免费文献传递   相关文献

An effective Transformation System of Sophora japonica

国槐遗传转化体系的优化*


采用根癌农杆菌介导的国槐叶盘转化法,在建立修饰的豇豆胰蛋白酶抑制剂基因(sck)转化体系过程中,对影响农杆菌转化频率的各种因素进行研究。结果表明: 国槐叶片需在黑暗条件下在MS培养基上预培养5天,农杆菌菌株选用GV3101,农杆菌共培养3天较合适; 农杆菌菌液浓度OD600约为0.7,感染时间10 min; 侵染前的菌液和共培养基中添加200 μmol·L-1乙酰丁香酮(AS)对国槐遗传转化有明显的促进作用; 抑制农杆菌生长的抗生素浓度以头孢霉素(Cef)500?mg·L-1最好。PCR和Southern blotting检测证实 sck基因已整合到国槐基因组中。

This paper investigated several factors affecting transformation frequency of Sophora japonica in establishing the modified sck gene transfer system process with Agrobacterium tumefaciens-mediated leaf disc transformation method. The results showed that S. japonica leaves were required to pre-culture on MS medium for 5 d in the dark. Agrobacterium strains, GV3101, were selected, and the Agrobacterium was needed to co-cultivation for 3 d. Agrobacterium bacterium concentration was about 0.7 in OD600, and the infection time was 10 min. Addition of 200μmol·L-1 acetosyringone (AS) to the co-culture medium before infection significantly promoted S. japonica transformation. Antibiotic, 500 mg·L-1 Cef, was best for inhibiting Agrobacterium growth. PCR and Southern blotting confirmed that sck gene had been integrated into the S. japonica genome.


全 文 :! !"" ! "#
# $ $ %$ " %
& ’ ( )
&’()*+(, &(-.,) &(*(’,)
./01!"**/1"
234*
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
# $ $ %
XbLM)}uc5‘}"
+&<5$# = >6 ?@A5 B C! D525
E51 FG&’H)hi()A\])v FG 5$$$76W #1 FG]cqde FG 5$$$6%W
61 FG]d&e}f FG 5$$$#%W !1 vX()vLMAxNhi)GH> FG 5$$5$5d
! "# KgUga>?)@5XbWh)}n*S>bij5klm=,2n~{34E !"#d)}uc$[
v*rvwa>?)}z{5=O40|}GHM#$9%aXbW&(SopAqBS 2&qr3oIqr "
R*a>??s*g 8.65$5*a>?0qr 6RF1MWa>??rs9 9:;$$6) $1<*tu^k 5$ =>?Wvu-5
?rl0qr3vw< #$$!=/08-
@ 5{UxQyE,&drXbLM)},%Q5z|AgWn~a>?hz5{h
0s9z|}~0E’ABd"$$ =C8-@ 5c!MD’El &/FGHAI? J0/GG>?Cw~"T !"# 34‘.1xXb34evM
$%&a XbWUg#$a>?W{UxQyWLM)}Wb%&W{’34
’()*+#&<571!;,K%!61# -./01#, -23+#5$$5 @ TUV#a#$$7 @ 5# @ #6M
3WXYaXYd\]GHxN/lE7;6/ldhJXYE#$$5,,#!!$!5dM
"([<)lmAgM
!" #$$#%&’(# )*+",$-*.+&’-" /0,&#. -$ !"#$"%& ’&#"()*&
LH3?C M>3/4>?C5*# 83? N>?C6 O>? PA>0F?5 LHF LHA?! P3?C QF3B3?C5
E5 R $%&&’(’ %) *+%&%(+",& -"+’."’ ,./ *+%0’"1.%&%(2**’+3+.( 4%5’!052 6.+7’5!+02 *’+3+.( 5$$$76W#R *’+3+.( 8,.(!1,. 9:5!’52 *’+3+.( 5$$$6%W
6 R *’+3+.( ;,5/’.+.( ,./ ;5’’.+.( *:5’,: *’+3+.( 5$$$#%W!R <.!0+0:0’ %) ;’.’0+"! ,./ =’7’&%>?’.0,& *+%&%(2*$1+.’!’ @",/’?2 %) -"+’."’! *’+3+.( 5$$5$5d
!1,&*+%&a +H>S T3TAI >?UASG>C3GAV SAUAI30 B3WG/IS 3BBAWG>?C GI3?SB/I=3G>/? BIAXFA?W4 /B -%>1%5, 3,>%.+", >? ASG3J0>SH>?C GHA
=/V>B>AV !"# CA?A GI3?SBAI S4SGA= TI/WASS Y>GH @(5%A,"0’5+:? 0:?’),"+’.!Z=AV>3GAV 0A3B V>SW GI3?SB/I=3G>/? =AGH/VR +HA IASF0GS
SH/YAV GH3G - R 3,>%.+", 0A3UAS YAIA IAXF>IAV G/ TIAZWF0GFIA /? 2& =AV>F= B/I " V >? GHA V3I[R @(5%A,"0’5+:? SGI3>?S*8.65$5*
YAIA SA0AWGAV*3?V GHA @(5%A,"0’5+:? Y3S ?AAVAV G/ W/ZWF0G>U3G>/? B/I 6 VR @(5%A,"0’5+:? J3WGAI>F= W/?WA?GI3G>/? Y3S 3J/FG $1<
>? 9:;$$*3?V GHA >?BAWG>/? G>=A Y3S 5$ =>?R ,VV>G>/? /B #$$!=/08-
@ 5 3WAG/S4I>?C/?AE,&dG/ GHA W/ZWF0GFIA =AV>F= JAB/IA
>?BAWG>/? S>C?>B>W3?G04 TI/=/GAV - R 3,>%.+", GI3?SB/I=3G>/?R ,?G>J>/G>W*"$$ =C8-Z5 ’AB*Y3S JASG B/I >?H>J>G>?C @(5%A,"0’5+:?
CI/YGHR D’E 3?V &/FGHAI? J0/GG>?C W/?B>I=AV GH3G !"# CA?A H3V JAA? >?GACI3GAV >?G/ GHA - R 3,>%.+", CA?/=AR
2#0 3-*4,a -%>1%5, 3,>%.+",W@(5%A,"0’5+:? 0:?’),"+’.!W3WAG/S4I>?C/?AWCA?AG>W GI3?SB/I=3G>/?W-’?+%01+!, "+.’5’,5+,W
>?SAWGZIAS>SG3?G CA?AS
XbE-%>1%5, 3,>%.+",d*l(E-ACF=>?/S3AdbT
’_i*qzvXFmE)**5%",g8Kg8-.(Iu5‘7TO*yX=AZ,
/qMSFG*iS^$zo5Xb~ic@*%&
Gb#":FG015%KM[!z#zoXb2
*’3F)4R*rs./, 5!OzoE5&Oe*
5%7"d*63FH5,b%&E -’?+%01+!, "+.’5’,5+,d8
7’E@>1+! ?’/+",(+.+!deM’389Xb5hih
z*J^Dvw:];-MY-SXb5/_v*Z
(^*‘(r08?8TI5
t"hz#s89*Cb"sO4R5’ @+>R
<=@uM
9:_ihi\]5‘AxN*l$34Z[
y’@’,K{’34qCqNxXb{’B*c
ss)6fMZq D3IS/?SeE5%7;d"TO-T6
z|}LM)}{b,K34fS&IZbv9
:*9B5&I34Z[GH_%LH|NMG‘
r-TEB%>:&:!d8ITEB+.:!d8@TEC:",&2>0:!d8=
CED:(&,.! 5’(+,de #$ @:TO|}O)}GH*
\vHQC‘-%O)34_sE(O&e*#$$#dM
[!*1(XbLM)}$[vD;5vw4-E
@*vuB,_u‘’C}*,OXb{’)345
GHFGr#HIMPdS-#@pIzU{+5
3noGHvwXb)}z{5=X40*‘})}c.*zJ|Isfg34Z[y’rXb|
}{’BLMo7M
5 KrAmn
565 6789
>gXbm?d_ZFG&’H)_ihi])(TUV*1XbO-KxB*SL< !"#$
%&’ ()8*+ %l ,$$ ’&% ()8*+ %5 -.E-/01234)5 !"
#$ 6*%7!8dqr3o|IsMa‘$%x*ddZ)
%&’ 9(zoM
!"# wHIJK
+, i j 5 %&"’ 3 4 5 _ i 9 : g u
:#4;!.<=1Uga>?E ()*+,#%"!*’-. "-.!/#%’!01d
?s >?@%’%N~*?sluO :#4;!.<=1vX(
)vLMAxNhi)GH>FPTUV#>{Q
RM>+\S34) 0&""EB~0T?)E23
4d*0&" lj$ij5klm=,2n~{34
CL*JUVJWWXE91/A4BACD50 (C2149 E40/2d5
@F.YM-)~*\ G"HI$.’34###B %M
B % :#4;!.<=uOBY
J4)6 % H41)01( CB :A12(4K :#4;!.<=
!"$ LMJ;NO
_xg()u5a>??O*S+,]Z~0
E=(*F’ ()8*+ %dAf[7EL4B*F’ ()8*+ %d8O{h
0577EMN#qr3dol]*q( 8O P\]^*
qr*hzxh05ru MN#qr3*( 8O P\]B‘aqr
E8’’ 08(4;+ %d$bM
!"% PQRS!TU
_hzcd5m?dW& Q%! e*0SIqr
qr3oIqr8 R @RMgWhnESC0293 !" #$ 6*
%7TFdsIqr5m?W&*S?rvfu %’ R
%F (4;*_xW&*gm?g#hZB*)’0qr
qr3v( 8F Ppqr8 R @RB*cBsW&)’
C}(*qr3v*8F P5iAqBqrMMa*
+x5{Bid)’hU(*qr3vj@hUM
W&Iqrqr3#ma-. U !"#$ @&’ ()8*+ %
U ,$$ ’&% ()8*+ %WW&0qrqr3#ma-. U
!"#$ @&’ ()8*+ % U ,$$ ’&% ()8*+ % U $. %’’
#(CA8*
+ %WW&C}(*qr3#ma-. U !"#$
@&’ ()8*+ % U ,$$ ’&% ()8*+ % U >Q%T T ()8*+ % U <5B
F’’ ()8*+ %WhU(*qr3a%V8-. U ,#$ %&’
()8*+ % U >Q%T %’ ()8*+ % U <5B 8’’ ()8*+ %M
!"& >?VWLMJS!-.;VX
_hzcd5m?dW&*j$vuB(*{
B_sMzUCLqvw)}($5 O:O:4-
’y*z#Sf%x{B9:_s53noo|=
:)}4-*%xI:d(8kA5)}c.M
%&F&% )*+,- vu-sW&CLSqr3
-. U !"#$’&F ()8*+ % U ,$$’&’F ()8*+ %oIqr ’*
@*Q*F*!*OR*sIqr5m?W&*S?rvfu
%’ R %F (4;*_xW&*gm?g#hZB*)’0q
rqr3v( 8F Ppqr 8 R @RB*cBsW&)
’C}(*qr3v*8F P5iAqBqrMMa*
+x5{Bid)’hU(*qr3vj@hUM
-l‘J5Iqr^kr)}z{5vwM
%&F&8./01 S WH!’’mCL) ’&@*’&F*’&O*
’&7*%&85?rvvu %’ (4;*gm?g#hZB*
CL)’0qrqr3v( 8F Ppqr 8 R @ R
B*cBsW&)’C}(*qr3v*8F P5i
AqBqrMMa*+x5{Bid)’hU(*
qr3vj@hUM-l‘J5?rs9r)}z
{5vwM
%&F&@ 23..4 4g #,W"L$H:;5Jmn
s_i9:gu :#4;!.<= @’Uga>?
*#$QQ’Q*>?@%’%*NS$%’Fv*HumnxOz*k
%o|}MsXbm?dW&*CLf’;}!5
@O?rvfu %’ R %F (4;*_xW&*gm?g#
hZB*CL)’0qrqr3v( 8F Ppqr
8 R @RB*cBsW&)’C}(*qr3v*8F
P5iAqBqrMMa*+x5{Bid)’h
U(*qr3vj@hUM-l‘J5a>??s
r)}z{5vwM
%&F&Q 56,- S WH!’’) ’&O5?rvvu 8*
F*%’*%F*8’ (4;*_xW&*gm?g#hZB*CL
)’0qrqr3v( 8F Ppqr 8 R @ RB*c
BsW&)’C}(*qr3v*8F P5iAqB
qrMMa*+x5{Bid)’hU(*qr3
vj@hUM-l‘J5vu^kr)}z{5
vwM
%&F&F 789:; %dSUga>?qr$[
v*govu5?rv*-l<’ F’’#(CA8*
+ %{U
xQyE@*F"K4(5X3CYZ"Q"3ZK0C YZ195XC :35;C;5*$.dl
‘<{UxQyr)}z{5vwW8dS0qr
$[v*-l0qrqr3v<’ 8’’#(CA8*
+ %{
UxQyl‘<{UxQyr)}z{5vwM
%&F&! <*+,- W&vu-BCL0qr %*
8*@*Q*F R*sW&CL)’C}(*qr3v*
8F P5iAqBqrMMa*+x5{Bid)
’hU(*qr3vj@hU*-l‘J50qr
^kr)}z{5vwM
%&F&O =.*+ XbW&0qr @RB*CL)
uxL<|}~0E95BCX1Y4;5*<5Bdlpqr~0
E910[5;494AA4;*<10[d‘Js9E’*@’’*Q’’*F’’ l !’’
()8*+ %d5(*qr3o|}qr*F! RB-lC
%8! F# isteaXbLM)}uc5‘}
}pq{./uveN5j@pqM
!"#"$ >?@A?BCDEF _hzcd5m
?dW& %&’e*0SIqrqr3oIqr #RM
gWhnsIqr5m?W&*Sw< &((
!)*+8,
- !{UxQyb ./0(() ("’ 5?rvfu
!( )12TU*_xW&*gm?g#hZB*)’J
|+ &((!)*+8,
- !{UxQy50qrqr3v(
&# 3pqr 4RB*cBsW&)’+|}~05
C}(*qr3v*&# 35iAqBqrMMa*
+x5{Bid)’hU(*qr3vj@hUM
J^**+rjvu5 !!0 em?W&*qr[!
JoM
!"# SUV6K; $%&TN
Kg 5678nE9*:;<=>1 !" #$ ?*!@@’dM_ #s)
34_s /A7*z B812"C5D)FBrO*zr)}
_sW& /A7)wBrO*NO %&’ 34yz?/
4ia #E Y) #E 77776F77F7F57557656654EW
4EY) #E6567F7F6657656665657657654EM
S &#!, (f5ucv<’34e( /A7 =
("! G )]7*4is9Z) ("% B)*+*HA69 = &((
!)*,*(#) ;12 ! IMf5[!) @# 3I3B
# )12*B @% 33B ! )12*## 3@G ! )12*’& 3rs
! )12*40:@ig ("$Jab7cdJew~M
!"’ SUV6K; ()*+,-./CDTN
g *+,H#2ouO B812"C5D*qcdvDT6
%!# E!K4&9dH569E!(!518!,
-!d!!, 0y\S{|Ms
95Lw~FB5_s /A7|*|Isf}*Pz|
*_ !(!Gg *+,H#2o6 !0 MB|}ab7cd
Je*cds9) !J*4# NJe6 !# M*z{|J)
Ens/A7)EhO1yz&’A){|$K4& 9\SB 0# 3xy$bM(
- ’( 30yZQv ’R*!" Ri&{O&SW#
$M
& #$ACD
0"! YVB:S!-.;>?
&"!"! GH)*+,-IA?BCDJK Xb
5W&J+=,u*WuF#*IqrrXbW&C
}!,$5*6z%hv39&*".ZbhPL
q*qC,f(a>?5tuWJ^6z%Tja
>?vuB5’()}ES;TU !" #$ ?*!@@#dMqzU
?q5rO#$E9 !d9%*‘|}Iqr*tug
a>?tu5W&ph{B*5z{) (*Iqr 4
R5)}z{) &"%J*Qod(rj$Iqr5
W&M.Iqr #R^*W+‘/N,Guv}*m
^5,_uj(*qr*-%5{B*5~ic@*
)}z{) !%"4JMIqr 0 V ’RB*)}z{-
*lx ’"!JM%9%Iqr^k5M.rz4%
XbW&.*a>?vu5fnHH;k*[!$
z^k5Iqr-;ZAiM
^ ! XYuNOZ[:J\]^_X‘da412/.5;>?
3245! 6/78*-/9- )7 :.-;9*8+*.- <*.2+=)/ )/
7.->*-/9? )7 12/. @,))+ :.)<*9+=)/
Iqr^k
9:;KWX+TX:;
T1);YH
,_u~
AX)<;: *Z
;[B+P2T=
{B*(~
6*TP+ 2X)<;: *Z
:;=1=TP2T =M**T=
)}z{
6:P2=Z*:)PT1*2
Z:;\X;2W]YJ
( ’& ( (
4 !&0 4 &"%
% !!% !% !("’
# !&( !’ !%"4
0 !&( !# !!"@
’ !!’ $ ’"!
&"!"& GH./01L56,-IA?BCDJ
K 19 &6]*?rs9r)}z{5vwLH*
.?rs9 ./0(() ("4 ^*Xb)}z{()
0"4J*. ./0((m;d*) !&"@JW. ./0((m;G*l*%k%?rs9$dLvu^k$z*"@
ra>?‘$$9*r_iKr"sX3Ag*@r
,_u)0**l)}({M?rs91lLf2
^k13*uOxv455a>?~i1h*S0q
r^)}({lMvu^kr)}z{5vwJ|
RF*.vu & V # )12^*-%5{B*FhW.v
u^krzh !( )12^*{B*zh5z{cd*)
!("’JWvu &( )12^*67>,,_uSqr$[
v)0*L8-%{B*M%9%XbW&vF9
-*r,ovwI):t*‘MAq5|P-IHA
*l\phz{*4m>bM@5a>?vumn
!s:)}5RF-MM
^ 0 XYJbcd>efZ[:S!-.;>?
32450 6/78*-/9- )7 429+-.=28 9)/9-/+.2+=)/ 2/< =/7-9+=)/
+=A- )/ 7.->*-/9? )7 12/. @,))+ :.)<*9+=)/
|P
6:;PT);2T
,_u~
AX)<;: *Z
;[B+P2T=
{B*(~
6*TP+ 2X)<;: *Z
:;=1=TP2T =M**T=
)}z{
6:P2=Z*:)PT1*2
Z:;\X;2W]YJ
?rs9
5*2W;2T:PT1*2 *Z
vu^k
^2Z;WT1*2
T1);Y)12
("4 @# 0 0"4
("# !!# !! @"0
("’ !&( !0 !&"@
("@ !!# % 4"!
!"& !!# & !"’
& $% % %"$
# !!# ’ 0"4
!( !&( !4 !("’
!# !(’ # %"&
&( @’ ( (
&& & ’ ( ) %#"
!"#"$ GHMNOP23..4IA?BCDJ
K $ O?s %&’(()(**+’#),*-.$#)# CLA)
uO /&01!234 5gug()}XbW&*Sw<
{h05qr3o(*qr 5 ;B./)}({
E9 $dM#$9%*a>? -.$#)#)@5)}{:
x #6")7**+’#), 5)}({)ve*C %&’(()(
S)}v9G5XBFk*a>?5hzF8n~*
SLH[9ovwOXbW&uveN5jslC
}*\)}{() 5"$$7M)}({Rp) -.$#)#
8 *+’#), 8 %&’(()(*k%a>?‘J?srXb
)}({,%QvwM
^ ! LMJXYJK:S!-.;>?!
"#$%! &’()*+’,+ -( ./((+0+’1 210#/’2 -( !"#$%&’()#*+, (+,)-&’*)./ -’ (0+3*+’,4 -( 5#’0 26--12 70-.*,1/-’
?s
29:;01<
,_u~
=>?@A: BC AD/E;19<
{Buv{
F:AG>A1HI BC :A<0<9;19 H;EE0J7
)0{
&:BK101L C:AG>A1HIJ7
C}{
M0CCA:A190;90B1 C:AG>A1HIJ7
)}z{
N:;1A1HIJ7
%&’(()( #() #O ($ $P 5"$$
-.$#)# #,! $5 $) P) #6
*+’#), #(, !$ $) (5 #(
"eNqr ,PR5./#$M{Buv{ Q{Buv~JuO(~ R #))7W)0{ Q)}5,_u~JuO(~ R #))7WC}{ QC}
5uv~Juv(~ R #))7W)}{ QC}5{B*~JuO(~ R #))7MN0<<>A H>E9>:A ,P S :A<>E9A1HI BC :A<0<9;19 H;EE0 Q =>?@A: BC :A<0<9;19
H;EE0 J NB9;E 1>?@A: BC AD/E;19< R #))7W&:BK101L C:AG>A1HI Q =>?@A: BC @:BK101L AD/E;19< J NB9;E 1>?@A: BC AD/E;19< R #))7WM0CCA:A190;90B1 C:AG>A1HI Q
=>?@A: BC S0CCA:A190;90B1 H;EE0JNB9;E 1>?@A: BC H;EE0 R #))7WN:;1A1HI Q =>?@A: BC :A<0<9;19 ?@A: BC AD/E;19< R #))7 T
!"#"( 789:;IA?BCDJK a>?
NVM=’.5N0uOo5 !"# 34785:f=,-sEW;?A< $%
&’ T*#66$dM4m*a>? !"# 3459:;\9:
E7tuvwx)}({MCxQyE’2de*j@ N0uO !"# 3459:*|C4
B NVM=’5)EE29;HUAE $% &’ T*#65PdMPzUKg
L<{UxQyGH\rXbW&)}z{5v
w*#$E9 (d9%aXbW&Svu-5?rl
0qrqr3vL< !))#?BE8%
X #{UxQy*f
pMd)}z{M
^ 8 ghijk49:5:S!-.;>?
"#$%8 &’()*+’,+ -( #+1-240/’;-’+49:5-’
(0+3*+’,4 -( 5#’0 26--1 70-.*,1/-’
,_u~
=>?@A: BC
AD/E;19<
{B*(~
NB9;E 1>?@A: BC
:A<0<9;19 @>S<
)}z{
N:;1C:AG>A1HIJ7
vu^< ’2
Y1CAH90B1 K09U ’2 ##! #! #)"O
vu^‘< ’2
Y1CAH90B1 K09UB>9 ’2 65 ! !
0qr^< ’2
3BVH>E90Z;90B1 K09U ’2 ##( #( #!"$
0qr^‘< ’2
3BVH>E90Z;90B1 K09UB>9 ’2 #)5 ( $"O
!"#", GH<*+,-IA?BCDJK 0q
r^k5z3*tuvwxY5345.1;)}
Zb5~i*qCvw)}z{MzU#$E9 ,d
9%*qr # [ $RrWh)}vw‘H*Cr{B
*5-%vwLH*0qr $R>-%5)}z{
!0qr #R5 ,[@M![*90qr^krz*
x*,_u5~Y;@*[!H@S(*$[v3
283,*cB0=W0qr^kF35pqB*H
@~) 4;1: **/FB:uFhMzUv1xG*
0qr^k>z*Wh95a>?‘$>@*(*q
r^a>?>8En~*?0qr^kz $R)@M
^ < XYlNOZ[:S!-.;>?
"#$%< &’()*+’,+ -( ,-=,*)1*0+ 1/>+ -’
(0+3*+’,4 -( 5#’0 26--1 70-.*,1/-’
0qr^k
3BVH>E90Z;90B1 90?AJS
,_u~
=>?@A: BC
AD/E;19<
{B*(~
NB9;E 1>?@A: BC
:A<0<9;19 @>S<
)}z{
N:;1C:AG>A1HIJ7
# 6P ! !"#
! 6P O O"$
$ #(( #, #)"O
( #!P ## 5"$
, 5, ! !"(
!"#"P =.*+IA?BCDJK Sga>?
)}_iZb^*"g|}~0EHACB9;D01A*3ACdlp
qr~0EH;:@A10H0EE01*3;:@d)~a>?5$ih
zM19 P6]*3ACs9$l*‘f-y)~a>
?hz*qC‘f,(A%x{BuveNW.s9
) ,)) ?L8%X #^*ra>?5n~c%Q*,_u0
={cl*) !O"(7*uveN5j@{cd*)
!)",7Ws9p;<-’(4Bv4Zb)}0
=*n~uveN5j@MC 3;:@ ,)) ?L8%X #^*0
={![D!cl*) !("(7*bv 3AC50={1
Fl*[m^uveN5j@{Fl*) #!"$7M
@CABx=O40*Xb)}^*+|}~0A
)n?{*s9) ,)) ?L8%X #M9Maqrp~5
;<*a>?)+0=*>g|}~0s9D6)+
*lh $)) ?L8%X #M
!"#"O QR>?@DA?BC SrvwXb)
}($5E@40|}‘}GHB*>bOICX
$!! ,# isteaXbLM)}uc5‘}
^ ! XY‘\X:LMJmJnoPpqS!;>?
"#$%! &’’()* +’ ,-’’(.(/* #/*-$-+*-)0 +/ *1( -/1-$-*-+/ +’
!"#$%&’()#*+, #/, *.#/0’+.2#*-+/ ’.(34(/)5
|P
!"#$%&#’%
,_u~
()&*#" +,
#-./$’%0
0={
1#$%2
"$%#34
uveNj@{
5$//)0 6’7)8%6+’
,"#9)#’8:34
5#,s9
5#, 8+’8#’%"$%6+’3
E&;8<= >d
5$"* s9
5$"* 8+’8#’%"$%6+’3
E&;8<= >d
?@@ AB >@@ @C>
D@@ >?B E>CF ?CG
B@@ EB HGC? >>CH
F@@ >>? ?GCB ?@CF
H@@ AB ?ECG >BCD
?@@ A@ >@@ @C>
D@@ >>H EH ?C?
B@@ >@? FFCE ECB
F@@ >?F ?BCB >?CD
H@@ A@ ?GC? ACH
bLM)}ucazIqr DR5XbW&),_
u*A I1H@@m) @CG 5a>??rvu >@ &6’*S
.Jm) FC@50qr3vpqr DREB8! = >d
B*j$(*j@EB8! = Dd8szEB8! = B*Fd
z;hUEB8! = Hde$[*-%{B_sEB8
! = A*Ed*rO_s)+0=EB8! = ?*GdMl$
RRzU%xXb)}z{7Z6:x ?>CD4E9
GdM1m"%PGH>b5a>?)}c.!kA
C&rd(5M
^ 6 r!z{;S!-.
"#$%6 ".#/0’+.2#*-+/ ’.(34(/)5 +’ *1( +7*-242 050*(2
,_u~
()&*#" +,
#-./$’%0
uveN~
()&*#"
+, 8$//6
{B*(~
!+%$/ ’)&*#" +,
"#060%$’% *)70
)}z{
!"$’0,+"&$%6+’
,"#9)#’8:34
zUe
!#0% ;"+). B?G ??> BG ?>CD
rOe
5+’%"+/ ;"+). >>H E @ @
B ? ) .K6’"L5M_s5 N5O w~
P6;Q ? N5O $’$/:060 +, .K6’"L5MR%"$’0,+" ./$’%0 ,+" %2# !"# ;#’#
> Q\DC-ui S+/#8)/$" &$"T#"W?QuO .K6’"L5M)FBrO
N+06%6U# 8+’%"+/WD QO)34_sA)wBrO (+’R%"$’0;#’68
8+’%"+/ ./$’%0WB V AQ)34_s .K6’"L5MR%"$’0,+" ./$’%0Q
898 s\6K;)B\7tuv
?C?C> N5O ST q{B_sM__i34e
1(W*zuO .K6’"L5MA)FBrO*zr)34
_sA)wBrO*|} N5OC;MJew~#$
KB ?>RMr)}_s34erxG N5O C;
&’*)345_sj N5OC;*%x > AlI#
z9I|H55 B>F *.5&’M
?C?C? L+)%2#"’ */+%%6’; UV CLq N5Ow~)
FB5Xb)34_sv9#*+ >@ s*g $%&7
#2o_i( 1(W*!"# 34&’A{|*|}
L+)%2#"’ */+%%6’;xyCD*#$9% Gs-%O%Q
5xyj!*CO)}rO34evrxGi2x
yj!EB Dd*|C"TO !"# 34‘.1|Xb5
34evMq)34_s L+)%2#"’ */+%%6’;5xyB
Yo<*|* F*G l >@ rxGj!*EEA)/F
B_s*\D&QROFk5xyj!*|C"TO
!"# 34‘.1|Xb534evM
B D Xb) !"# 34_s5 L+)%2#"’ */+%%6’; CD
P6;Q D L+)%2#"’ */+%%6’; $00$: +, !"# ;#’# 6’ %"$’0;#’68 ./$’%0
NQuO .K6’"L5M3$%&7#FBrO .K6’"L5M X$0 76;#0%#7 *:
$%&7# $0 .+06%6U# 8+’%"+/W> QO)34_s34e 1(Wg
$%&7#2o (+’R%"$’0;#’68 8+’%"+/ ./$’%*;#’+&68 1(W
X$0 76;#0%#7 X6%2 $%&7#W? V >>Q)34_s34e 1(Wg
$%&7#2o !"$’0;#’68 ./$’%0 ;#’+&68
1(W X$0 76;#0%#7 X6%2 $%&7# Q
D #QAwQ
,_uSga>?uOG-!_(F|}Iq
r*dFv5#$‘IrMI50)Iqr4_i
eNaG;H*6z|ZbCI*CILA5ZbJ
’.1,K 1(W*4CMd,K3453K9:l
kA.1{*J^6z%Ta>?>@r5’()
}E(Le*>EEEWS#%Y ’( )* Q*>EEFdMPdzUD"
TO%I#QM,_u5Iqr^kA\z{,%
QOc*PIO,_uZ,\cMIqr^kMI
qr^k1z*l,_u5)}{*I5z ? V DR
)@*N K O P QE +’(,&%) -./0%1) d8R S
E 2."34’0!%",5 ’!",*’&(,5 d8B C DE 60)/%134!%!
(-)*%)&)d8TVE70)!!%") ")54’!(0%!de5)}vZ( ?
RIqrEL%$82#/ ’( )* Q*>EAHdM[!*1(PdG
Hvvu5*uKroZzQd*W&Fi#*ra
>?5.*fnFn*4m*%x5#$!Iqr F
R5($FMM[S_i34Z[v*D,&f5
#$*,5_iIqrmAg*KU"E8%"3(%)&)
()/)",5d8klE 9%:&) ,&:,%",*)()dlVWE;’1%"):3
!)(%<) dW&Z‘(FIqr*6h1,5_ijI
B? & ’ ( ) BF"
qrfC-*l)}{*KTRSE !"#$%&’()*’
+,-’.,’d8XYCE /.-0(0)0’ .$0(,(101dE!"#$"% ,- ’2 &*
’()*deM(G*!_(FIqr*Iqr@z^k*
FUS‘J_i8‘J,_u8‘J_iOl‘Jq
rc.CAM
?rs9lvu^kr)}z{5vwLH*
\D_i5)}zUDGHOZgr)}z{5v
wE[O\e*+,,-Wd]^e*+,,-d*PzU#$A
mIrM?rs9$dLf2^k$z*"@ra
>?‘$$9*r_iKr"sX3Ag*@r,_
u)0*L8%x{B*W?rs91lLf2^
k13*uOxv455a>?~i1h*S0qr
^)}({lM4m>bM@5a>?vumn!
s:)}5RF-MM
‘JIP_iAa>?5&8Ag=,nP*
K_$E3’241 #4&02’do ./0’,’ ‘( 1!022,28
3-)3’E!"%4$ ,- ’2 &*’((2dW-TE5%#4241do 3-)‘
( 1!022,2E5678##6$ ,- ’2 &*’((-dM4mS_i)}
v5‘‘JTO*+,(5a>??sM
a>? 9:;<0.54e*nF1 98uOo5 60* 34785:f=,
-sM4m*a>? 60* 3459:;\9:E7
tuvwx)}({M_i*vZbCa5S&<
B}1ira>?3459:,j@AgE!"#$"% ,-
’2 &*’()*W=>?6@ ,- ’2 &*’((,dMPzU#$9%a
SXb534)}v*%A-0S_$8-TeIP_iv5GH#$&
IrE=>?6@ ,- ’2 &*’((AWB"4C8% ,- ’2 &*’((’WDC6%@
,- ’2 &*’())WE$>FG6# ,- ’2 &*’()*W!"#$"% ,- ’2 &*’()*W
H6$I8 ,- ’2 &*+,,2dM[D,GH0)*S?rv<’
{UxQy‘fMd)}z{E3"%J>#"%86I8 ,- ’2 &*
’((-WKG>%L ,- ’2 &*+,,,dM
,_uAUga>?0qr!-%)}u5n
FbcMS0qrd’*N~,Y53459:;<0
Sa>?u*-s|_i34eEE$>FG6# ,- ’2 &*’()*dM4m*0qr
^k5z3*tuvwxY5345.1;)}Z
b5~i*qCvw)}z{M
#Z0qr5,_u95;ekeNv*iS
Hia>?*)-%cf5ph_s*"g|}~0
lpqr~0)~a>?5$ihz*CbZgl
n~a>?hz,*1vw_i5hzxNM
="6I@M"eE’((*dHI*|}~0z|hU*n~*5
C}WCpqr~0-z|*C}*n~hUM4
mUS‘JKrS‘Jhzd’5L<‘J5n?
{M
>bd(kA5eNqrphc.!ga>?
vu_iZb-%)34_s5RF-MMPzU
S{+Xbd(phuc5GHv*\phz{6
: ),NzoW[ja>?vuB*phz{cdg
, +’OANM%9%Xb,_uO"9-*‘MAq
5vw-IHA*l\phz{*4mXbJ-h
Jd(5LM)}uc5>bi(|IsGHM
E F - .
) * & ’(-P &vXTICB) &FGa()x89 &
5&O Q 1*8jbj R&’()-&3’k1^P@Q &FGa()x8
9 &
d]^*Llm &+,,- &‘}a>?)@5%n)}uc &FG&’H
))H*+PE2da*, S *2&
(O&*mop &+,,+ &_i34Z[ &FGa()x89 &
( L*&Iq & ’((( &a>?)@5IP_i)34 &hi\]l
m*’*E*da+A S +P&
[O\*ri[*sta*e &+,,-O a>?)@duv)}uc5>
b &=a)H*’(E-daA2, S A2*&
!"#$"% B T*<6@$6I . T*B"I4"% Q H& ’()* & H#>%$ UG6%"#8F F"?U"7%4@
8%47F6 6VUI6@@8"% "J $G6 /7*%+’.-,*04& -4&,8’.0,(1 #"F8 %66464 J"I
W8I7#6%F6& EF86%F6*+A+a()A S ()-&
!"%4$ 0 ;*.LL6I?"%$ X*;I7>I$ H*,- ’2 & ’((2 & /7*%+’.-,*04&:?648>$64
$I>%@J"I?>$8"% "J >UU#6 >%4 >@@6@@?6%$ "J J>F$"I@ >JJ6F$8%L 8% L6%6
$I>%@J6I 6JJ8F86%FY 47I8%L 6>I#Y $I>%@J"I?>$8"% @$6U@& H#>%$ 36## R6U*’Aa
-)P S -(A&
3"%J>#"%86I8 Q*!>#6@$I>ZZ8 0*!8@"JJ8 E &’((- & 5>F$"I@ >JJ6F$8%L /7*%+’.-,*04&
-4&,8’.0,(1:?648>$64 $I>%@J"I?>$8"% 8% @6W6I># M#>F[ U"U#>I F#"%6@& H#>%$
36##*98@@76 >%4 DIL>% 37#$7I6*2Aa+’- S +++&
5678##6$ 3*1>7W6IL6>$ \*;6@C>I$6 3*,- ’2 & ’((- & 98@@76 >%4 F6## @U6F8J8F
6VUI6@@8"% "J > F8%%>?Y# >#F"G"# 46GY4I"L6%>@6 UI"?"$6I 8% $I>%@L6%8F
U"U#>I U#>%$@& H#>%$ Q"# !8"*+Pa*-’ S **P&
B"4C8% ]*9"44 B*5"I4:1#"Y4 !*,- ’2 & ’((’ & 9G6 6JJ6F$@ "J >F6$"@YI8%L"%6
>%4 U/ "% /7*%+’.-,*04&:?648>$64 $I>%@J"I?>$8"% W>IY >FF"I48%L $"
U#>%$ @U6F86@& H#>%$ 36## R6U"I$@*(a*P’ S *P-&
/"I@FG R !*5IY = .*/"JJ?>% < 1*,- ’2 & ’()- & 0 @8?U#6 >%4 L6%6I># ?6$G"4
J"I $I>%@J6II8%L L6%6@ 8%$" U#>%$@& EF86%F6*++Pa’++( S ’+AA&
=>?6@ ; =*H>@@6Y 0 =*!>M>I> =*’((,O /7*%+’.-,*04&:?648>$64 $I>%@J"I?>$8"%
"J $G6 F7#$8W>$64 @$I>CM6IIYE 9*’7’*0’ ^ ’(’(’11’ ;7FG&d 7@8%L
48@>I?64 M8%>IY W6F$"I@& H#>%$ EF8**(aP( S (2&
=>?6@ ; =*_I>$@7 E*3G6%L =* ,- ’2 & ’((A & 0F6$"@YI8%L"%6 >%4
"@?"UI"$6F$>%$@ #8[6 M6$>8%6 "I UI"#8%6 @Y%6IL8@$8F>##Y 6%G>%F6
/7*%+’.-,*04&:?648>$64 $I>%@J"I?>$8"% "J >UU#6& H#>%$ 36## R6U*’+a
--( S -*A&
="6I@M" Q*D[[6#@ 5 9& ’((* & 0 %"W6# UI8%F8U#6 J"I @6#6F$8"% "J $I>%@L6%8F
U#>%$ F6##@aU"@8$8W6 @6#6F$8"%& H#>%$ 36## R6U*’*a+’( S ++’O
Q6$Z 9 ;*;8V8$ R*.>I#6 . ;& ’((- & /7*%+’.-,*04& -4&,8’.0,(1 ?648>$64
$I>%@J"I?>$8"% "J MI"FF"#8E:*’110.’ %2,*’.,’ W>I & 0-’20.’d>%4 F>MM>L6
E: & %2,*’.,’ W>I & .’#0-’-’d& H#>%$ 36## R6U*’-a+)P S +(+&
Q7I>@G8L6 9*E[""L 5&’(*+ & 0 I6W8@64 ?6487? J"I I>U84 LI"C$G >%4 M8">@@>Y@
-+! -# isteaXbLM)}uc5‘}
!"#$ #%&’((% #"))*+ (*,#*-+. /$0)"%, /,’1#*23a456 7 485.
9!+1) : ;*<=">%(?" @ A.28BB . C-’1)D%-=’#"%1 %D )%0&+’1 (+,,) *)"1> ="E+F
)#-’"1) %D !"#$%&’()#*+, (+,)-&’*)./ ’1F G$+1%,"( (%=G%*1F). /,’1#
/$0)"%,%>0*BBa35H 7 356 .
/’-)%1) C I*<"1?’- J /*<#+##,+- K L*)( &0 .28BM . C-’1)D%-=’#"%1 %D G%G,’- &0
!"#$%&’()#*+, (+,)-&’*)./ . N"%OC+($1%,%>0*4EMda366 7 36M .
/+#-" A*@,&*-P*+-P*+ Q*R’-("’OA’)#",,% <. SHH4 . L’(#%-) ’DD+(#"1> >+1+
#-’1)D+- +DD"("+1(0 #% ’G-"(%# ,+’T+) F*-"1> +’-,0 !"#$%&’()#*+,O=+F"’#+F
#-’1)D%-=’#"%1 )#+G).U%-#"( <"( N"%#+($*58a5H4 7 52S.
/%-+&)?" <*N’",+0 R*N’*= N K.2885 .V%F"D"(’#"%1 %D AC@N ;Q@ +E#-’(#"%1
G-%#%(%, D%- G,’1#) (%1#’"1"1> $">$ G%,0)’(($’-"F+ ’1F G%,0G$+1%,
(%=G%1+1#). /,’1# V%, N"%, K+G*23aB 7 23.
<#’($+, < W*X’=&-0)?" / A. 28BM . J"-@ ’1F T"-R (%1#-%, #$+ G,’1#O"1F*(+F
’(#"T’#"%1 %D #$+ CO;Q@ #-’1)D+- G-%(+)) %D ! . (+,)-&’*)./ . A+,,*4Ma
6S3 7 666.
<#’($+, < W*V+))+1) W*V%1#’>* J*)( &0 . 28BM . Y1F+1#"D"(’#"%1 %D #$+ )">1’,
=%,+(*,+) G-%F*(+F &0 !%*1F+F G,’1# (+,,) #$’# ’(#"T+ CO;Q@ #-’1)D+- "1
!"#$%&’()#*+, (+,)-&’*)./ . Q’#*-+*62BaMS4 7 MS8.
X$’1> L :*C’?’$’#’ Z*[’#’1’&+ V*)( &0 . SHHH\ !"#$%&’()#*+,O=+F"’#+F
#-’1)D%-=’#"%1 %D (%#0,+F%1’-0 +EG,’1#) %D A$"1+)+ (’&&’>+E 1#&//*’&
’&,2)/(#*/ :. ))G. 2)3*.)./*/d. /,’1# A+,, K+G*28a3M8 7 353.
!"#$% & ’(
MS & ’ ( ) 43"