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 166 0 H26 166 2,41 H240 166 0,6 H95 165 4,24 H20 166 0 th y chuy ng n A tumefaciens s d ph thu mang gen H26 T nh ng k t qu nh gian t S sinh x n gen ch ng m thi Th i ti cs d tu c ch n l c hai tu n: ng ch a 100 mg/l kanamycin, tu n th ch cho k t qu t u qu u ti p theo nh m c i ti i vi c 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 K t qu i n quan tr ng u chuy n gen t ng h p tinh b Ti p t cho hi u qu chuy chuy n c cho t t c n U THAM KH O Ti ng Vi t -30 Brittle (Bt2 -Glucose pyrophosphorylase -6 oa Nam, 2007 -227 Ahmadabadi M, Ruf S, & Bock R (2007) A leaf-based regeneration and transformation system for maize (Zea mays L) Trans Res 16(4): 437-448 Anami SE, Mgutu AJ, Taracha C, Coussens G, Karimi M, Hilson P, Lijsebettens M.V, Machuka J (2010) Somatic embryogenesis and plant regeneration of tropical maize genotypes, Plant Cell Tiss Organ 102:285-295 Bhave MR, Lawrence S, Barton C, and Hannah LC, (1990) Identification and molecular characterization of Shrunken-2 cDNA clones of maize Plant cell, (6): 581-588 Cobb BG, and Hannah LC, (1998) Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm1 Plant Physiol., 88: 1219-1221 Denyer K, Dunlap F, Thorbjrnsen T, Keeling P, and Smith AM, (1996) The major form of ADP-Glucose pyrophosphorylase in maize endosperm 1s extra-plastidial Plant Physiol., 112 (2): 779-785 10 Dickinson DB, Preiss J, (1969) Presence of ADP-Glucose Pyrophosphorylase in Shrunken-2 and Brittle-2 Mutants of maize endosperm Plant Physiol 44 (7) 1058 1062 11 (1992) Transgenic maize plants by tissue electroporation Plant Cell 4:14951505 12 Figueiredo DA, Sine LF, Chantereau B, Mestres J, Fliedel C, Rami G, Courtois JF (2010) Variability of grain quality in sorghum: association with polymorphism in Sh2, Bt2, SssI, Ae1, Wx and O2 Theo Appl Genet., 121, 1171-1185 13 Frame BR, Shou H, Chikwamba R, Zhang Z, Xiang C, Fonger T, Pegg SE, Li B, Nettleton D, Pei P, Wang K (2002) Agrobacterium-mediated transformation of maize embryos using a standard binary vector system Plant Physiol 129: 13-22 14 Frame BR, McMurray JM, Fonger TN, Main ML, Taylor KW, Torney FJ, Paz MM., Wang K (2006) Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts Plant Cell Rep 25(10):1024-1034 15 Giroux MJ, Hannah LC (1994) ADP-glucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize Mol Gen Genet 243: 400 408 16 Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH (1980) Genetic transformation of mouse embryos by microinjection of purified DNA Proc Natl Acad Sci USA 77(12):7380-4 17 Hannah LC, Shaw JR, Giroux MJ, Reyss A, Prioul JL, Bae JM, Lee JY (2001) Maize genes encoding the small subunit of ADP-glucose pyrophosphorylase Plant Physiol 127: 173 183 18 Hannah LC (2005) Starch synthesis in the maize endosperm Maydica 50: 497 506 19 Hannah LC, Futch B, Bing J, Shaw JR, Boehlein S, Stewart JD, Beiriger R, Georgelis N, and Greene T (2012) A shrunken-2 transgene increases maize yield by acting in maternal tissues to increase the frequency of Seed development Plant Cell 24 (6): 2352 20 63 Huang B, Chen J, Zhang J, Liu H, Tian M, Gu Y, Hu Y, Li Y, Liu Y and Huang Y (2011) Characterization of ADP-Glucose pyrophosphorylase encoding genes in source and sink organs of maize Plant Mol Biol Rep, 29:563 572 21 Ishida Y, Hiei Y, Komari T (2007) Agrobacteium-mediated transformation of maize Nature Protocol l.2 (7):1614-1621 22 James MG, Denyer K, and Myers AM, (2003) Starch synthesis in the cereal endosperm Plant Biol 6: 215 222 23 Joersbo M, Brunstedt J (1990) Direct gene transfer to plant protoplasts by mild sonication Plant Cell Rep 9: 207 210 24 Li N, Zhang S, Zhao Y, Li B, Zhang J (2011) Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize Planta 233:241-250 25 Luo ZX, WuR (1989) A simple methods for the transformation of rice via the pollen-tubepathway Plant Mol boil Reporter 7: 69-77 26 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with T0 bacco tissue cultures Plant Physiol 15: 473-479 27 Ombori O, Muoma JVO, Machuka J (2012) Agrobacterium- mediated genetic transformation of selected tropical inbred and hybrid maize (Zea mays L.) lines Plant Cell Tiss Org DOI 10.1007/s11240-012-0247-1 28 Prioul J Ll, Jeannette E, Reyss A, N, Giroux M, Hannah LC, and Causse M (1994) Expression of ADP-glucose pyrophosphorylase in maize (Zea mays L.) grain and source leaf during grain filling Plant Physiol 104 (1): 179 187 29 Russell DA, DeBoer DL, Stark DM, Preiss J, Fromm ME (1993) Plastid targeting of -glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells Plant Cell Rep 13: 287 292 30 Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics Proc Natl Acad Sci USA 81: 8014-8018 31 Sairam RV, Parani M, Franklin G, Lifeng Z, Smith B, MacDougall J, Wilber C, Sheikhi H, Kashikar N, Meeker K, Al-Abed D, Berry K, Vierling R, Goldman SL (2003) Shoot meristem: an ideal explant for Zea mays L transformation Genome 46:323 329 32 substances Sanford JC, Klein TM, Wolf ED, Allen N (1987) "Delivery of into cells and tissues using a particle bombardment process" Particulate Science and Technology (1): 27 37 33 Schell J; Montagu MV (1977) "The Ti-plasmid of Agrobacterium tumefaciens, a natural vector for the introduction of nif genes in plants?" Basic Life Sci 9: 159 79 34 Sidorov V, Gilbertson L, Addae P, Duncan D (2006) Agrobacterium-mediated transformation of seedling-derived maize callus Plant Cell Rep 25: 320-328 35 Takava S, Rahnama H, Rahimian H, and Kazemitabar K (2010) Agrobacterium-mediated transformation of maize (Zea mays L.) Journal of Sciences, Islamic Republic of Iran 21(1): 21-29 36 Tsai CY and Nelson OE (1966) Starch-deficient maize mutant lacking adenosine diphosphate glucose pyrophosphorylase activity Science 151: 341 343 37 Vega JM, Yu W, Kennon AR, Chen X, Zhang ZJ (2008) Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays L.) using standard binary vectors Plant Cell Rep 27: 297-305 38 Wang Z, Chen X, Wang J, Liu T, Liu Y, Zhao L, Wang G (2007) Inreasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants Plant Cell Tiss Org 88: 83-92 39 Wang TT, Ji J, Wang G, Guan CF, Zhang L, Jin C (2012) Study on Agrobacterium-mediated transformation of psy gene into shoot meristem of maize China Biol 32(8): 36-40 Nguy h, Tr n Th n Th Zea mays L.) 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