THE GENETIC TRANSFORMATION OF STRAWBERRY WITH WINTER FLOUNDER ANTIFREEZE PROTEIN GENE
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- Χρύσηίς Κεδίκογλου
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1 2009,23 (3) : Journal of Nuclear Agricultural Sciences 423 : (2009) (11, ;21, ; 31, ;41, ) : 2, pre pro mature 3 (AFP), 7 Km PCR PCR2Southern,6,, pre pro mature AFP 2 DNA,pre pro mature 2 1, 1156 % 1149 % 1167 % ; mature 3,4105 % : ;;(AFP) ;;PCR2Southern THE GENETIC TRANSFORMATION OF STRAWBERRY WITH WINTER FLOUNDER ANTIFREEZE PROTEIN GENE SUN Rui2fen 1 LI Tian2ran 2 AN Yu2lin 1 LI Kun 3 SHI Hui2qin 3 NIU Su2qing 1 ZHAO Jing2hui 4 (11Biotechnology Research Center, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Huhhot, Inner Mongolia ; 21 College of Life Science, Inner Mongolia University, Huhhot, Inner Mongolia ; 31 Institute of Horticulture, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Huhhot, Inner Mongolia ; 41Vegetable Technology Extend Station of Huhhot, Huhhot, Inner Mongolia ) Abstract :Antifreeze protein (AFP) of Pseudopleuronectes americanus gene encoding pre, pro and mature was introduced into strawberry ( Fragaria ananassa cv1 Xingdu 2 and Allstar) by mediated Agrobacterium gene transfer of a binary vector,and the axenic leaf discs and lower part of petioles of strawberry were used as explants. The results showed that the foreign AFP gene encoding pre, pro and mature was integrated independently into the genome of six transgenic strawberry plants by PCR and PCR2Southern bloting analysis of seven Kmamycin2resistant transformed plants. Three regenerated Xingdu 2 lines were positive for pre, pro and mature, and their transformation efficiency was 1156 %, 1149 % and 1167 % respectively. Three regenerated Allstar lines were positive for mature and its transformation efficiency was 4105 %. Key words :strawberry ; winter flounder ; antifreeze protein (AFP) gene ; gene transfer ; PCR2Southern blotting ( Fragaria ananassa Duch. ),,,,,,,, (antifreeze protein,afp),,, : : :(NK ) : (19632),,,,, E2mail com
2 AFP AFP, AFP Cultur [1 ] AFP, 118 Strizhor [2 ] AFP cdna AFP, Geroges [3 ] AFP,, CAT Western, AFP ( Pseudop leuronectes americanus), [4 ] AFP [5 ] AFP, AFP, - 615, AFP [6 ], A FP,, AFP (Pseudop leuronectes am ericanus) 3,mRNA cdna cdna pre pro mature 3, mature 5ml ( Km 50mgΠL), MS ( 189bp,pro ( mature ) 252bp, pre ( pro mature ) 310bp 3 AFP (expression cassette ), ppcv002, AFP E. coli S 1721 (pre pro mature AFP p PCV002, ) GV3101(p Mp90Rk), AFP 12A B C pre pro mature 3 173bp, :Primer 1 :5 GAC ACC GCC TCT GAT 3 (15mer) ; Primer 2 :5 TCC GTC GAT CCT TAA CCT CTG 3 (21mer) E. coli S 1721 GV3101 E. coli S 1721 ppcv002 ( pre pro mature), (mob),e. coli, RK 2 [7 ], ppcv002 Vir (pmp90rk) GV3101,, : (1) GV3101 ( Km r ), 5ml YEB ( Km 50mgΠL Rif 20mgΠL), rΠmin 2d ; (2) pre pro mature 3 E. coli S 1721, 5ml Amp 100mgΠL LB, rΠmin ; (3) GV E. coli S rΠmin 10min,, 10ml YEB LB (4) 3 E. coli S l, 50 l YEB ( ), 28, ; (5) 1ml ddh 2 O YEB Tip,; (6) 100 l, ddh 2 O ; (7) 100 l, YEB ( Amp 100mgΠL Km 50mgΠL Rif 20mgΠL),28 2d, GV3101 S 1721 ; (8) Amp 100mgΠL Km 50mgΠL Rif 20mgΠL YEB,28 2d, ) OD 600nm 015, 2 5min,, MS(B 5, MS B 5 ) + 62BA 213mgΠL + IAA 118mgΠL, 3d d Km 25mgΠL cef 500mgΠL, MS + 62BA 110mgΠL + NAA 0101mgΠL ( Km 25mgΠL)
3 AFP Fig. 1 Plasmid and expression cassette of AFP gene A :P NOS : ;P AOCS : ;NPT2 : ;B L B R :T2DNA S ;Pg5 :T L 2DNA B :vir :Ti ;tra Ti :Ti ;ori Ti :Ti ;tra 1 3 trf 2 : RK 2 C :4 En :4 ;TLE:TMV ( ) ;P 35S :CaMV35S ;pa :CaMV35S polya Figuer A :P NOS :promoter of nopaline synthase gene ; P AOCS :polyadenylation sequence of octopine synthase gene ; NPT2 :neomyein phosphotransferase gene ; B L and B R :left and right border sequences of vecter T2DNA S ; Pg5 :truncated promoter of T L 2DNA gene. Figuer B :vir :toxic origin of Ti plasmid ; tra Ti :transfer site of Ti plasmid ; ori Ti :replication origin of Ti plasmid ;tra 1 3 and trf 2 :transfer gene from auxiliary plasmid RK 2. Figuer C:4 En :4 enhancers ; TLE: TMV (translation enhanced sequence) ; P 35S :promoter of CaMV35S; pa :polya pause sequence of CaMV35S, 4 5, MS + IBA 015mgΠL ( Km 25mgΠL) ppcv002 DNA PCR () 215 % [8 ] 3 3 E. coli S 1721, 110 % Membranes(Mandel ) [9 ] pre pro mature Detection Starter Kit PCR E. coli S 1721 pre pro mature PCR : min,94 1min,56 1min,72 015min,30,72 10min 215 %211 Km Amp Rif YEB Km DNA,,,3 Plant Genomic ppcv002 DNA Kit, mature E. coli 3 DNA, DNA,,PCR GV kb ( pre) kb (,PCR pro) kb( mature), PCR2Southern E. coli S 1721 ( 2), ppcv002πmature PCR,, Roche DIG High Prime DNA Labeling and Detection Starter Kit DNA PCR DNA PCR (), Southern Total BLOT + TM Nylon DIG High Prime DNA Labeling and 3 GV pre pro mature PCR
4 pro mature 5min, 3d, MS 62BA 213mgΠL IAA 118mgΠL Km 25mgΠL cef 500mgΠL 15d, 2 ( 42a b) 2 Fig. 2 Agarose gel electrophoresis of recombinant plasmid in Agrobacterium 1 8 : DNAΠHind ;2 4 6 : pre pro mature ; : pre pro mature 1,8 : DNAΠHind Marker ; 2,4,6 :recombinant plasmid with pre, pro and mature gene in Agrobacterium ; 3,5,7 :recombinant plasmid with pre, pro and mature gene in E. coli pre pro mature PCR, ( 3), E. coli, 3 PCR Fig. 3 PCR analysis of recombinant plasmid in Agrobacterium 1 :50bp DNA ( ) ; : pre pro mature PCR ; : pre pro mature PCR 1 :50bp DNA Ladder Marker ;2,4,6 :PCR product of pre, pro and mature gene in Agrobacterium ;3,5,7 :PCR product of pre, pro and matur gene in E. coli 173bp, mature, pre 2 3cm,, pro mature 3 ( Km 25mgΠL),, pre 42c d 4 (a,b) (c,d) Fig. 4 Km2resistant shoots (a, b) and clone lines(c,d) MS IBA 015mgΠL Km 25mgΠL, ( 5),, 3 A FP, pre pro marture 2 ;,mature 5 Fig. 5 Transgenic plants for inducing roots,7 ( 1) 214 PCR
5 3 427 target gene varieties of strawberry 1 3 AFP Table 1 The transformation results of strawberry with three different A FP genes total number of explants explants regenerated shoots regenerated shoots number of Km2 resistant shoots rooting Km2resistant lines PCR2Southern PCR2Southern positive lines frequency of target gene transformation ( %) pre 2 Xingdu Allstar pro 2 Xingdu Allstar mature 2 Xingdu Allstar pre pro marture mature, pre pro marture AFP DNA ( 6), 6 (173bp), 4105 %( 1) 7 PCR2Southern Fig. 7 PCR2Southern blotting detection of transgenic plants 1 6 :;2 3 4 : pre pro mature 2 ; 5 10 :;7 8 9 : mature 1,6 :positive control ; 2,3,4 :transformed lines of Xingdu 2 with pre, pro and mature ; 5,10 :negative control ;7,8,9 : transformed lines of Allstar with mature 3 6 PCR Fig. 6 PCR analysis of transgenic plants M:50 bp DNA (TaKaRa ) ;1 6 :; : pre pro mature 2 ; 5 10 :;7 8 9 : mature M:50bp DNA Ladder Marker ; 1,6 :positive control ; 2,3,4 :transformed lines of Xingdu 2 with pre, pro and mature ; 5,10 :negative control ;7,8,9 :transformed lines of Allstar with mature 215 PCR2Southern PCR, PCR, ppcv002πmature PCR PCR2Sonthern,,PCR ( 7),,, 6, pre pro mature 2 1, 1156 % 1149 % 1167 % ; mature 3,,,,,;,, 30min [9 ] 5min, 30 60min 1 2d,,, 2d [5,10 ] [11 15 ],,,,, [5 ] AFP Km, Km [14 ] Km M14
6 428 Journal of Nuclear Agricultural Sciences 2009,23 (3) : , 50mgΠL Km [16 ] Km 20mgΠL [17 ] TCS RIP, Km 20mgΠL,25mgΠL [18 ] gus,,: Km, 20mgΠL [19 ] CBF1 25mgΠL Km 50mgΠL Km, 25mgΠL Km,,,, [18 ],,,, (1d),, ; 4d,,,,,, Km Nehra [20 ], 10d,, 3 % 7 %, [16 ], 2,, 3 AFP 2, PCR PCR2Southern, pre pro mature DNA,,,, AFP :, : [ 1 ] Culter A J, Saleem M, Kendall E,Lawrence V C, Georges F. Winter Flounter Antifreeze Protein Improve the Cold hardiness of plant Tissue [J ]. Plant Physiol, 1989,135 : [ 2 ] Strizhov N. Report to the Scientific Advisory Board of the Max2Planck Institut fur Zuchtungsforschung[J ]. Cologne,1991,147 [ 3 ] Georges F,Saleem M,Cutler A J. Design and cloning of a synthesis gene for the flounder antifreeze protein and its expression in plant cells[j ]. Gene, 1990,91 (2) : [ 4 ],,,,. (afp) [J ]., 1997,13 (4) : [ 5 ],,. [J ]. ( ),1998,29 (1) : [ 6 ],,,,,,. [J ]., 2002,17 (4) :49 53 [ 7 ] Csaba Koncz, Jeff Schell. The promoter of TL2DNA gene 5 controls the tissue2specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector[j ]. Mol Gen Genet, 1986,204 : [ 8 ] J, E F, T.,. ( 2 ) [M]. :, 1998,19 22 [ 9 ],. [M]. :,1998,224, [10 ],,,,,,. (PLRV) [J ].,1995,11 (4) : [11 ] Nehra N S,Chibbar R N,Kartha K K,Datla R S S,Crosby WL,Stushnoff C. Agrobacterium2mediated transformation of strawberry calli and recovery of transgenic plants[j ]. Plant Cell Reports,1990,9 :10 13 [12 ] Nyman M and Wallin A. Tranient gene expression in strawberry ( Fragarina ananassa Duch. ) [J ]. Plant Cell Reports, 1992,11 : [13 ],. Tudla [J ].,1998,6 (2) : [14 ],. [J ].,2000,17 (2) : [15 ],,,,,. GO [J ].,2005,3 (6) : [16 ],. [J ].,2007,21 (5) : [17 ],,. TCS RIP [J ].,2008 (2) :14 18 [18 ],,,,. [J ].,2008,22 (1) :36 40 [19 ],,,,. CBF1 [J ].,2007,27 (2) : [20 ] N S, Chibbar R N, Kartha K K, et al. Genetic transformation of strawberry by agrobacterium tumefaciens using leaf disk regeneration system[j ]. Plant Cell Reports, 1990 (9) :
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