(A ntifreeze p roteins, A FP s) (afp ),

Transcript

1 29 1 ( )V ol. 29 N o Jouṙ of N orthw est Sci2Tech U niv. of A gri. and Foṙ (N aṫ Sci. Ed. ) Feb α 1, 1, 2, 3 (1, ; 2, ; 3, ) []A utum n K ing, CTAB DNA, PCR (Polym erase Chain R eaction) (afp), pu Cm 2T V ector afp pta F, E cor g pta F, DNA g K lenow, X bag,, (afp)pb I121 X bag Sm ag,, T 4 DNA, afp pba F [] ; ; []Q 789[]A [ ] (2001) ,,, (A ntifreeze p roteins, A FP s) (afp ), 1 A utum n K ing DNA CTAB, [ 8 ], afp PCR ( Polym erase Chain R eaction ) ( [1 ], Georges [2 ], H igh tow er [3 ], W allis [4 ] ), A utum n K ing afp cdna, 1, : A T G A A T A T T G A A T C A T C T T T C T G C C C T; : G G T A A C T A G C, A T T C T G G C A A T G G A G C A, afp [5, 6 ] York W orrall [7 ] afp, afp, afp, cdna CaM V 35 S,, afp DNA, PCR (Polym erase Chain R eaction) afp,, DNA : 9410 m in (941 m in 631 m in 722 m in ) m in 30, T aq P lus g DNA Polym erase (T aq+ Pfu)PCR M inicycler TM PCR, DNA ( ),, B am H g P stg, DNA 15 ΛL + 10Buffer 2 ΛL + BSA 2 ΛL + B am H g (P st g ) 1 ΛL 37 4 h α [ ] [ ] (91K122G19 (11) ) [ ] (1956- ),,,,

2 1 : 7 (Sangon) pu Cm 2T V ector PCR,,, : 2 ΛL + 5 ΛL PCR + 1 ΛL 10L igation Buffer+ 1 ΛL (50 m ggl ) pu Cm 2T V ector+ 1 ΛL T 4 DNA L igase 16 DH 5Α, [ 9 ] X2gal IPT G 2, 2 ml LB, rgm in, 8 ml LB [7 ] DNA B am H g, X bag + E cor g DNA 14 ΛL + 10BufferD 2 ΛL + BSA 2 ΛL + X ba g 1 ΛL + E cor g 1 ΛL, 37, 4 h Sanger, PE 377 DNA pta F E cor g, DNA 15 ΛL + 10 BufferH 2 ΛL + BSA 2 ΛL + E cor g 1 ΛL. 37, 3 h DNA g K lenow fragm ent : DNA 17 ΛL + 10Buffer 2 ΛL + 10 mmolgl dn T P 0. 5 ΛL + K lenow (10 Λmolg(m inλl ) ) 0. 5 ΛL, 169 h,, X bag DNA 15 ΛL + 10BufferD 2 ΛL + BSA 2 ΛL + X bag 1 ΛL, 374 h afp, pb I121 Sm ag, DNA 15 ΛL + 10 BufferJ 2 ΛL + BSA 2 ΛL + Sm ag 1 ΛL, 254 h,, X bag, X bag, pb I121 afp, T 4 DNA L igase, afp DNA 5 ΛL + pb I ΛL + 10 Buffer 2 ΛL + T 4 L igase 1 ΛL, 1922 h Kanam icin 100 m ggl,, B am H g DNA PCR PCR PCR, bp (1) 5363, B am H g P st g, B am H g 600 bp 400 bp, P stg 900 bp 100 bp (2) 2. 3 T aq P lusg DNA Polym erase PCR 3 A, pu Cm 2T V ector, 9 cm 1 000, 50% (A B ) DNA B am H g, 600 bp bp ; X ba g E cor g, bp2 700 bp (3), A pta F (4), B 2. 5 pta F afp

3 8 () 29 1PCR 1. PCR ; 2. DNA F ig. 1E lectrophoresis of PCR p roducts 1. PCR p roducts; bp DNA ladder 2 1. DNA ; 2. ; 3. P stg ; 4. B am H g F ig. 2R estriction enzym e digestion of interest fragm ent bp DNA ladder; 2. Interest fragm ent; 3. P stg digestion; 4. B am H g digestion 3 1. A B am H g ; 2. A X bag E corg ; 3. B B am H g ; 4. B X bag E corg ; 5. DNA F ig. 3R estriction enzym e digestion of recom binant p lasm id 1. D igestion of clone A w ith B am H g ; 2. D igestion of clone A w ith X bag and E corg ; 3. D igestion of clone B w ith B am H g ; 4. D igestion of clone B w ith X bag and E corg ; bp DNA ladder 2. 6 pb I121,, 13 kb, CaM V 35S, 4(pTA F) afp. ; Amp ṙ ; L acz. Β2; rep. F ig. 4D emonstration of recom binant p lasm id pta F GU S ; NO S N PT g, Kanam icin afp : E cor g

4 1 : 9 pta F,, DNA g K lenow, X bag,, (afp)pb I121 X bag Sm ag,, T 4 DNA, afp pba F (5) 5 afp (pba F) afp. ; GU S. Β2; CaMV 35S. 35S ; N PT II(kanr). ( ) ; NO S p rogteṙ g F ig. 5P lant exp ression vector of carrot afp 2. 7, pba F, B am H g, 600 bp pba F DNA PCR, bp, afp pba F, (6) afp R T 2PCR (R everse T ranscribed PCR ), polya + RNA, oligo dt, polya + RNA cdna, T aq DNA DNA, DNA, PCR afp DNA PCR, R T 2PCR, afp 6 afp (pba F) PCR 1. B am H g ; 2. PCR ; 3. DNA F ig. 6Identification of p lant exp ression vector of carrot afp w ith enzym e digestion and PCR 1. B am H g digestion; 2. PCR p roduct; bp DNA ladder, : (1),,, T 2 ; (2), ; (3) (OR F), OR F (pu C18, pu C19 ) PCR, PCR 5, PCR,,,,,, T 2T 2 T aq, T th,amp lit aq K lent aq DNA polym erase PCR, PCR 3 2A, T 2 3 2T, pu Cm 2T V ector, 9 cm 500,

5 10 () afp pb I121,,, 3 (X bag, B am H g Sm ag ),, GU S (GU S S acg ), afp B am H g, pb I1121 X bag Sm ag 2 T 2 E cor g afp,,,, GU S T 2,, T 2, pb I121 [ ] [ 1 ],,,. (afp) [J ]., 1997, 13 (4) : [ 2 ]Georges F, Saleem M, Cutler A J. Design and cloning of a synthesis gene for the flounder antifreeze p rotein and its exp ression in p lant cellsj [J ]. Gene, 1990, 91 (2) : [ 3 ]H ightow er R, Cathy B, Ranela D. Exp ression of antifreeze p roteins in transgenic p lants[j ]. P lantm olecular B iology, 1991, 17 (5) : [ 4 ] W allis J G, W ang Hongyu, Guerra D J, et al. Exp ression of a synthetic antifreeze p rotein in potato reduces electrolyte release at freezing temperatures[j ]. Plant M olecular B iology, 1997, 35 (3) : [ 5 ],,,. [J ]., 1999, 41 (7) : [ 6 ],,,. [J ]., 1998, 25 (3) : [ 7 ]W orrall D, E lias L, A shford D, et al. A Carrot L eucine2r ich2repeat P rotein That Inhibits Ice Recrystallization [J ]. Science, 1998, 282 (5386) : [ 8 ],,. [M ]. :, [ 9 ][]J, E F, T. [M ]. 2.,. :, ; C loning of a carrot gene encoding antifreeze p rotein and construction of its p lant exp ression vector Y IN M ing-an 1, CU I Hong-wen 1, FAN Da i-m ing 2, GUO L i 3 (1 College of H orticulture, N orthw est S cience and T echnology U niversity of A g riculture and Forestry, Y ang ling, S haanx i , China; 2 X ij ing H osp ital, the Fourth U niversity of M ilitary M ed icine, X iθan , China; 3 Colleg e of L if e S cience, N orthw est S cience and T echnology U niversity of A g riculture and Forestry, Yang ling, S haanx i , China) Abstract: Genom ic DNA in the seedlings of carro t cultivar A utum n K ing w as ex tracted w ith CTAB m ethod. T he carrot antifreeze p rotein gene (afp) w as amp lified by PCR (Polym erase Chain R eaction). C loning vector pta F of carrot afp w as constructed w ith pu Cm 2T V ectoṙ PTA F w as digested w ith E cor g and becam e lineaṙ Its ends w ere filled w ith DNA Polym erase g K lenow fragm enṫ T hen it w as digested w ith X bag and a designed fragm ent (afp) w ith a cohesive end and a blunt end w as released. P lant exp ression vector pb I121 w as digested w ith X bag and Sm ag and a linear p lasm id w ith a cohesive end and a blunt end w as obtained. T he linear p lasm id and the designed fragm en t ( afp ) w ere directively ligated w ith T 4 DNA ligase, and the p lan t exp ression vector of carrot afp w as constructed. Key words: carrot; antifreeze p rotein gene; molecular biology