*oPf A
ZPf B <=M|*hihEkdzZPsu]g
JR 4<9ifU;A{U|HfY*moUQs&^Hg
K]/s7K9>#’ *- ". %*8KKPQw[K|r*
XEf 8L‘AK‘AJ QHIQCZ*t IRKK s KOR S
KOPi*2T T4:U<=-#CZ B @!*wpJD*
o !VU ?H?Z$+TjJ!*]g*|NBT9VkQ*Ekn5’+2 T4:U-#*f-#i
bUn5Xbde{s 8POK E’*G >MJB!! A# ‘({qk94LbrrI 1 W D53n5|H !"#$%&’( U|rGibXe
V ! K|rJR "#$%&’( ! "#$ )*+,-./0 1&/2 0&3’45 .4+36 .25.75,
86 545./*+)2+*50&0
9*:*;kK|rJR <&3’45 .4+36 )*+,-./L!kdzu] "4=0>&,
.+3/*+4L?kPu] @:A .+3/*+4L
Bk9 78Xbe"8 9 78 >+45.-4=* >=*75* (
mT^st9CD248*=3,/ !" #$ (*9EE!Q*+Vgh5_
4y)gjBg
so ;F GCZ*f-#CZiXE2T H"IJ
9*3_Q9K*:?*;K*;?*!K*!?*?K*9KK*9 KKK
!>+4IL
M 9*5hBTJl H"IJ3_UW2Ekn5
UibejB^(Qlmn9VkQ*f:h2T9*
3_Q9K!>+4IL
M 9 H"IJgFUibIh_?*Ek
n5ibe‘*‘{Un5ibe_bg
h;A3VDdz9)NI:;=QU|rsu]*^
hCZHIQ9;F G*:!K *I>&3M 9QCZ ; 2!*o 9K
!>+4IL
M 9 H"IJ9*3_Q<=-#CZ9;F G*9FK *I
>&3M 9Q*XEf-# 9*:*;*! 2!+g<=n5<
KXe*5h9V ?QBT;AEkdzVDU|r*
f{Un59:OPK 7-QUibeJli4U%sV
jBW2U*‘B‘Lou] )NI:;=f :OPK 7-*AH6*&.j2ibifU4:4g
! jb
%#&’()*+ |HIU $)%&, S|H@a*f?T
Un5G."Kbr1f94U QRSK:!T!‘PJ
9 -./0# 1#"23#QU RQQKK?;F‘2 9 m 39 42$256
$7892:$7.56QU QRA:KO?:‘+h2:9;51# #%5628#"#Q
U QR%FK9?F h3."Fbr1fq(9<2%7"2#8#
"#&#%56QU ":F!E‘;W9 =6#.#8")51 )/>7%)78?.2#%51Q
U #QQ?:F?K‘r[9(#8#@ 9281!89QU RQN!T;O;‘?
NO9 (!"582# )/&.2?#QU "9:KT:‘<=9 A7$#856
$/%7>!.12%56QU "9!:FOUst46f OTUh|*bX
BT2|H@af<{|U[_4gfhs1f:
$)%&, AoR|Hwx*f?TUn5da4AN+
V ? )NIV.=8V"2N9V>=/-*5fct6J:Uib
%&’( ? NW)*500&+3 +X )NIV.=8V"2N9V>=/-*5 &3 * ( %7$2
9*!k04|r2 H"IJ-# "+0&/&Y5 .4+35 &3,-.5, 86 H"IJL:*?k0
4|rg2 H"IJUu] "+0&/&Y5 .4+35 .+3/*+4 1&/2+-/ H"IJL;*Tk
2 H"IJ-#U )NI:;=u] )NI:;= &3,-.5, 86 H"IJLBkn5d
Xbde"8 "*+/5&3 >=*75* (
Uy)9Q4453 !" #$ (*9EE:Q*+Vn5U56Gm+
D.fs:Uy)ghV."94U $)%&, S|H
%#&’()*+ G %#&’()*,9N%:KF:;KQfn5P?Ust
4s ETU*5;V!.f56Zm+|Uy)*f
y)VjUf?hURDq9gh;|+iFU2
>*SVjgh|HT?Ti&U*&Guf.&UX]‘6{<=g
%#&’()*+ |HUk37)4ib5hij*f
r@k3&X9rf‘r8ZoFQ%#&’()*+ |HU
ibM_d0‘*ofh:D3oM|gjg %#&’
()*+ |H.sLb@I53n5|H*fibVf
b3.oUb_Jn?###rrDUSxD:
9Q4453 !" #$ (*9EE:Q*iGrrDfghk3:UX
Y^stg %#&’()*+ |Hfct6J RH:9 ZN;Q:
-#ibi@(*gh5_9:?*;K*;F GQHIQ’
+‘Iib*i3A7UEkn5U6{gf<=:@(JlCZi4U%s*gq
H"IJ-#U; %#&’()*+ |HUct6J:/+i
&{Un5H69V ?Q*ofCZ|:72QRk
U7K.lb|^ibU^Hoj*?i&U{U
n5H6IU|^P?ibg45*sG )NIVD
O!9 & ’ ( ) !?"
U-# !"#$3_f %&’ ( ) **+,I-. )a4*x<=
:-#_"3_s )%!*+,I-
. )*KKbU /0#VD
pGU-#3_*im+Gq|Hf 1!2)9304Q:
U|^ibP?0‘ALg
M N , -
‘({*4z?*V"d*q 5 2%%6& 4bLbrrI 7 8 953n5|
HNsU|rZ:6Xe 5 &’()*’494Qk4’ . 4:&
M9: 5 2%%2& US4q 5 ‘0kRt()$OL%& 5
y>K*e 6*4>>*q 5 2%%%75 ‘q1fbMC"Lb@In
5$3D56Gm+<=U/. 4%)&
y>K*4>>*9c?*q 5 2%%%95 @k -<=92|Hfct6J:
U‘Iib3fibif@IUEk 5:;()*4%9’Qk4>4 .
4>;&
u H*{ r*AU&*q 5 );;:& TVZBW -?@"U?^5/
3f56Uq&Xe 5:;()*2:9’Qk4’4 . 4’:&
A,,BC D 3*EF7B"7CFBCC7 /+,L/B/FGMBK GC NH7C7 7CM KFH+*7 *B*9H7CBK +J K/GC7=<5
",7CF "&
@7JJ7HHG E*@H+=B P*@7FFGQB,,G -* !" #$ 5 2%%’& A ,++R SGFMGJJBHBCFG7, 7C7,LKGK +J F7CFBCC7 =+*/,BU +J 6
. ;’6>&
$7+ X& AC BJJB=FGQB *BF<+M J+H F+F7,
PZA GK+,7FG+C JH+* 97*9++5 @O94QkO2 . O’&
D,G**BR [*EW\MGC A*Z+VFK+K @* !" #$ 5 2%%>& A9VCM7CF,L 7CM H7HB,L
BU/HBKKBM -<= /H+FBGC NBCBK BU/,7CFK5 ",7CF "D]<,9H7CMF ^*^ 7CN 3 Z*[VWGL+K<7HQBKFGCN =+*/,BU 9L B,B=FH+C =HLKF7,,+NH7/6k>)’ .
>2)&
-GV @7C7,LKGK +J F<7HQBKFGCN =<,+H+/KGFBTMGHB=FBM *VF7NBCBKGK5 a+VHC7, +J 1G+,+NG=7, @. ’;O5
-GV XK/GC7=< *7W+H ,GN’2:9):Qk2:6 . 2;2&
"7V,KBC ?*PV,BH c*PVGNBH ^5 );;%& PB=+CKFGFVFG+C +J /GN*BCF =+CF7GCGCN
=+*/,BUBK JH+* ,GN+QBHBU/HBKKBM GC %&’(!)*’(*# ’+$* 5 ",7CF7*):)k2%’ . 2))&
P+9BHF -*$HIBN+HI a5 2%%>& #/GN*BCFT9GCMGCN /H+FBGCK +J /<+F+KLKFB*" 5 A=F7 1G+=O49’Qk>;4 . 6%:&
!"#$% V W)
;’)! 4# ‘({qk94LbrrI 7 8 953n5|H ’#,-.(%/ U|rGibXe