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Chuyển gen Bt2 vào một số dòng ngô trồng bằng sử dụng mô phân sinh và Agrobacterium tumefaciens (Luận văn thạc sĩ)Chuyển gen Bt2 vào một số dòng ngô trồng bằng sử dụng mô phân sinh và Agrobacterium tumefaciens (Luận văn thạc sĩ)Chuyển gen Bt2 vào một số dòng ngô trồng bằng sử dụng mô phân sinh và Agrobacterium tumefaciens (Luận văn thạc sĩ)Chuyển gen Bt2 vào một số dòng ngô trồng bằng sử dụng mô phân sinh và Agrobacterium tumefaciens (Luận văn thạc sĩ)Chuyển gen Bt2 vào một số dòng ngô trồng bằng sử dụng mô phân sinh và Agrobacterium tumefaciens (Luận văn thạc sĩ)Chuyển gen Bt2 vào một số dòng ngô trồng bằng sử dụng mô phân sinh và Agrobacterium tumefaciens (Luận văn thạc sĩ)Chuyển gen Bt2 vào một số dòng ngô trồng bằng sử dụng mô phân sinh và Agrobacterium tumefaciens (Luận văn thạc sĩ)Chuyển gen Bt2 vào một số dòng ngô trồng bằng sử dụng mô phân sinh và Agrobacterium tumefaciens (Luận văn thạc sĩ)
VI VI T NAM VI T NGUY N TH THU CHUY N GEN BT2 D TS NG B NG S AGROBACTERIUM TUMEFACIENS LU C -2015 VI VI T NAM VI T NGUY N TH THU CHUY N GEN BT2 D NG M TS NG B NG S AGROBACTERIUM TUMEFACIENS C TH C NGHI M LU C NG D N KHOA H C PGS TS Nguy g 11-2015 L uc k t qu li u, Nguy n Th Thu L IC c t i PGS TS Nguy t ng d n, ch b om u ki c bi nt om p th cv u ki n v v t ch m t tinh th n k thu t c hi o Vi n S t Vi Vi u ki n thu n l Cu ng nghi p su t th o c DANH M C T , C M T VI T T T DNA : Deoxyribonucleic acid RNA : Ribonucleic acid bp : C p base (base pair) CTAB : Cetyltrimethyl amoniumbromide dNTP : Deoxynucleosid triphosphat EDTA : Ethylene diamin tetra acetate EtBr : Ethidium bromide kb : Kilo base PCR : Ph n ng chu i polymerase (Polymerase chain reaction) Rnase : Ribonuclease TBE : Tris base, Boric acid, EDTA TE : Tris EDTA MES : 2-morphlinoethanesulfonic acid PPT : L- Phosphinothricin AGPase : ADP-Glucose pyrophosphorylase MS : Murashige and Skoog Medium BSA : bovine serum albumin fraxtion BCIP/NBT : Bromo Chloro toluidine salt/Nitro Blue Tetrazolium SDS : sodium dodecyl sulfat idolyl phosphate, p- DANH M C B NG B ng 1: K t qu ch n l ng ch n l c 24 B ng 2: S b n gen chuy ct m i 29 B ng 3: S ng th m 30 DANH M M t s enzyme quan tr ng q tinh b t n gen pCambia2300-Ubi-Bt2; 15 nc -Ubi- m ts 23 t qu chuy n gen t ng s nl 25 K t qu ki m tra s t c a gen Bt2 m t s n gen b ng PCR v i m c hi u 26 K t qu ki m tra s t c a gen Ubi m t s n gen b ng PCR v i m c hi u 27 7: K t qu ki m tra Southern m t s n gen; 28 M U 1 tv M N u I LI U 1.2 Tinh b u ng h p tinh b t ng s d ng p nh vi khu n Agrobacterium tumerfaciens 1.3.1 Chuy 1.3.2 Chuy n gen tr c ti p 10 u chuy n gen 1.4.2 11 u chuy gi i 11 u chuy c 14 T LI 2.1 V t li U 15 u 15 2.1.1 V t li u .15 t 16 n gen b ng PCR 19 n gen b ng lai Southern 20 T QU 3.1 Ch n l O LU N 23 n gen 23 n gen b ng PCR 25 3.3 n gen b ng lai Southern 28 T LU N NGH 32 4.1 K t lu n 32 4.2 Ki n ngh 32 ng th quan tr iv gi i sau is i cung c i, c cho nhi c ta Hi c a cv h t Vi c bi iv gi ng Hi tr i ti c nt o y c d ng gi ng m i d n gi l c n gi t o gi ng m u ng di truy n ngu n gen, di truy n l c nh ch th ngh ng thu h thu ut y u ph p, s ng h t, kh ng c a h t ng d c ng h p tinh b t, ho ng c u ng h p sinh kh i Nhi ng h p tinh b u n t ng h p tinh b t u cho th y ADP- h u i gen Brittle (Bt2 m ti u ph n nh i gen Shrunken (Sh2) Chuy n gen Sh2 ph n l t nh b u nh ti u Bt2 ng h p tinh ng ng d om ts tumefaciens u: Chuy n gen Bt2 ng b ng s d Agrobacterium Khi chuy [11] n ng ch a 200 mg/l kanamycin K t qu c cho th y h u h ng ch n l c ch a kana ng (b ch t ng) nl n Bt2 ycin Sau 8- ng ch n l kanamycin, m t s 3C), m ts nt r gen chuy k t lu cl n Bt2 ng ch n l ng r t c chuy ti p t c 3E) n gen kh u T0 STT S h t S h t b S S T l non nghi m n y m S tri m m m nhi m tri n ng chuy n ch n ng (%) l c H95 500 495 490 490 37 7,55 H26 500 492 490 490 29 5,91 H240 500 494 490 490 12 2,44 H20 493 490 490 18 3,67 500 gen) n gen T0 t c b t qu b ng cho th y t l ng ch n l 7,55% T l ng t n gen cao nh nh n t l th p l l 10% c c ch n l ng n gen 0,1% glufosinate S c u 3.2 n gen T0 ch ng ch a 200 mg/l c chuy n tr 85,42%) c ti (chi m th t t d thu m t DNA t ng s M DNA t ng s n gen.M: DNA marker kb 1-8 n n di ki m tr v s d ki m tra s t Bt2 b c DNA t ng s c hi u Bt2 DNA c c PCR ki m tra s d ng c p m c mang gen s c kho t qu c hi u Bt2 s n ph m PCR s d ng c p m : n di Bt2 - T n th ts pv n n gen chuy n Sau i T0 n gen t qu nh t qu Bt2 c n chuy b Bt2 nl cv m tra t qu ng ch n l i cho k t qu c hi u Bt2, Ubi v ic pm chuy nh ki m tra ir ,c huy nl c chuy , c chuy ng ch n l c DNA t ng s c Ubi b ng PCR s d ng c p m DNA c gel agarose 1% H v ic pm c ti p t c ki m tra s t c a gen c hi mang gen sau PCR b ng m t qu c hi u n di s n ph m PCR DNA t ng s c a c hi : Ubi NA - T gen n th pv i T0 ts n n gen chuy n gen Bt k t qu y mang gen Bt2 Ubi i th y r ng c 22 u mang gen Ubi t t qu nh t qu m tra b ng PCR s d ng c p m k t qu ch ng t s i s d ng c p m c hi biR cho c hi u Bt2 t c a vector chuy n gen n c sau ki m tra gen Bt2 gen T0 PCR s Ubi b ng u ti p theo 3.3 m tra gen Bt2 b t c ti p ng lai Southern DNA t ng s c n gen c c t b ng enzyme SacI, th m truy DNA t ng s n gen Bt2 c c t b ng enzyme SacI c nh n bi t lai v n gen Bt2 K t qu cho th n3b chuy 7 th cg n n gen t qu lai Southern m t s (t (t n T Bt2 b ng PCR, sau ki m tra b ng lai Southern nh n gen t Bt2 S m gen chuy b n y b ng 2 S b n Bt2 S m gen H26 H95 H240 3 3 10 1,22 2,04 0,20 T ng Hi u su t chuy n gen (%)* *Hi u su t chuy nh ng t l n gen mang gen Bt2 ct m n gen c a Qua b ng th y b n gen chuy n S n gen nh n ng t gen chuy n chi m n chi m 1,22 n chi m 0,2% K t qu nh cho hi u su t chuy so v u su t chuy n gen c n th y k t qu c cho th So u so v gi i uc V ic ng s 10% Omb i u su t chuy n gen ng s u su t chuy n ng s : 12% - ng s : 50% A188, 15% t c a gen Bt2 K t qu y th m t t nh t cho chuy A tumefaciens d th ng PCR (B ng 3) m gi STT S Th nhi m (gi ) 2 16 D T l (%) 166 0 H240 165 0 H95 165 0 H20 166 0 H26 166 1,2 H240 166 0 H95 165 1,82 H20 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Thu, H Th chuy ng tinh b t TC Sinh h c h c 37(4): 496-502 Bt2 qua Agrobacterium tumefaciens -698 Shrunken 2(Sh2 -Glucose qua Agrobacterium tumefaciens Nguy n Th -109 c, Nguy gen Shrunken (Sh2) t 1301-Ubi-Sh2.T t k vector chuy n gen pCambia c 35 (3se): 122 128 ... s tumefaciens th pv it c c ch n l chuy h hi ng kh m d ng A i gian T nh ng k t qu u chuy Bt2 n gen t c a gen t lu n sau: Bt2 cs tc a gen Bt2 t n gen c b n3 u su t chuy n gen cao nh t c th A tumefaciens. .. p Bt2, Ubi DNA t ng s cs d u cho PCR ki m tra s n gen Bt2, Ubi m tc Ki m tra s t c a gen Ubi, Bt2 Ph n n gen c th c hi n v i h n h p ph n ng g m X polymerase (5 U/ 80C (gen Ubi 720 C (gen Bt2) ;... S m gen chuy b n y b ng 2 S b n Bt2 S m gen H26 H95 H240 3 3 10 1,22 2,04 0,20 T ng Hi u su t chuy n gen (%)* *Hi u su t chuy nh ng t l n gen mang gen Bt2 ct m n gen c a Qua b ng th y b n gen