Floral dip Transformation of EXPA1 to Arabidopsis thaliana

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1 Crop Biotech. Vol.2, No. 2, Spring and Summer 2012 (39-47) 1391 $%" &#!!" # EXPA / 6. 1 *./ '()!* + *,)!" #$% &' () /0 1 ' -'!",.),+ (90/12/2 :;: 4-90/11/1 :/2 4)!+ 3 Floral dip Transformation of EXPA1 to Arabidopsis thaliana M. GHADERI MOZAFARI 1, A. R. ABBASI 2 * AND A.H. SALMANIAN 3 1, 2, M.Sc. Student, and Assistant Professor, University College of Agriculture and Natural Resources, University of Tehran, Iran, 3, Associate Professor, Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran (Received: Jan. 21, Accepted: Feb. 21, 2012) Abstract 89 Drought is by far the most important environmental stress in agriculture and many efforts have been made to improve crop productivity under water-limiting conditions. Expansin is a protein super family consisting of 4 families in vascular plants. Expansions are cell wall proteins which mediate acidic cell wall loosening through breaking hydrogen bonds between cellulose and glycan matrix. In this study, a construct named pbiexpa1 containing nptii and AtEXPA1 genes under the control of CaMV35s promoter was designed and fabricated. Arabidopsis plants were then transformed by pbiexpa1. Transformation was done by floral dip which did not need any tissue culture process. Transformed seedlings were able to remain green on MS medium containing 50mg/L kanamycin. Transgenic plants were confirmed by PCR and as expected two bands of 753 and 1080 bp were observed for transgenic plants. Keywords: Arabidopsis, Cell wall, Drought, Expansin Protein, Floral dip and AtEXPA1 ( ) #$ #*&'.! " #$! 6 *"5".,-. / 01 ( $ #:8!1 *. :,"* ;!< 8 =8 $ :,"* >? #*;!< $ #, *"5".:8:4 8@! :* #*:8<,"4 AB $ >?,:,"! :4 # AD.: *- *?. (4! C>?. $ :, :4 #,- E #,F *.!1,cDNA +!,4! :. HF, 7 RNA 8> G"<!: 1! :. # /, #*.$I1 62! :4.:4, pbiexpa1 8@$ G5! :. 8"2* 3!:L #*7K8 $:8 +, #!L $ $ M, G"<!: 1,F +D,8?L CaMV35s #!BI 6! =,!.1 $ M,,F 72N.:. $8 -.K! 7L 6 -. / O17. :4! P,8.:4 T 1 7"8 1 $ :1 *.! :8 #!L 3 #! *1 $ 7/L #*R*. S* Q $ 8@$ 7/L *.,F.:8:8 K C( "8,> =.? 50 [ $?< #P8$! $ #$ M) 1080! 753 :8! V *. "4! 5- ].:. :\ DNA 8 #*@ $ 7/L #* ^ 2* :1 7/L.-. :* K 7? CP! "5" S"5" ;!< SG"<!: 1 : AtEXPA1 SO17. #!BI S>? rezabbasi@ut.ac.ir #$% &'()% :! *

2 1391 #%4,.(5 67+, #-./0 1 #)% 2)3 40 cp- L2 T 6.) 1 (Rayle et al. 1970;.# -! 6(4 2,4 642.;(d=4.Cosgrove. 1989) '! 2(, 2=. #S) 4 6.;('0 ;9 f 'L,4 #S) 4 2 ( ()5 29$ NS) ;( #!W(. 6.!(0 (McQueen Mason et al. =# 8.4'7 -! ;(d=4 #S4 Z()? 2 N3.1992) 3.# ;( ;('0 ; 4 2=.# 4 6/)5 Q 2 4 ;('0 2= 2!-! (d0 ;( Q,4 ;('0 g:,4 O =# F ;('0,4 #E0!I29$ 2 I 6/) (15KDa) #(I 6!4 ;(.#.,!=)5!4 45 4!: #9((S= ;( 6 2= (10KDa) #)(='= 6!4 ;( # #a)%.(5 '52!4 6.W'SI 4!: 2 i' +4 -! 2:.I <=,. 2= # #('0 4!:'4 M.;(d=4.(jS4-1 Z9) Z(9-4!: Y,4 6!I.(5 2=. H ;(d=4 I ;(d=4 4!:.!4 <= 2=#ST!4 4 4 D'5 +()U 2 H ;(d=4 2$ I ;(d=42$ 'Q 4!: 2 +.$ F.+4 -! # #: ;(d=4 64' # N!I +(SO7 =.,!=)5!4 '=4,I ;(d=4 ku4.+4 -! 2: 4.I 6NS) 6.Z '$(7'9( l>,4.=)5 (H-1Z9),# I # N!I 2)T 64' W ( <.=.(Sampedro et al. 2005) 2 '3 m#!i D,4 a4 I ;(d=4 (Cho and Cosgrove. '5#,4 +O! 6 ' I;(d=4 ( - 2=#ST 2000) D,\I Z( 2 '3 2U #!4 8 ' 1. Acid Growth 2. EXPANSINS 3. Domain 4. Signal peptide )) = 2= +4 #>(? < ; '8 #9-:.+4 '= 2 B? $ +F 6, 6.DE = K'L M,4 +4 HI $= J 4' 6.8(!9 < N5 +F #O($L HP!4 'Q K'L,4 +4 R 65, '9)% < 4N74 +F #TEG4 6.+(SO '5 3!4 +-= +? 6.2!5 6BU4 < 4N74 2 '3 #TEG4 +(SO7 80,4 <( #9-: J 4' #%4, 6.2!5,4 6( '9)% T ;(..+4 #9 WS N(7 #S9S X0 #Q!QY,4 6,4 2)G7 == 4!4 I +2 HI $= 2 (5 < J 4' I 4 J 4' '9)% ;( 6, < 4N74 2)G7 ; 4!ZU4T 2.4 F 4[\ ;(] +F < J 4' '9)% 64 0 (Cattivelli et al. +4 6'& ( I 64' 4 6( 6.@: X L '-.2008) -=.+4 Z? #S9-: 2F4 _B:2.+4 $! ^ %U ; 4,4 N(! 4' 4,4.+4 '6F 4'? ; 4 '(: #9-: 2 Z? R.(5 ; 4 8(3T 2-.(5.# # 4N +(.4 #9-: 2 +$! 6'-( Z? 644 #!AL 2-(. #(GB: ;(Y.(5 ; 4,4..2!5 #:' 4.!4 5=`EG4 H)> 644!4! F 8(3T 6.2- U4 M=2 Z(S ;(. 2.#! H)> +(a(= #U4 ;(Y ; 4 + # M(!W# 3!4 4# 4 ; 4 2= #.= 2)F,4.'= Z(: 6.;('0 =(S 6.W R!4 4 4#.;('0 ; 4 2)F,4.+4.) (Ghaderi. '= '=b 4 EXPANSIN 4!:.2010)

3 41...,4!(S m(0(4i (5 2EXPA1 W R!4 :#$% 6'an 6U # n!4 23(! 4 <R! 2- Z L 'o!4 #9-: 2 '-( Z? +F R!4 <./0 ; 4 ; 4'.(Wu et al. 2001) +7'5 4'U 'n! m(0(4i (5 2 EXPA1 W 6O 6.<./0 2= #)(9 6..= 3!4 < 4N74 2-2O I <R! 4'U 2OS> (5 #9-: 2 Z?.'(5 Zenoni et.(pien et al. 2001) '5# q' (5 PhEXP1 ( <..= (2004) al. l>.q'$)5,4!4 2=!!'=.- #)L4 r(=' Z <..= (04 6.).+4 '((s Y N(! #S) 4 4!:'4 ]% ; 'K'O 6 ' 2= EXPA1 W # ;(d=4 W ; 4 2=.# -! j)p.(5 &' 29.#S) 4 ;(d=4 ;('0 Z%?! (H.I.Z(9-6.;( ;(d=4 ;('0 (js4-1 Z9 xpansins.#s) 4 F NS)#. NS) ;( #!W(. 6.!(0 ;7Z5 2 +F 4PS #- 2 (,4.( +% 16 2, L I $%!"#,+ ()*!+ ), 6,2!. 64'DH 5 α 2 E.coli 6'= 6(E0 6.#P 4' 'n! #!W, 2 Agrobacterium tumefaciens m(0(4i 2 W R!4 +F LBA4404 pgemt-easy ZU!,4 f(r? ; 4. a4 2 +R W 6T (Q'0 +=' +:) T/A 6,2!. ZU! M 4% %2 ;()(#di 2 +R W 6T pbi121 ZU! a4 :! *) ()*!+ + ), ), 1 ($)= 8U m(0(4i (5 f(r? ; 4 [ H4: +9 +F.+7'5 4'U a4 2F 4 6 #9 m(0(4i 6.[ #7G [\= 6 ' +% 2 4'5#! 6T 4)5 [ I,4 m0 7'5 4'U m(: MU4 md 4 4'U t-+(0 # +% 12 6! H 2F 23 6,4.! 642Q! #9 +% 12 m0 +4 ), (5 M m( (0(4I 2=# FI 1. Arabidopsis thaliana

4 1391 #%4,.(5 67+, #-./0 1 #)% 2)3 42 0A D 2 : +7 ' #% ( # 3 45! 9:% 7,$! ( 6'=.(5 29 4,4 Z$U, 'n!, ZT '='5I,4 #!)= M M 6T k +-= J(? '(S#)( 10 ('(S ' '5#)( 50) ;((da 6.M((#!I. '5 +-= ('(S ' '5#)( 50) ;(!= 28C 9!4 +% md md. 4 4'U 2R(U = J(? '(S#)( 50 4R 6'(S#)( 250 ;S4 J(? 64' '=b 6.M((#!I 27&4 2 k,4 '(S#)( 2. ', md. '5 2( Z$U 27&4 J(? '. 2 #)$U +-= J(?.! 4 4'U Z$U J 4' 9!4.;S4 2 1/75 ;( v$ 'R.6'=OD 600 2= #!, I.! a4 I.6'= # k! Wa '! 2R(U 6T )?!#U H md. 2P @)( +!97 %0/05,'= %5 T 6'= #! OD 600 2=! ZT 642!5 D,4 m(0(4i.(5 l(r) 64'. 0/8 D ; 4. a4 ; biz5 62L\ 2P '- M I 6.6'= 7'5 4'U '- 6A 2!4 L2.4)5 md 6'= Z:4 (5 ; biz5 2=6L2 ;(. 2(!o 2L\.(5 62L\ Q..! 24 2Q! +ST #:2.Z5! 4 9 ; Y md! 2-\I '='5I 6T )? #!)! <0 ', +% 2.(5 +% 24,4 O.7'5 4'U #9 6.[ (,4 O.! ZR '(7 +F < N5 n2 +4'.I.(5 6. Dellaporta 1 #.(5 8( #!( ZU! 4%2 ;(!=. '5 a4 2 AtEXPA1 W == #S4 3 ()=! 1665 L AT1G #'.'5,\I. +7 NCBI #%EL4M! ; 4,4 == #S4 5u 6!4 3u 6!4 6., t4 ' 4N74'!,4 a4.! 2:.'5,\I # #S4 cp- NEBCutter #S4 L SacI BamHI EcoRI 6.8 N!I '5,\I 5u 6!4 2 23(!.4! F == 5 6!4 2 BamHI 8 N!I # #S4 '-(0 27&4 SacI 8 N!I # #S4 #-5' '5,\I `' ; 4 2 a4 6.'5,\I #S4. '5 F-EXPA1: # ggatccatggctcttgtcaccttcttg R-EXPA1: gagctcctaacgtagctgcgcacctg.! '5 2( W( +=',4.'5,\I ; 4 2= cdna 0+ /RNA 4 /DNA +-. ( 1" m(0(4i : +7 '5#)( 100,4 B? RNAx plus g4'p4 +(=,4 a4 RNA,4. '5 g4'p4 RNA W( +=' 4'+! 8 N!I,4 a4 I+2 5 2: cdna,'7 +=' B? t9o,4 a4 2: cdna 2O>U. J AtEXPA1 W #GB:4 6.'5,\I #)0 64 '(3!, <=4 #L High fidelity8 N!I 27 '(^9 2O>U 3u 6!4 2. '5 '(^9,4',4'#)0 M 8 N!I,4 a4 'Q #-=4 #L (Sambrook, miniprep '5 27&4A ()=! #)( 100,4 a4 N(! DNA g4'p4.2004) 1. nptii 2. Coding sequence 3. Accession number 4. Polymerase Chain Reaction(PCR) 5. RevertAid M-MulV Reverse Transcriptase

5 43...,4!(S m(0(4i (5 2EXPA1 W R!4 :#$% 6'an 6U 6.'5,\I,4 a4 'n! 2O>U +4 cdna 6,4 AtEXPA1 W #GB:4 g-2 Z9 I y! 2= '5 '(^9 2: M.Y 2= #-=4 B?.+ +4 I W 6,4 6,cS: +F 4 4'U M 'Q!-!.-! 2 M.Y. '5 HP!4 753 T 27 '(^9 2O>U.+4 #S9S, W CDS 2O>U L 2= 4 L, +af = AtEXPA1.4 +R>. a4 +P 4' '.(5 2 #( ;< = EXPA1 3cDNA >?@ 0+ (5 #- 6.+7,4 RNA 44,4 g4'p4rna. '5 g4'p4 m(0(4i,5i G M W 6 ' m(0(4i : I js4-2 Z9 I y! 64[5 F RNA,4 cdna +: y!.+4 I H-2 Z9 dt Q(S4 6.'5,\I,4 a4 ; &',4 RNA g4'p4 y! M.Y.,5I G M W 6 ' RNA 64[5 y! (js4) -2 Z9 cp- E= ) A ' R F Z(S2 18srRNA 28s rrna..# -! 4 m(0(4i :.# -! 4 cdna +: y! 1 M.Y.G M,5I W 6 ' cdna 64[5 y! (H).#, +af 753 T L 2 642O>U.,5I G M W 6 ',4'#)0 64 '(3!, <=4 B? 64[5 y! (g).#ambdadna/ecori+hindiii a4 '= N +4 cp- 1 M.Y 2=.+4 I H-3 Z9 (E0 D' y! 2= +4 ' (E M.Y.+4 (6, +af 753) 'n! 2O>U 6T 2O>U B! +4 :D' ( (E0 A!,4 Z$U (E0 4 M.Y..# -! 4 27 D'..# -! 4 D' W 644 F pbi121 (E0 8 N!I I D' 2= +4,4(!=A5 <!+ ) (-,=# ( + A@ pgemt-easy Vector ZU! 2=# FI,4 : Z:4 4SacI 8 N!I #GB:4 D' Z? B? 4 +F (js4-3 Z9) 44 pgemt-easy ZU! 2,4'#)0 64 '(3!, <=4 44 ; 4' +4 $! cp- vector SacI 8 N!I 'n! 2O>U 6T 6.(E0 2=! 'BamHI 8 N!I md #>:

6 [ Z(9-,4 m0. 3!4 ; biz5 G + 4 ' [ ( 2!4 '0 F MS J(? 6 ' #!! a% & ZG T [.! +-= ;(! = ' (S '5#)( # 50 6 T Y 4!, 2!4 F. [ <,I 64 4 y! 2 G +-= -= # P!4 J(? -! # P!4 J(?. [ +-=,4 I+ 2,4 O J(? ; 4.2z. (5,4 # #]O 2= 4. -! 2=!! #U N$ # #5' 2 2)T' ; 4.! (a.2z. (5 #]O ]O!I # P 4' [ 4 O Y ;( 0 # P 4' # 4 = D Z U +P 4' 6.; A 4 O # (S 6, 12 <,I,4 2)T' ; 4. '5# ZG T # F 4 ;(! = ' (S '5#)( 50 Z? #!4 (5. +P 4' 6.; A # 'n! 'n 2 2= 4 #%4,. (5 6 7+, #-. /0 1 # )% 2)3 44 K[T,4 (! =A 5 W SacI BamHI,4 2 7 D' 2O>U D' [0 I ZG T ( E0 3!4,4 m0. '5 pgemtexpa1 pgemtex ( E0 ]T ({ + F E.coli 2 R!4 B 4 < =4 ( E0 pbi121 ( E0 'n! 2O>U ' BamHI SacI # ' 8 N!I pbi121!.+ 4 I -3Z9 I y! 2=, +af T L 2 7 D' ( E0 A 4, +af 753 L 'n! 2O>U B! 'n! 2O>U g-3 Z9 2 2F.. # -! ku4.+ 4,4 (! =A 5 W ; NQ F 2 R!4 I 2: # #!W, 2)T' ; 4. # ' = '5I B # 8 -# % # 34 5! 9 :% 6 2L \ D C! 7,$ W R!4 f(r? ; 4 &' (H).+ 4 cp- SacI 8 N!I D' Z?. I F D' 6.Z? pgemt ( E0 2-R! (js4) -3 Z9 'n! 2O>U B!. :D'' ( E0 A!. G M, 5I W 6 pgem-et ( E0 D' 6 4[5 y! 2=. # -! 4 pbi:expa1 2( 2, (g).. # -! 4 D',4 Z$U ( E0 4 M. Y., +af 753 L A!.'n! 2O>U 6 T pbi121 ( E0 D' ( ) ! N(! # ' 6.8 N!I N D' Z? I.. # -! G 1, 5I W 6 ' 4, +af 753 L 'n! 2O>U B! 2 7 D' ( E0

7 45...,4!(S m(0(4i (5 2EXPA1 W R!4 :#$% 6'an 6U 6T 6'='5I,4 <=4 ; 4.#! a4 +$^ '= <=4 +F 'n! #!W, #T4'L ; 4. '5 1 AtEXPA1 W == #S4 t4 '.'5,\I 642.!W F W,4!4 2= 4'Y # #T4! -!'(^9 Z(S2 4 #, +af 1665 =[ 6.'5,\I 5 UTR 3 UTR 27 '(^9 2O>U,4!4,4'#)0 64 '(3!, <=4 6T pbiexpa1 ZU!.#, +af 1080 f$l 2= # AtEXPA1 W == #T4!., +af '(^9!,4!4 GmEXPA1 W 2= FI,4 2(S Z L 2R> #GB:4 - (Lee et al. 2003) # ( 2!o #F ZU L2 GmEXPA1 ( < 4N74 23(! 2= +4 4 < 4N ; 4' # 2- (04 6.) L,4!4 4,4 f(r? ; 4 Y r)> ; 4 2 2F AtEXPA1 W #! '= +FCaMV35s +P 4'.(5 # n!4 +4 a4.-..(5 2 +$! 6'Z L 6.2- #!W, AtEXPA1 W F. CaMV35s,4!4 4 '= +? 2: N(! 2= # n!4 # #'..- +P 4'.(5 :.(5 : 2- 'Q 4'.2 W'0 244 ZGT +P 4' ;(d=4 4!: 6.W ',4 ZGT 6.; A.+7'5.4: 4'U Z()? 2 N3 #' AtEXPA1 W ( - +$^ '(o} 2 4 W ; 4 4# 2- Z L ' ZR N(! 4N)=! m(0(4i 4!: 8. #%4,.'= 1. Coding Sequence (CDS),4 a4 WR!4 O = ZT4' #L +7+-= (T 0 ) 'ag Z! I +2.(5.# 6'-(, K'G 2,(! ;(z. # 6.D.# in planta 6.D 2 +$! K[T = 2,(! in planta W R!4 #7'O Z.D ; 4.+4! ZU4T 2 2 8(R R!4 6'='5I 2)(2 DNA,4 #$,! ; S.(5 2=6L2 (5 #5!, 2:'Y # \4 #F 6.) DNA D ; 4 Z=.# r(=' 27!, ;(F '. m(0(4i 2= FI,4 4 4 #({0 # 4= 3!4 +! =# (S [ T (5 2 WR!4 D ; 4.,# R 4 Z% ; N 644 #7+-= ZT4' 2,(! % Z(S D ; , +4.4P! F # +7+-= D D 2 +$! 6'=, 2,(! ;(z. D ; 4,4 ZGT.(5 E% = (Primrose et al..# Z(9-4 Z! T ) '=.(5 #P 4' '-( ( 64' ZGT.(5,4 ;(!= '(S '5#)( 50 'n! 2O>U '(^9 3!4 DNA g4'p4 #GB:4 6.'5,\I,4'#)0 64 '(3!, <=4,4,4'#)0 64 '(3!, <=4 : 2 2= m(0(4i N$ 6.2z.(5 DNA,4 a4 (H-4Z9)! ZR I g-4 Z9 AtEXPA1 W 6.'5,\I 6, +af 1080! Z9 ; 4 2= +4 ; 4 2= # (5 F 2(S4 W 2 i' 753! M 4 # 8.. (5! i' 2= #.(5 #]O 6, +af. 2!! +4 4 #!W, 2

8 1391 #%4,. (5 6 7+, #-. /0 1 # )% 2)3 46 &' #U N$ ;(! = ' (S '5#)( 50 6 T J(? 2= m( 0 ( 4 I #S T4 +P 4' 6.2z. 2z (5 (js4) -4 Z9 6 T J(? N$ 6.2z. (5 (H).!4! (a +P 4' '(\ 6.2z. (5.!4 cp- ~! ( ' 4!4! '= N.1M. Y.m( 0 ( 4 I DNA,4,4' #)0 64 '(3!, < =4 y! (g) : 2 R!4,4 m0 ;(! = 1080!.#a ' =.9 M. Y. 2!! 8 M. Y +P 4' 6.; A M.. Y +$^ + ' =.2 M. Y.+ 4 (5 2 4 r(=' (='! W i ' 6, +af 753! + 4 (5 F 2(S 4 W 2 i ' 6, +af REFERENCES Cattivelli L, Rizza F,, Badeck Bad W, Mazzucotelli E, Mastrangelo Mastrang A, Francia E, Marè C, Tondelli A, Stanca A (2008) Drought ttolerance improvement in crop plants: pla An integrated view from breeding bree to genomics, Field Crops Research. R 105(1-2): Cho HT, Cosgrove DJ (2000) Altered expression of expansin ansin modulates modul leaf growth and pedicel dicel abscission abscis in Arabidopsis thaliana.. Proceedings Procee of the National Academy cademy of Sciences. S USA 97: Cosgrove DJ (1989)) Characteriz Characterization of long-term extension xtension of isolated cell walls from growing cucumber hypocotyls. Planta. 177: Ghaderi M (2010) 10) Overexpr Overexpression of At. EXPA1 into Brassica sica nap napus. Lee DK, Ahn JH, Song SK SK, Choi YD, Lee JS (2003) 003) Expression Expres of an expansin gene ne is correlated correla with root elongation in soybean. soyb Plant Physiology. 131: McQueen-Masonn S, Dur Durachko DM, Cosgrove DJ (1992) Two endogenous proteins that induce cell wall expansion in n plants. Plant Pl Cell. 4:

9 47...,4!(S m(0(4i (5 2EXPA1 W R!4 :#$% 6'an 6U Pien S, Wyrzykowska J, McQueen Mason S, Smart C, Fleming AJ (2001) Local expression of expansin induces the entire process of leaf development and modifies leaf shape. Proceedings of the National Academy of Sciences. USA 98: Primrose SB, Twyman RM (2001) Principle of gene manipulation and genomics. Blackwell publishing. Rayle DL, Cleland RE (1970) Enhancement of wall loosening and elongation by acid solutions. Plant Physiol. 46: Sambrook J, Russell D (2004) Molecular cloning: a laboratory manual. Sampedro J, Cosgrove DJ (2005) The expansin superfamily. Genome Biology, 6, 242. Wu J, Meeley RB, Cosgrove DJ (2001) Analysis and expression of the_ expansin and _ expansin gene families in maize. Plant Physiology. 126: Zenoni S, Reale L, Tornielli GB, Lanfaloni L, Porceddu A, Ferrarini A, Moretti C, Zamboni A, Speghini A, Ferranti F, Pezzotti M (2004) Downregulation of the Petunia hybrida _ expansin gene PhEXP1 reduces the amount of crystalline cellulose in cell walls and leads to phenotypic changes in petal limbs. Plant Cell. 16:

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