免费文献传递   相关文献

Isolation, Expression and Single Nucleotide Polymorphisms Analysis of PtDREB2A in Populus tomentosa

毛白杨PtDREB2A基因的克隆、表达及单核苷酸多态性分析


利用基因特异的PCR引物由毛白杨受干旱胁迫后构建的cDNA文库中扩增获得一PtDREB 2 A cDNA克隆,并进行测序和序列分析,结果表明: PtDREB 2 A cDNA片段总长为946 bp,基因内部含有完整的开放阅读框架,大小为864 bp,可编码长度为287个氨基酸残基的蛋白质,所推导的蛋白质氨基酸序列在AP2/ERF结构功能域与拟南芥AtDREB2A和水稻OsDREB2A蛋白的同源性分别为80.3%和83.3%。组织特异性Realtime-PCR结果显示, PtDREB 2 A在杨树根、茎、叶片和顶端分生组织中均有表达,但其表达模式不同: PtDREB 2 A在叶片表达丰度最高,在树干皮层及根部组织表达丰度中等,而在顶端分生组织及主干韧皮部、形成层与木质部表达丰度最低。非生物胁迫及激素诱导差异表达显示,PtDREB 2 A不仅受高温、低温、干旱与盐诱导表达,还受到激素IAA,NAA及GA3的上调表达,但与ABA的诱导无关。组合利用MEGA4.0和DnaSP4.50.4软件对毛白杨45株基因型个体的PtDREB 2 A序列进行比对和分析,共检测到49个单核苷酸多态性(SNP)位点,SNP频率为1/18 bp,多样性指数π为0.010 48。 在外显子区域,共检测到46个SNP位点,其中19个为同义突变,27个为错义突变。

A cDNA clone encoding DREB2Awas isolated from a cDNA library prepared under drought stress by the gene-specific PCR amplification from Populus tomentosa. The cDNA was 946 bp in length with an open reading frame (ORF, 864 bp in length) which is capable of encoding a protein of 287 amino acids. The deduced protein sequence of the PtDREB2A shares 80.3%, and 83.3% identity with functional domain of AP2/ERF of Arabidopsis thaliana AtDREB2A, and Oryza sativa OsDREB2A, respectively. Tissue differential expression detected with realtime-PCR indicated that PtDREB2Atranscripts had their mRNA products in roots, stems, leaves and apical shoot meristems with varied levels. The PtDREB 2Atranscripts were the most abundant mRNA products in leaves, with medium expression in stem bark and roots, but a weak expression was detected in apical shoot meristems, and in the phloem, cambium, and xylem of stem. We analyzed the expression patterns of PtDREB2Aunder abiotic stress conditions and phytohormone treatment, and revealed that expression of PtDREB2A was induced by heat-shock, cold, drought and high-salt stress, but also by IAA, NAA, and GA3, not by ABA. The genomic sequences of PtDREB2A in 45 individuals were aligned, compared and analyzed using the software MEGA4.0 and DnaSP4.50.4. A total of 49 single nucleotide polymorphisms (SNPs) were detected and the frequency and diversity of SNPs were 1/18 bp and 0.010 48, respectively. In total, 46 SNPs were detected in the coding regions of PtDREB2A, of which 19 and 27 were silent and missense mutations, respectively. The results, therefore, would provide the important foundation for gene-assisted improvement of tolerance to abiotic stresses in P. tomentosa.


全 文 :!!"
" !
!
#
# $ % %
$
!
%
& ( )
&()*+(, &(-.,) &(*(,)
./01!"!*/1!
,234!# $ % %
¹º¯
!(R=;EW3
«??Հ
%
‰=Ö
%>é¥[N;qz
!
J
5
v
5
gwx
5
gyC
5
&z{
5
3|}
"
gÖ&E) &ɹ†X¯OP*+, &ÉQØÞ¹†¸¹ºRS*+,
5

%$$$>6#
!
5
"
!5
…ñ«?f+?
gI
‘€3¹º¯öβ1ùšYV?
OB*,
–«\ä.-:³
!(R=;EW3OB*,
Õ
€
!
˜{¼i&n&vqz
!
‡ˆ‰_
!(R=;EW3OB*,
Ó–I¥
9!7 V2!
«?Ôº—<¹T?dՂƒSc
!
E2¥
>7! V2!
5H„I0¥
#>"
âw«¥c«?Nºú
!
þ—g?Nºúw«¥&v¡
,g#r)IC
‡Yòó.y
zU…
,HBI)@#,
n`†
[QBI)@#,
Nº?.Ý;qr¥
>$16_
n
>616_&
`úf+;
IDG0HJ‡ˆ
~
! !(R=;EW3
¡¯Ñ5
%
À
%
Fn£ŽqM`ú\<‰=
!
Т‰=^_-.
!(R=;EW3
¡F‰=R0Q
ý
!
¡ÑÎu¨Ö5º`ú‰=R0\ç
!
Ú¡£ŽqM`úÖ£ÎYuº
%
¯Þ¨yÉúº‰=R0Qž
&
™M
€1ù֜9.g*+‰=~
!!(R=;EW3
-Œöý/
%
ž/
%
βyD.g‰=
!
Üö÷œ9
(,,!*,,
Ö
i,
6
?K€‰=
!
Ðy
,@,
?.gÛ/
&
`…ñ
Y)i,!1$
n
BEG&g!18$1!
!p7¹º¯
!8
»«?¤âj?
!(R=;EW3
&v{¼Ê7nqz
!
¾çi÷
!9
â%>é¥[N;
" &*g#
WS
!&*g
`i¥
%r%> V2!
[¬;џ #
¥
$1$%$ !>&
¡ý@N.
!
¾çi÷
!7
â
&*g
WS
!
¢%9
â¥.J€8
!#"
⥇J€8
&
#$%
5
¹º¯
$ R=;EW3
«?
$
™M€1ù
$
%>é¥[N;
&()*
! &"%>1!7$ m9!61#555
+,-./
!,555
+01*
!%$$% ?"!>>"#$%%#$! ?$$!9 ?$>
¦§¨#
#$%$ ?%# ?$>$
©ª¨#
#$%% ?$% ?##&
«¬­®

X¯&fÑ;¼(lÒ­/ˆ­®
"#$%$$!$$9# $
¸¹ºÈ¹Ö̾æ¶HkéÒ­«¬
"*)+^ $" $^$>!# &
!µ!>¥ÄÅÆÇ
&
T.&2-%(&)! !L<,#..(&)-)01()52#N=$2#&%(0#O&2@;&,<>(.;./)-2@.(.&"
+1789:W;()+/-$#$%1/621/%"
iN/mJ5ZGEF@G/0DJ5 ZGEF@/LDE5-J@GJ0JGE5APGEFBDoJGEF
"X1(.$21+;2,.2&&%.2, 819$%1($%):$%I%&&E%&&C.2,57&)819$%1($%)$:K&2&(.*12C E%&&C.2, .2 #$%&(I%&&
12C U%21A&2(1+!+12($:G.2.(%)$:;C0*1(.$25E&.J.2, #$%&(%)42.5&%.()5E&.J.2, %$$$>6#
/7.%,-$%5,OB*,O0/EDDEO/RJEFR=;EW3LGQJQ/0GHDR T3/FDED^Q2DOJTJOgIG<20JTJOGHJ/E T3/"[IC! >7! V2 JE 0DEFHP# LPJOP JQOG2GV0D/TDEO/RJEFG23/HDJE /T#>" GgHBI)@#,QPG3DQ>$46_! GER >646_ JRDEHJHSLJHP TNEOHJ/EG0R/GER U%)>1 1(.51 [QBI)@#,! 3DQ2DOHJUD0S4+JQQNDRJTD3DEHJG0DW23DQQJ/E RDHDOHDR LJHP 3DG0HJ!(R=;EW3H3GEQO3J2HQPGR HPDJ3!(R=;EW3H3GEQO3J2HQLD3DHPDVNHGLDG] DW23DQQJ/E LGQRDHDOHDR JE G2JOG0QP//HGEG0S\DR HPDDW23DQQJ/E 2GHD3EQ/T!(R=;EW3NERD3GVJ/HJOQH3DQQO/ERJHJ/EQGER 2PSH/P/3HPGHDW23DQQJ/E /T!(R=;EW3LGQJERNODR VSPDGH^QP/O]! O/0R! R3/NFPHGER PJFP Q^G0HQH3DQQ! VNHG0Q/VS(,,! *,,! GER
i,
6
! E/HVS,@,4+PDFDE/Q/THLG3DY)i,!4$ GER BEG&g!48$1!1,H/HG0/T!9 QJEF0DENO0D/HJRD2/0ST3DoNDEOSGER RJUD3QJHS/T&*gQLD3D%r%> V2 GER $1$%$ !>! 3DQ2DOHJUD0S1(E H/HG0! !7 &*gQLD3DRDHDOHDR JE HPDO/RJEF
3DFJ/EQ/T!(R=;EW3! /TLPJOP %9 GER #" LD3DQJ0DEHGER 23/UJRDHPDJ<2/3HGEHT/NERGHJ/E T/3FDED^GQQJQHDR J<23/UDB#@ C&,0.5!$<0+0($A&2($1$ R=;EW3FDED$ GVJ/HJOQH3DQQ$ QJEF0DENO0D/HJRD2/0S& ( )
!"
"
5
55
β
%
ýDÖHŽ/01ùù@Aß&M˜F
nº‚&M˜?£ã™M€1ù?9
!
ÚM€O
P+Þ¥®¹"€„^™M€1ùQ¥‚‰nQ<
:’?†‡
!
ö÷¹º›XŠ‹?ý0Rø
&

ýlm‰_
!
"€?š²
%
HŽ/0
%
DŒçš
ß;±ù3[«?‚?Ÿ‘;±
!
ù[†å
%

în[’x«.Æñ?‡ˆ
!
<限`Ú?€

"APGEF&(1+4! #$$!$ MND&(1+4! #$$7#&
ã:?
lm£ãþÖβ
%
ýDÖHŽ/01ù1
!
"€¡
NºœÕ
%
`Ä"€#
%
ö‚€#€úÞ
%
tÆ
ñ€#
%
Ž(¹ï#RSNºç†å(M?8•
6ÖÂ/«??Հnٕ
!
…‰lmވEE—
=Z"€šÎ²
%
HŽ/0
%
DŒ†å?ØÞ®
¹{P
"PGUDQ&(1+4! #$$!$ IGQP/HD&(1+4! #$$7 #&
›
!
+ՀZŸ²âyβ
%
HŽ/0nD1ù.
g‰=Â/?«?
!
t*Zö‚€#€ú‡‘I
%
w’\¸Œ
%
’ûHnˆiHç¡ýç"€šÎ
²
%
HŽ/0
%
DŒ†å<ö‚G&y€#Æ
ñ
!
Ѕ‰€ú%&7šß;±?Æñ˜-ž
"YGHQN]N3G&(1+4! #$%$#&
±Ú
!
Q*?lm‰_
!
"€›†.g¤«??
‰=£ãöfwÙ\?@¡Ù\`CK?€
",FG3LG0&(1+4! #$$7#&
ÚH„R`A^p‡

" RDPSR3GHJ/E 3DQ2/EQJUDD0DBI)@#
?Ù\?@«?
!
ù³†
BI)
4_Æñ
p
!
?@¥¡ŸÎ²
%
ýDž/1ù^“«?? 
=@!
7¡…‰1ùop1?«?.g‰=’€
Æ ñ
" ,FG3LG0&(1+4! #$$7$ YGHQN]N3G&(1+4!
#$%$#&
ÄÒÊ
R=;E
«?qrÙð^_"€‡z

"3%19.C$<.(/1+.121#
6Ö£ãDƀ‡`†
"U%)>1 1(.51#%
³´
"F&1 A1)#
n2¼
"I%.(.*0A
1&(.50A#
ç?E‘lméqt_Z
R=;E
?ґ‰
=5<þœ:˜@A”«??‰=`C
!
˜óž
÷ýٕ"»7β
%
DŒÖýž/í,?•P
0
!
©Ú.>"€7[†ß,?–šn„^óF
!
¥
"€šß;±?q@¹†vwZRã?«x
"YGHQN]N3G&(1+4! #$%$$ mJE &(1+4! #$$"$ &G]N1+4! #$$7V$ KN &(1+4! #$$>#&
?Y
!
¡&É
!
frù¡ñ„ц\qí
R=;E
«?¯—ÞK6Öïðlm4—7β
%
ýDÖH
Ž/01ù?q@ØÞ)€=‚
!
©Ú¥Ä¹š
²
%
DnóŸ¡HŽ/01ðñMI?šßц
÷ˆq@«x
&
frùQ*˜M?«ŸÄ«?Ô
%>é¥
"QJEF0DENO0D/HJRD2/0S?
ëØÞ)lm™<ÀŸ&ɹ††˜?‚w
"+PN<Ú ¹ º ¯
" !$<0+0
($A&2($1#
ùOXf" &DOH48&0*&#
ц
!
qÖ¡OX•š’[.–
%$$
x
]<
#
?k1
Ô
!
¡OXg†
!
ý¢¡•Z[.&M˜nMNí
,Vj\È&
¡I#{•ÒP!
¹º
¯†Ô¯ÞZc[8+Š¤
!
څ†G—?8+ó
tñ!lm‚®Û;±?q@ØÞ)«x
"APGEF&(1+4! #$$"#&
hŸ¹º¯|M;>
%
ØÞ8
+RS
%
{•IüIçfS
!
5óm„ñŸde&
ɟ‘;±Â/«??
&*g
(¤nëØÞ)l
m
&
¥Y
!
&lm©¹º¯/β1ùšYV?
OB*,
\qí:÷³
R=;EW3
«??
OB*,
&v
!
˜
7¢{¼Z`úf+;
!
֙M€1ù
%
"€œ9Ö
H.g?*+‰=qz
&
¡Y«xK
!
çiZX«
?¡¹º¯°±_j\?
&*g
[¬;
%
%j¤
" PG20/HS2D! cG2 #
n á › - C D
" 0JE]GFD
RJQDoNJ0JV3JN‡Yqz
&
&lm¥qí¢£Ñ
†
R=;EW3
ÞK6Ö{¼X«??á›-CDÆ"
n«?œÀÑɹ†÷ˆZRã?3ÿ
&
%5
„…y†‡
FEFG
ÿñŸ
!(R=;EW3
«?Ô
&*g
[¬;qz?„…
Š°7X¹º¯«?«\óŸQEP0ê;<¹º
¯™±qÖk1?
!8
»«?¤âj
"
ݝžç
!
#$$9#$
ÚñŸ
I*,
÷Š?„…Š°
%
$M¹º¯
Û;à
&
FEDG
!°"#$%TUrÿ<&ٍ
¡:#E‘‰+?«xK
!
ŠwZü¥‚
?™M€1ù֜9.g‰+àá
!
n
!
7Ÿ™M€
1ù‚
!
ѳ‚Ê/ÒÛ;à•?¹º¯
%
$M4ià
6
»
!
qr/Òý/
"!# :!7 P#%
ž/
"! :!7 P#%
β
"%
™
#%
D
"#8$ <?%
!8 P#
Ö

"%
™
#$
Ú7Ÿœ9.gLqrÊ؂°0¥
%$ ?%
!%$$ ?%
!%
#
?%
y
8$ ?%
?
R $ ¥
" GVQOJQQJO GOJR! ,@,#%
   ¥
"EG2PHPG0DEDGODHJOGOJR!*,,#%
¡¢¥
"JER/0D^6^
GODHJOGOJR!(,,#
Ö7£9
"FJVVD3D0JE!i,
6
#
’l¤
К
7 P
{¼F«d
&
ÊK¤þ<‚š«d?
F`úڟlB\â¥Ùñ
&
FEHG
K
9N/
@()
–
B*,
? ÷ Š Ï
B*DGQSg0GEHYJEJXJHQ
"mJGFDE! (EO4!.G0DEOJG! ,! j&,#
.¤?†‡

&
$8
5!
!
# §
5


¹º¯
!(R=;EW3
«??Հ
%
‰=Ö%>é¥[N;qz
FEIG*N/
()$9N/
@”Ý
Ê/1ù‚y"€œ9.g?„…
!
Ö¹º
¯5º`ú
%
Yuº
%
¯Þ¨
%
gÞnÉúº
%
ÞnÉ
úº
%
¥FnÞnF„…–
I*,
?÷Šn
OB*,
?ÞÏ
APGEF
ç
"#$%$G#
.¤?†‡{¼
&
FEMG+1789:<;
*+,‡
¥Zqí¹º¯Î²1ù*+‰=?«?
!
Æ
Çþ¡¦š`YVZ¹º¯Î².g?
OB*,
–
«
!
˜7
)&+Q
Հ{¼Zè=i&yÊ7
!
çi÷
Z³7I
R=;EW3
«??
OB*,
&v
&
FEYG
-<`ÎO6*
./
5ÿþ:?
!(R=;EW3OB*,
&v
!
¡¢5ó
?dՂƒS3Žjk
%
7¢i>饑€

!(R=;EW3C 8a^++iii++i,i,+i,i,, 6^a$
!(R=;EW3I 8a^i,i++,,+,i+,i,, 6^a&

#8
#
-B*,
iÕ§_;^
"gI#

!
6¹º¯ÞnÉúº
OB*,
¥^Ó
!

#18
#
-
%$ dVNTD3! %1>
#
- #8 <?%
YF0
#
! %
#
-
%$ <?%
R*+g!I1QB*,
iÕ
%1$ j"
6K‰
>¨°
g3/f$
#! %$
#
?%
êsn;s‘
€›
%
#
-!
3„‘Gö`
#8
#
-&
Ÿ
9! : 6 -
"96: 6$ Q
-
78 : 6$ Q
-
"#: % âìí
-
"# : 8 •ìíop1
!
ä.ïI–
# "$$ V2
?
OB*,
Ó
&
FEUGO6*
0<@12

‡3rÎ4G(Y
Ê
gI
ä.:÷?®?«?Óª¦šáA
Ÿ
2i)Y+^
K
&
áA˜€Ù•E©ª×
Bc8

!
Ø
į;Հ
!
Øf$i&
&

B*,Y,*71$
!p
n
-j&+,-K
!pšP&—gïw«¥&v˜{
¼
*@(
çsqz
!
±šqrìÿni—gNº
ú?q@‘nçaS
&
FE[G*#-2%(;#O6*

+1789:<;
*+@^56
7rP8m9:;
«ñ
,@(g3J!p¡
!(R=;EW3
«?ÔjkóŸä.I0–¥
76 V2 OB*,
Ó?
IDG0HJ‘€
!
¢&v‡1
!(R=;EW3I+C 8a^
,,,ii+,,,ii,iii,i, i, 6^a$ !(R=;EW3
I+I 8a^,++,+i+++i++ 6^a&
6«=Nº«?
3*(.2
Æ¥ÔB
!
‘€&v¥
3*(C 8a^ +,+,+i,,,+ii,+i 6^a$ 3*(I
8a^++iiii+,i+i+i,i,++ 6^a&

gI
;
^Ï 
APGEF
ç
"#$%$V#
.¤?†‡{¼
!
Qšñ
X«?¡8i¬Þ\?ä.‘yÔB
3*(.2
«?ä
.‘?ʪ
!
nÂ7‰=‘qz¢‰=^_
&
FE\G+1789:<;
*+@
1NO
 NE(Y
3ÿ+Հ?
!(R=;EW3
«??>é¥&vj
k«?f+?‘€
!
6Ċ?
!8
»âj?–
B*,
¥^Ó{¼
gI
ä.
$
Ê
gI
ä.˜€{¼¬Q
H­®a¯qí
!
ª¦
%
ù•®?Óšy
gi)Y^+
°jáA
!
ٕš±Š¯;%Հ{¼&viw
&
ѳ¯;ՀR`
6
îi&
!
©Ú-:܊?i&
‡ˆ
&
¡ Y « x K
!
`  … ñ
Y)i,!1$
n
BEG&g!18$1!
!p7ѳ«??
!8
â&v{¼q
z
!
Ûï
&*g
WS
%
kÿ
&*g
`iÖ[¬;џ
$
qz.J€8
%
‡J€8nÛJ€8¤
&
#5
‡ˆyBA
DEFG
<=!
789:<;
¸„
$9N/
@12r3>
(Y
¡7¹º¯/β1ùšYV?
OB*,
–«?
)&+Q
Հè=i&?«xK
!
çi÷Z³
R=;EW3
«??
OB*,
&v
&
¡Y«xKjk«?f+?‘
€
!
6X–«?
OB*,
¥^Ó{¼
gI
ä.šy
2i)Y+^
°jáAšÕ€i&
&
‡ˆ‰_
!
Հ?
…³
!(R=;EW3OB*,
–I¥
9!7 V2!
qrš—I0
¥
#% V2
y
7% V2
?
8aj+I
Ö
6aj+I"
«?0²q
¥
cm778$$7#$
¡
!(R=;EW3
&v!
«?Ôº—
<¹T?dՂƒSc
!
E2¥
>7! V2!
5H„I0
¥
#>"
âw«¥c«?Nºú
&
·ñ
B*,Y,*71$
!pìÿ—g?XNºú?q@‘–¥
6%17 ]N!
¢
çaS¥
81##&
Êü
!(R=;EW3
?
OB*,
y«?`
B*,
&v~
!
X«??H„N.-¥¡Ô—@
&v
&
hŸ"€
;=#"DHPS0DED^3DQ2/EQJUDTGOH/3#
ù³
mE?«?¯—
!
^_"€zU…Ö`†7«?`
qr—<
%##
Ö
%69
â
;=#
ÞK
!
öq”Ÿ
%#
Ö
%8
âV¯—
"*G]GE/&(1+4! #$$7#&
¥Z-4Հ
?¹º¯
!(R=;EW3
”Ÿl†Š¤?
;=#
ÞK
!
Æ
Ç3zU…ѳV¯—\Ċ
% b#
â
;=#
ÞKÖ
++tòó?þ<
;=#
«?¾
68
âÞK
!
y¹º
¯
gHBI)@#,
Nº{¼Z.Ý;{•qz
"
"
%#&
3"
%
5F
!
6zU…
,H,g#
¥Šý_
!
þ)IC
ÞKi¡³’
!
Ú¹º¯
gHBI)@W,
yzU…
V¯—
(UG
,HBI)@#
Š¤?
)IC
ÞK”Ÿ.³
V¯—
!
öyzU…
,HBI)@#,
?³Ÿ/àQ*
!
´Ê¢ä]¥¹º¯
!(R=;EW3
ù‚?
&
ÄÒ
(EHD3EDH

*@(!
Ê
!(R=;EW3
«?&v{¼
*@(
çs
!
‡ˆÇ|‰_
!(R=;EW3
ù¹º¯\â
îՀ?
R=;E#
Š¤?Ù\?@«?
&
¥Zqz
gHBI)@W,
Nºú?³Í‡Yfg
!
%8
& ( )
!"
"
5
"
%5gHBI)@W,
yzU…
)ICQ

?àá{•/à
CJF4%5+PD2PS0/FDEDHJO3D0GHJ/EQPJ2 VDHLDDE gHBI)@W,
GER )ICQT3/<3%19.C$<.(/1+.121
Ê
!(R=;EW3OB*,
—g?w«¥&vyzU…
,HBI)@#,
Ö`†
[QBI)@#,
Nºú&v{¼Z
.ÝÊün‡Yqz
"
"
##& gHBI)@#,
w«¥&
vyzU…
,HBI)@#,
Ö`†
[QBI)@#,
\?
,g#r)ICR/&vý0.Ý
!
.Ý;qr¥
>$16_
n
>616_&
3"
#
~?Nºú³Í‡Y
5F
!
y^_"€?
BI)@#,
Âõ
!gHBI)@#
—<

BI)@#,
þû!
‡¡
gHBI)@#
w«¥&v?\º!
">*%6>
w«¥c«
WڗI+rBI)
‡?
,g#r)IC
‡Y.
"-JN &(1+4!
%99>#$
$Zý0GÍ?
,g#r)IC
‡Y.ý
!
¡
*
Ž¥¡Â7GÍ?>wW¼q‡
XXIX"
WŸw«
¥&v?
%9*87#!
¡

Ž¥;N.—<Ù\œ:
.
"

#8#*#>"#!
¢£ãòót¤¥ù¡"€1
ùop1
!
óŸ>ô
,g#r)IC
‡Y.y
B*,
?
I+rBI)
‡
!
©Úœ:1L«??.g‰=
"&G]NDEDG+1789:<;
*+@^567rP8m9(Y
¥Zçi
!(R=;EW3
«?¡¯Ñ-.`úy_
S\?‰=^_
!
3ÿX«??
&v
!
jk
%
7óŸä.ï
76 V2
?
OB*,
Ó?‘€
!
6ßÙ
\Þ?
OB*,
!
%
§¥^Ó
!
6
3*(.2
«??ä.
‘Æ¥ÔB
!

IDG0HJä.
& !(R=;EW3
«?¡-.`ún_S\?ä.‘yÔB
3*(.2
«
?ä.‘?ʪ
!
nÂ7‰=‘‡"
6
þ~
&
3"
6
5F
!!(R=;EW3
«?¡¯Ñ5
%
À
%
Fn£Žq
M` ú \  < ‰ =
!
Ð ¢ ‰ = ^ _ a - .

!(R=;EW3
¡F\‰=R0Qý
"!17##!
Ú¡Ñ
Îu¨Ö5º`ú‰=‘\ç
!
¡£ŽqM`úÖ
£ÎYuº
%
¯Þ¨yÉúº‰=R0Qž
&
¡ß
,±N1
!
FùT4HŽ/0
%
R`ÖýDçšß
¼qü¥ST?_S
!
Ú5ºLù¡™M€1ù1
wAµöß,9,?`ú
!
?Y3X«?¡¯ÑF
y5º`ú?ýR0‰=5—i
!(R=;EW3

yß,1ù&
̱Հ?
!(R=;EW3
«?¡Nºú³Í‡Y
KûÙZ"€
R=;E#3
Š¤?³r‡Yfg
!
Ð-
.€†¡{•ÒP\¯ÞZ¢f+?šß=‚
!
f
rù[$MÑÉy
%
$M6&"€5óû<-.?
«?€=‚
&
?Y
!
m<ªã¡Ù\`CKçi
!(R=;EW3
«?7›†™M€1ùùaû<€‰
=Æñ
&
¥Y
!
…ñw‘
IDG0HJµpçiZ
¹º¯
!(R=;EW3
«?¡-.ß,op֜9‚
.g1?
I*,
Ù\`C
!
˜kÿ
!(R=;EW3
«??
ä.‘yÔB
3*(.2
«?ä.‘?ʪ
!
nÂ7‰=
‘
"
"
!#&
3"
!,
5F
!
$Ÿ`Ÿ±N1
!
¡¢
£ß,‡ý/
%
β
%
ž/ÖýDop1
!!(R=;EW3
«?¡Ù\`C<-.P0?K€‰=
&
y7 
ÂÊ
!
‰=‘qr÷ýZ
%1> b!>1>
¸
!
¢\X«
?7ý/1ù?A^Q¥_
&
?Y
!IDG0HJgI
çi‡ˆ~
!
þqí?
!(R=;EW3
ùöHŽ
/0
%
βÖýD
"*G0#
1ù.g‰=?«?
&
"€œ9
!
‡R$¥
",@,#%
¡¢¥
"(,,#%
 ¥
"*,,#
y7£9
"i,
6
#
çù"€MI(¹
֖àß,1ù?Rã€#>
!
5óBy€šß
«??‰=
&
Ú&–Õ€?
!(R=;EW3
«?ùaö
ýݜ9@A¶
!
Öm·l†œ97X«??.g
û3
¥Zª“…³cš
!
ÆÇ¡¹
º¯FK¤ÐZýݜ9‡
,@,!(,,!*,,
Ö
i,
6
!
.¬«ñ
IDG0HJµpçiZX«??
#8
5!
!
# §
5


¹º¯
!(R=;EW3
«??Հ
%
‰=Ö%>é¥[N;qz
"
#5gHBI)@W,
yzU…
,HBI)@#,
Ö`†
BI)@#,
Nºú³Í‡YÊü
CJF4#5/<2G3JQ/E /THPD23J"
65!(R=;EW3
«??`ú‰=õqz
CJF465ID0GHJUDH3GEQO3J2HGVNERGEOD/T!(R=;EW3
JE 3//H! 2P0/DI
5
I//H$ @
Ñu
@G3]$ g
ÑÎYuº
gP0/D
Ñίި
GÑÎê¡(¹?Éú
º
BDUD0/2JEFWS0DÑÎÞnÉúº
YGHN3D
WS0DF
-DGT$ ,
£ŽqM`ú
,2DW1
Ù\`C
"
"
!@#&
3"
!@
~?‡ˆ5
F
!
ýݜ9‡MI9
(,,!*,,
Ö7£9
i,
6
?
Ð3ž÷ýZ
!(R=;EW3
«??‰=
!
Ú
!(R=;EW3
yR$¥
,@,
?.gÛ/
&
ã:+¡^_"€‡zU…Ö£ãDƀ‡`
†
%
2¼y³´\7
R=;EW3
Š¤?«?{¼Zü
¥3k?™M€1ù.g‰=^_lm
!
t_ZX
Š«?¡-3ñŸ
,@,
?1ù¼qÙg’x!
Ä
"
!5!(R=;EW3
?™M€1ùÖ
œ9.g*+‰=qz
CJF4!5IDFN0GHJ/E /T!(R=;EW3DW23DQQJ/E
JERNODR VSGVJ/HJOQH3DQQGER P/3Z-

ZGHD30/FFJEF$ c+
ý/
cJFP HD<2D3GHN3D$
-+
ž/
-/LHD<2D3GHN3D$ B
β
B3/NFPH1
Òy
BI)@
?Â2Æñ
!
œ:1L«?
=RWY3
?
‰=!*¤¢šßòó
&
‡
-JN
ç
"%99>#
¡…ñ¸
68
& ( )
!"
"
5
y%Ú5µpqíï
3(R=;EW3
«??«xK
!
.
g‰=‡ˆ~
!
zU…
3(R=;EW3
öR`ÖýD
"#8$ <?%
*G0#
1ù.g
!
ÐyRBÖ
,@,
?.gÛ/
&
¡zU…\ê;J‰=
R=;EW3

@‘’βnDA^«??‰=
!
ܑ’Z•œÂ
/«??‰=
!
êJ‰=
R=;EW3
ž.3ZÙ«
?"»?•;
!
Ú;J‰=?"»LPžZ•
;
"&G]N¡`†\¾çi÷
7
â
R=;EW
Š¤?«?ÞK
!
7¢8
â{¼Z.g‰
=qzš~
!
y¹º¯.Ý;Qý?
UR=;EW3
?‰=öβnýD?.g
!
ÚyRBÖ
,@,
?.
gÛ/
"YGHQN]N3G&(1+4! #$%$#&
-lzU…y`
†\?
R=;EW3
«?
!
2¼y³´R=;EW3

=-ŒöβyýD.g
!
öyRB?.gÂ/
"mJE &(1+4! #$$"$ KN &(1+4! #$$>#&
?Y
!
-.۠
R=;EW3
Š¤«??‰==‚5óyX€†¡I#
(•ÒP\¯Þ?„^;{•!
Ú&–lm‡
ˆÇ|‰_
!(R=;EW3
«??‰=-Œö÷ý/
%
ž/
%
βÖýDç™M€1ù?9?@A
!
Üy"
€œ9‡
*,,!(,,
Ö
i,
6
.g&
DEHG+1789:W;
*+?
1NO
@ NE(Y
«??ØÞ[¬;ùr耆I#{•ÒP¯Þ?†Ô>é¥WS?28+
!
Úlm«?Ô
&*g
?[¬;ù7X«?{¼_jØÞ)n
(
«Ÿ
Ä«?Ô
&*g
á›-CDÆ"
)
?«x
&
¥Y
!
¡qz
!(R=;EW3OB*,
&v?«xK
!
6Š°¹
º¯°±qÖN6
â_j
!
š—
9
âQ
%
9
%
°¹
N
!
óŸQEk1P^¹º¯MIN.?
!8
»«?
¤âj¥„…
!
7š—
#% V2
?
8aj+I!>7! V2
?H
„Nn
7% V2
?
6aj+I
ç¾
9!7 V2
?>é¥&v
{¼Z%>é¥[N;qz
"
‰
%#&

%
5F
!
¡¹º¯€†(•ÒP!(R=;EW3
«?Ôqr(
MZ
%
îÁð
%%
î™êÖ
!9
â%>饀8
&
¢!
Áðn™ê(M¡H„N
!
I0qr¥
6 V2
y
66 V2!
3Y5#ï
!
¡X«?Ô(M?Áðy™
êggh‰„€8
&
?Y
!

!(R=;EW3
«?
!
5555
¡I0¥
>7! V2
?N.Ô
&*g
(M?`i¥
%r%>
V2!&*g
[¬;
!
n
!
kI3&v9Ž>é¥*+?
CŸ #
+
h$1$%$ !> q$1$$$ !%&
¡Y«xK
!
qz
Z
&*gQ
¡_j\?`i
!
‡ˆ~
!
¡çi÷?
!9
â
&*gQ
!
<
%!
┟F
&*gQ!
n¢ï¤`i

%$_!
Ú
6!
┟ºF
&*gQ&
yŠÂÊ
"&NPG&(1+1! #$$8#!
¡¹º¯&*g
(M?`iü
ý
!
…£ã3ŸÑÉ|M;>
%
ý0Ú
!
¡¢€†
I#(•ÒP!
¥Z„^°±º[8?ý/
%
R
B
%
βç™M€1ùn–àÝaçM€1ù
!
¡°
±ÄQ?Æñ1¯ÞZc[8+Š¤
&
¥Zçi
!(R=;EW3
«?H„NÔ>é¥WS
?®8ùa@AZ¢H„?w«¥&v
!
¡qz
&*g
[N;WS?«xK{¼Zw«¥?[¬;q
z
&
7
!(R=;EW3
«?H„NÔ?
!7
â
&*gQ

Z.J€8
%
‡J€8nÛJ€8qz
!
‡ˆ‡‰
%
þ~
&

%
5F
!
¡
!(R=;EW3
H„NÔ?
!7
â
&*gQ
!
<
%9
┟.J€8
!
4—WŸs„@
?!
6
â>é¥K
!
Ú
#"
┟‡J€8
!
<<ç
i÷ÛJ€8
&
hŸ‡J€8\F
&*g
7Ÿ
Ä«?Ô
&*g
á›-CDÆ"?Rã;
!
Ïã7‡
J€8\?F
&*g
{¼34qz
&
¡
#"
â‡J
€8!
<
"
┟F
&*gQ&
¢!
<
#! !
y
%
âqrWŸs„@?!
%! #
Ö
6
â>é¥WÚK
&
‡
!
¡H„N?!
8#
Ö!
"8"
WÚKqr(MZ!
%
Ws„@?€8
!
3v!?
,+;i+
y
+,+;
i,+!
©Úqrgh¢H„?w«¥3«w¥
"+P3#
;
-w¥
",0G#
y»w¥
"+S3# ;
™5Yw¥
",Q2#&
¡H„N!
.J€8y‡J€8?>é
¥[¬;qr¥ #
QSE
h$1$%! 98!
#
E/EQSE
h$4$$> 9$!
™.J€8y.J€8?Êi¥
$17$&
?Y
!

!(R=;EW3
«?
!
3¢H„NÔº(M?™.J€
8y.J€8?Êi
$17$ ;%!
~Z¡¹º¯€
†(•ÒP!
ùù•ÄQ
!
Ú-ù°±ÄQ7
!(R=;EW3
«?Ô.J
&*g
WS’Z£ã?{•>
=F
&
m
FG+1789:<;
*+?
1NO
 NE
:-7RFG1NO.0(3#,.(%@ ()+1789:<;
«?
iDED
&*g
Ÿ‘
mNGEHJHS
&*g
`i
C3DoNDEOS
&*g
[¬;
BJUD3QJHS
8aj+I
H„N
/RJEF3DFJ/E
.J€8
&SE/ES‡J€8
YJQQDEQD6aj+I
!(R=;EW3 !9 %> $1$%$ !> q$1$$$ !% $ %9 #" 6
55
¥Zqz¹º¯
!(R=;EW3
«?Ô>饀8
?(Š¤
!
7çi÷?
!9
â
&*gQ
{¼8+Š¤
ák
"
‰
##&

#
5F
!
¡…‰
&*gQ
!
<
6>
â
”ŸÙۊ¤
!
qrš—
%>
â
i
.
,
n
#$
â

.
+&
7Ÿ¼Û(Š¤
!
¾çi÷
%%
â
!
qrš›
#
â

.
,!6
â
i
.
+!!
â
,
.
+
n
#
â

.
i&
?Y
!
Ù
!8
5!
!
# §
5


¹º¯
!(R=;EW3
«??Հ
%
‰=Ö%>é¥[N;qz
Û
r
¼Û¥
61!8 =#1$!
ºÞ…³‡ˆ5óù
!&*g
¡
i
&vKï¤Q¥`O
!
Úö[ù

-
+!
v?
ù
i

nн¾¥ý«•?
%
°(êRwš
nÞ¥c¿½¾
"&NPG&(1+4! #$$8#&
m
DG
<=!
+1789:<;
*+?Ð
1NO
@Ä@)A
:-7R#5:>#.=7.%(%=%(&)%@<#&"1NO."&,+1789:<;
«?
iDED
Ÿ‘
mNGEHJHS
(Š¤
&NVQHJHNHJ/E HS2D
ÙÛ
+3GEQJHJ/EQ
¼Û
+3GEQUD3QJ/EQ
,
.
i +
.
,
.
+
.
i ,
.
+ i
.

!(R=;EW3 !9 %> #$ # % " #
DEIG+1789:<;
*+B<=!CD‚?@EF
(J
¹º¯”OX?î8ц
!
¡I#?€†(•
ÒP!
¯ÞZ
6
âÂ7&ð?œyê‚N.
!
m
<ªã…ñ«?Ô
&*g
ØÞ[¬;!lmN.Ô
555
ÖN.Ž?ØÞq•P0
&
¥Y
!
ÆÇ7¢qr{
¼Z%>é¥[¬;
%+GsJ"
QR"+GsJ9#
n
CN GER -J
"
QR"CN &(1+4! %996#
?\;ç+
"
‰
6#&

6
5F
!
7Ÿçi?
&*g
[¬;џ‡ #
H/H
!
#
Q
n #
E
¡
6
â_jŽÌ<-.Н~ZÂõ?‡ˆ
!
‰_¡¹º¯{•ÒP6
âN.ö÷ZÂõ?Ä
Q¿
&
–jK#
!
…ñ
+GsJ"
QR
n
CN GER -J
"
QR
{
¼?\;ç+‡ˆ‰_
!R
!
¥ìª
!
…~Z¡T
â_jÔ
!!(R=;EW3
¡{•ÒP\¥¡Ò†?ºF
&*g!
ö[Ÿù<,8+
!
ºÞ?…‰8+3Ÿö÷
ìÄQÆñÚ¡†_\GHÊüž?ÊË
&
ˇ
!
¡
¹º¯Tâ°±qÖNÖ
6
âÂ7&ð?qÖN
!
º
F
&*g
?Êˏ[Ÿ
78_&

6
â_j!?
!

¥ #
E
;
#
Q
!
n™.J€8y.J€8?Êi
;%!
?
Y
!
¡¹º¯
6
âÂ7&ð?qÖNÔ
!
ù•ÄQù
7X«?Ô.J
&*g
WS?£ãÄQ¿
&
m
HG+1789:<;
*+B‚?@GHIÄ@
!
:-7RHG1=;;-,@ &")=$2#&%(0#3-,(-%(&)()H <&<=2-%(&)."&,+1789:<;
_j
g/2N0GHJ/E
âjŸ
2
WSŸ

#
H/H
#
Q
#
E
+GsJ"
Q
R
CN GER -J
"
Q
R
qg
*/3HPDGQHD3E 3DFJ/E %8 #> $1$%$ 78 $1$%7 !! $1$$> "6 $18#" 7> ?$1%6! "!

&/NHPD3E 3DFJ/E %8 #9 $1$%$ "# $1$%8 8% $1$$> >9 $1!%$ 8! ?$1"!9 6!
fg
*/3HPLDQHD3E 3DFJ/E %8 #" $1$%$ %! $1$%6 7> $1$$> "" $1!>$ #" ?$188> >!
–n
+/HG0 !8 !9 $1$%$ !> $1$%! 98 $1$$> 9$ ?$18#$ >8
?#19"% #9
!
55
!
2
i&?¬&Ÿ
*Nqí?WSŸ
*N#
H/H

Tâ«?N.%>é¥[¬;
,GUD3GFD
ENO0D/HJRDRJUD3QJHSJE TN0FDED$
#
Q

H„N.J€8%>é¥[¬;
,UD3GFDENO0D/HJRDRJUD3QJHS/TQSE/ES#
E

H„Nԙ.J€
8[¬;
,UD3GFDENO0D/HJRDRJUD3QJHS/TE/E Q^SE/ES!
!!;$1$81
65
‡A
%#
…ñ
I+^gI
†‡
!
âî3¹º¯öβ1
ùšYV?
OB*,
–«\ä.-:³
!(R=;EW3
OB*,
Հ
!
˜{¼Zi&n&vqz
!
‡ˆ‰_Õ
€?
!(R=;EW3
«?Ôº—<¹T?dՂƒS
c
!
E2¥
>7! V2!
5H„I0¥
#>"
âw«¥c«
?Nºú
&
—g?Nºú³Í‡Y~
!!(R=;EW3
—<"€
R=;E#3
þû!

¢w«¥&v?\º!
">*%6>
w«¥c«Wڗ
I+rBI)
‡?
,g#r)IC
‡Y.
!
Ú¡
*
Ž¥¡Â7G
Í?>wW¼q‡
XXIX"
WŸw«¥&v?
%9*
87#!
6Ö¡

Ž¥;N.—<Ù\œ:.
"

#8#*#>"#&
##!(R=;EW3
«??`ú_Sր‰=qz
‰_
!
X«?¡¯ÑF\‰=R0Qý
!
¡ÑÎu
¨Ö5º`ú‰=‘\ç
!
Ú¡£ŽqM`úÖ£
ÎYuº
%
¯Þ¨yÉúº‰=R0Qž
&
¡ß,
š›ý/
%
β
%
ž/ÖýDop1
!!(R=;EW3
«
?¡Ù\`CK<-.P0?K€‰=
!
‰=‘
qr÷ýZ
%1> b!>1>
¸
!
¢\7ý/1ù?A^
Q¥_
&
"€œ9.g‰=~
!
ýݜ9‡M
I9
(,,!*,,
Ö7£9
i,
6
?Ð3ž÷ýZ
!(R=;EW3
«??‰=
!
Ú
!(R=;EW3
yR$¥
,@,
?.gÛ/
&
6#
¡¹º¯°±_jÔ
!!(R=;EW3
«?Ô¥
¡RS?
&*g
8+
!
[¬;`C #ý=
$1$%$ !> q
$1$$$ !%! &*g
`i¥
%r%> V2$
7ŸH„N.
!
¡
¹º¯Tâ_jÖ
6
âÂ7&ð?qÖNÔ
!
¥
™.J€8y.J€8?Êi
;%!
—iù•ÄQù
¹º¯°±_jÔ
!(R=;EW3
«?Ô.J
&*g
WS
?£ãÄQ¿
&
L M + ,
ݝž
!
¯Ân
!
ÁÀÁ
!
ç
1#$$94
¹º¯789Õ«?
!(@&3!
?Հ
%
‰=Ö%>é¥[N;qz
4
&()
!!8 " 8 # %
?%$4
,FG3LG0gX! ,FG3LG0g! IDRRSY X! &(1+4#$$74I/0D/TBI)@
H3GEQO3J2HJ/E TGOH/3QJE GVJ/HJOGER VJ/HJOQH3DQQH/0D3GEODJE 20GEHQ4
g0GEHD0ID2/3HQ! #8"%## %#76 ?%#"!4
88
& ( )
!"
"
5
PGUDQY Y! [0JUDJ3GY Y4#$$!4 YDOPGEJQ3DQJ0JDEODH/LGHD3RDTJOJHQ 23/Q2DOHQT/3LGHD3^QGUJEFGF3JON0HN3D4
k/N3EG0/T)W2D3J!4
CN MNEWJE! -JZDEPQJNEF4 %9964 &HGHJQHJOG0HDQHQ/TEDNH3G0JHS/T
-JN mJGEF! XGQNFGY! &G]N4+L/H3GEQO3J2HJ/E
TGOH/3Q! R=;EZ GER R=;EW! LJHP GE )I)@gr,g# B*, VJERJEF
R/GER 0/L^HD<2D3GHN3D^3DQ2/EQJUDFDEDDW23DQQJ/E! 3DQ2DOHJUD0S! JE
3%19.C$<.4+PDg0GEHD0! %$"># %69% ?%!$74
YGHQN]N3G&! YJ\/Jk! M/QPJRG+! &(1+4#$%$4/<23DPDEQJUDGEG0SQJQ
/T3JODR=;EW H^S2DFDEDQHPGHDEO/RDH3GEQO3J2HJ/E TGOH/3QJEU/0UDR
JE HPDDW23DQQJ/E /TGVJ/HJOQH3DQQ^3DQ2/EQJUDFDEDQ4Y/0iDEDH
iDE/6"## %>8 ?%974
*G]GE/+! &N\N]JX! CNsJHPD;=#FDEDTG"## !%% ?!6#4
mJE C! XG]JGEG0SQJQ/TFAR=;EW3JE 3DQ2/EQDH/R3/NFPHGER PDGHQH3DQQDQJE
F&1 A1)-4g0GEHk! 8$"%# 8! ?794
IGQP/HD,Y! YGQ/E Yi! cNHOPJQ/E )! &(1+4#$$74,QNVQDH/T
3%19.C$<.,g# H3GEQO3J2HJ/E TGOH/3QO/EOD3HLJHP GHL/^O/<2/EDEH2GHPLGS4g3/O*GH0,OGR &OJj&,!
%$6"#9# %%$>% ?%%$>84
&G]N/TGE 3%19.C$<.H3GEQO3J2HJ/E TGOH/3! R=;EW3! JEU/0UDR JE R3/NFPH^
3DQ2/EQJUDFDEDDW23DQQJ/E4+PDg0GEHD0! %>"8# %#9# ?%6$94
&G]NTNEOHJ/E /TGE 3%19.C$<.H3GEQO3J2HJ/E TGOH/3R=;EW3JE LGHD3^
QH3DQQ^3DQ2/EQJUDGER PDGH^QH3DQQ^3DQ2/EQJUDFDEDDW23DQQJ/E4g3/O
*GH0,OGR &OJj&,! %$6"!9# %>>## ?%>>#"4
&NPGM! .JsFk4#$$84&*gRJQO/UD3SJE GQQ/OJGHJEFFDEDHJOUG3JGHJ/E
LJHP PN?864
+GsJ94 &HGHJQHJOG0PS2/HPDQJQVSB*,2/0S8 ?8984
+PN<@.221A$)+@$3%&C0*(1&"@@=# G3DGQQ/OJGHDR LJHP UG3JGHJ/E JE
?%#784
KN A&! *JAM! -JN -! &(1+4#$$>4PG3GOHD3J\GHJ/E /THPDI13?R#1
FDEDDEO/RJEFGI+rBI)^VJERJEFTGOH/33DQ2/EQJUDH/R3/NFPH!
PJFP Q^G0H! GER O/0R QH3DQQJE LPDGH4Y/0iDEDHiDE/$"7#
!9" ?8$>4
MND@! KNDZ M! KJ/EF-A!&(1+4#$$74iDEDHJOVGQJQ/TR3/NFPH
3DQJQHGEODGH3D23/RNOHJUDQHGFDJE 3JOD QD2G3GHJ/E /TR3/NFPH
H/0D3GEODT3/< R3/NFPHGU/JRGEOD4 iDEDHJOQ! %"# " # # %#%6
?%##>4
APGEFBDoJGEF! BN mJEF\PGEF! KN @G/PNG! &(1+4#$%$G4+PDGOHJE
2PS0/FDEDHJOGEG0SQJQ4Y/0iDEDHiDE/!"## %$8 ?%%94
APGEFBDoJGEF! MGEFKJG/PNJ! APGEFAPJSJ! &(1+4#$%$V4)W23DQQJ/E
GER ENO0D/HJRDRJUD3QJHS/THPD2/20G3@UE8 FDED4+3DDiDEDHJOQGER
iDE/APGEFBDoJGEF! APGEFAPJSJ! XGJMGEF4#$$"4(RDEHJTJOGHJ/E /T,C-g
($A&2($14&J0UGDiDEDHJOG! 87"%# #" ?6#4
APGEFkA! 3DD0jQJEFJET/3R3/NFPHH/0D3GEODJE O3/2Q4g0GEHgPSQJ/0! %68"## 7%8 ?7#%4
!
!"#$
5
Ž
5

"
78