Đề tài Tạo chủng vi khuẩn Agrobacterium tumefaciens mang gen kháng côn trùng để chuyển vào cây trồng

Öc biÓu hiÖn thµnh c«ng gen cry trong E. coli cã mét ý nghÜa v« cïng quan träng. §Æc biÖt khi vect¬ Ti-plasmit mang kÕt cÊu gen Ubi-cryIA(c) do chóng t«i thiÕt kÕ ®· vµ ®ang ®­îc chuyÓn vµo lóa vµ c¸c lo¹i c©y trång kh¸c. Ngoµi ra, c¸c nghiªn cøu chuyÓn gen cry trªn ®èi t­îng c©y ng« ®· ®­îc triÓn khai trong ch­¬ng tr×nh hîp t¸c víi ViÖn Nghiªn cøu Ng«. Ph­¬ng ph¸p ph¸t hiÖn protein tinh thÓ ®éc tè trong c¸c c©y lóa, ng«…chuyÓn gen cry cÇn ph¶i cã kh¸ng thÓ (s¶n xuÊt trªn c¬ së protein Cry t¸i tæ hîp). Nh­ vËy, protein Cry t¸i tæ hîp sÏ ®­îc sö dông ®Ó kiÓm tra c¸c c©y trång chuyÓn gen nµy. H¬n n÷a, Cry t¸i tæ hîp còng cã thÓ dïng trong m« h×nh thö nghiÖm ë quy m« phßng thÝ nghiÖm nghiªn cøu t¸c ®éng cña protein tinh thÓ ®éc tè lªn c¸c loµi c«n trïng kh¸c nhau. Qua kÕt qu¶ lai miÔn dÞch nµy, cã thÓ kh¼ng ®Þnh chóng t«i ®· thµnh c«ng trong viÖc biÓu hiÖn gen cryIA(c) ë vi khuÈn E. coli. 3.5.2 S¶n xuÊt kh¸ng thÓ S¶n phÈm protein CryIA(c) t¸i tæ hîp sau khi tinh chÕ vµ kiÓm tra ®· ®­îc chóng t«i sö dông ®Ó g©y miÔn dÞch s¶n xuÊt kh¸ng thÓ kh¸ng CryIA(c) dïng trong kiÓm tra c¸c c©y chuyÓn gen. §Çu tiªn, protein Cry ®­îc trén víi t¸ chÊt Freund toµn phÇn víi tØ lÖ 1:1 (1ml dung dÞch chøa kho¶ng 200 mg protein CryIA(c) tinh chÕ + 1ml t¸ chÊt Freund toµn phÇn) vµ tiªm d­íi da ë hai bªn h«ng gÇn sèng l­ng thá. Sau ®ã tiÕn hµnh tiªm nh¾c l¹i vµo c¸c ngµy thø 5, 10 vµ 30. §Õn ngµy thø 10 tÝnh tõ lÇn tiªm cuèi cïng, chóng t«i ®· tiÕn hµnh lÊy m¸u thá vµ thu huyÕt thanh. CÇn chó ý khi thu huyÕt thanh cÇn lÊy m¸u ë ®éng m¹ch, thao t¸c nhÑ nhµng tr¸nh lµm vì hång cÇu. §Ó kiÓm tra chÊt l­îng kh¸ng thÓ thu ®­îc, chóng t«i còng ®· tiÕn hµnh lai miÔn dÞch víi 3 lo¹i kh¸ng nguyªn: i) Kh¸ng nguyªn t¸i tæ hîp CryIA(c) do chóng t«i tinh chÕ; ii) Kh¸ng nguyªn CryIA(b) (nhËn ®­îc tõ TS. NguyÔn ThÞ Léc); iii) Protein CryIA(c) (nhËn ®­îc tõ TS. Vâ ThÞ Thø – ViÖn C«ng nghÖ Sinh häc). KÕt qu¶ lai ë c¶ ba mÉu thÝ nghiÖm trªn (h×nh 3.30 - cét 1, 2, 3) ®Òu xuÊt hiÖn mét b¨ng protein cã kÝch th­íc kho¶ng 68 kDa. Chóng t«i còng ®ång thêi tiÕn hµnh thÝ nghiÖm kiÓm tra chÊt l­îng kh¸ng nguyªn CryIA(c) chóng t«i tinh chÕ ®­îc b»ng c¸ch lai miÔn dÞch víi kh¸ng thÓ chuÈn kh¸ng CryIA(c) (do TS. NguyÔn ThÞ Léc cung cÊp). S¶n phÈm lai (h×nh 3.30, cét 4) xuÊt hiÖn mét b¨ng cã kÝch th­íc kho¶ng 68 kDa. KÕt qu¶ d­¬ng tÝnh nµy mét lÇn n÷a kh¼ng ®Þnh chóng t«i ®· tinh chÕ ®­îc protein CryIA(c) t¸i tæ hîp. H×nh 3.30. Lai miÔn dÞch c¸c protein Cry t¸i tæ hîp víi kh¸ng thÓ kh¸ng CryIA(c) vµ kh¸ng thÓ chuÈn 1. Protein CryIA(c) (VCNSH) vµ kh¸ng thÓ kh¸ng CryIA(c) (VCNSH) 2. Protein CryIA(b) (VL§BSCL) vµ kh¸ng thÓ kh¸ng CryIA(c) (VCNSH) 3. Protein CryIA(c) (TS. Vâ ThÞ Thø) vµ kh¸ng thÓ kh¸ng CryIA(c) (VCNSH) 4. Protein CryIA(c) (VCNSH) vµ kh¸ng thÓ chuÈn (VL§BSCL) Nh÷ng kÕt qu¶ trªn cho thÊy kh¸ng thÓ chóng t«i s¶n xuÊt ®­îc cã chÊt l­îng tèt vµ cã thÓ sö dông ®Ó ph©n tÝch c¸c c©y trång ®­îc chuyÓn gen cryIA(c). Nh­ vËy, nh÷ng c©y trång ®· chuyÓn gen cry ngoµi viÖc ®­îc ®¸nh gi¸ s¬ bé b»ng kü thuËt PCR (Polymerase Chain Reaction), kü thuËt lai miÔn dÞch protein cña c©y chuyÓn gen vµ kh¸ng thÓ ®Æc hiÖu cho protein Cry cña lo¹i gen ®­îc chuyÓn vµo c©y nhê sö dông kh¸ng thÓ chóng t«i s¶n xuÊt ®­îc ®· kh¼ng ®Þnh ë b­íc cao h¬n, ®ã lµ kh¶ n¨ng biÓu hiÖn gen chuyÓn thµnh s¶n phÈm protein cã ho¹t tÝnh kh¸ng s©u. kÕt luËn vµ ®Ò nghÞ kÕt luËn §· ph©n lËp, t¹o dßng ph©n tö vµ ®äc tr×nh tù nucleotit ®­îc ®o¹n khëi ®éng cña gen m· ho¸ sucroza synthaza (sucrose synthase) ë gièng lóa ViÖt Nam C71 (C71S-P) víi kÝch th­íc ph©n tö 1954 bp. Ph©n tÝch tr×nh tù cho thÊy C71S-P mang c¸c yÕu tè ®iÒu khiÓn biÓu hiÖn gen ®Æc hiÖu bã m¹ch (phloem) nh­ hép ASL, hép II, hép GATA. Ngoµi ra, C71S-P cßn chøa 1 ®o¹n tr×nh tù míi dµi 13 nucleotit cïng víi sù kh¸c biÖt ë 14 vÞ trÝ nucleotit, so víi ®o¹n khëi ®éng Rsuc1 do c¸c t¸c gi¶ n­íc ngoµi c«ng bè. Tr×nh tù C71S-P ®· ®­îc ®¨ng ký t¹i c¸c Ng©n hµng gen quèc tÕ EMBL/ GENBANK/ DDBJ ngµy 3 th¸ng 7 n¨m 2000 víi m· sè AJ401233. §· thiÕt kÕ ®­îc 4 Ti-plasmit t¸i tæ hîp míi, mang c¸c kÕt cÊu gen nh­ sau: i) §o¹n khëi ®éng c¬ ®Þnh Ubiquitin + Gen cryIA(c) tæng hîp ho¸ häc + ®o¹n kÕt thóc NOS trong vect¬ pCAMBIA1300 (ký hiÖu: pC1300-Ubi-cryIA(c)); ii) Gen cryIA(c) tæng hîp ho¸ häc + ®o¹n kÕt thóc NOS khuyÕt ®o¹n khëi ®éng trong vect¬ pCAMBIA1300 (pC1300-cryIA(c)) dïng cho nghiªn cøu chøc n¨ng cña c¸c ®o¹n khëi ®éng míi; iii) §o¹n khëi ®éng ®Æc hiÖu bã m¹ch C71S-P + Gen cryIA(c) tæng hîp ho¸ häc + NOS trong vect¬ pCAMBIA1300 (pC1300-C71S-cryIA(c)); (iv) §o¹n khëi ®éng c¬ ®Þnh Ubiquitin + Gen cryIA(c) tæng hîp ho¸ häc + ®o¹n kÕt thóc NOS trong vect¬ chän läc tÝch cùc pNOV2819 (pNOV-Ubi-cryIA(c)). §· c¶i tiÕn, hoµn thiÖn vµ sö dông ph­¬ng ph¸p xung ®iÖn vµ ph­¬ng ph¸p phèi ba bè mÑ ®Ó t¹o ®­îc 4 chñng A. tumefaciens míi trªn c¬ së LBA4404 (pGA3850) vµ EHA105, mang 2 Ti-plasmit t¸i tæ hîp míi chøa gen kh¸ng c«n trïng (pC1300-Ubi-cryIA(c) vµ pC1300-C71S-cryIA(c)) ®Ó chuyÓn vµo c©y trång. §· thµnh c«ng trong viÖc thiÕt kÕ vect¬ biÓu hiÖn gen cryIA(c) tæng hîp ho¸ häc trong vi khuÈn E. coli. KÕt qu¶ lai miÔn dÞch kh¼ng ®Þnh c¸c protein CryIA(c) t¸i tæ hîp cã ph¶n øng ®Æc hiÖu víi kh¸ng thÓ kh¸ng CryIA(c). §· tinh chÕ protein CryIA(c) t¸i tæ hîp vµ ®· sö dông ®Ó s¶n xuÊt kh¸ng thÓ kh¸ng CryIA(c) phôc vô cho viÖc kiÓm tra sù cã mÆt cña protein CryIA(c) ë c¸c c©y trång chuyÓn gen. §· chuyÓn giao 4 chñng A. tumefaciens míi t¹o ®­îc cho Phßng C«ng nghÖ TÕ bµo Thùc vËt, ViÖn C«ng nghÖ Sinh häc vµ Phßng C«ng nghÖ Sinh häc, ViÖn Nghiªn cøu Ng« ®Ó chuyÓn vµo c¸c gièng lóa vµ ng« nh»m t¹o nguyªn liÖu khëi ®Çu cho chän gièng kh¸ng s©u. ®Ò nghÞ TiÕp tôc nghiªn cøu thiÕt kÕ c¸c tæ hîp gen kh¸c nhau trªn c¬ së c¸c vect¬ Ti-plasmit míi t¹o ®­îc còng nh­ mét sè nguån gen ®· s­u tËp vµ ph©n lËp ®­îc. Më réng triÓn khai øng dông c¸c chñng A. tumefaciens vµ kh¸ng thÓ t¹o ®­îc vµo nghiªn cøu chuyÓn gen ë c¸c c©y trång kh¸c nhau. Nh÷ng c«ng tr×nh cña t¸c gi¶ ®· c«ng bè liªn quan ®Õn ®Ò tµi luËn ¸n Lª ThÞ Thu HiÒn, §inh Duy Kh¸ng, Lª TrÇn B×nh, N«ng V¨n H¶i (1999). Gen kh¸ng c«n trïng vµ øng dông trong c«ng nghÖ chuyÓn gen thùc vËt. Kû yÕu 1998, ViÖn C«ng nghÖ Sinh häc, Trung t©m Khoa häc Tù nhiªn vµ C«ng nghÖ Quèc Gia. Nhµ xuÊt b¶n Khoa häc vµ Kü thuËt, tr. 31-42. Lª ThÞ Thu HiÒn, Lª TrÇn B×nh, §inh Duy Kh¸ng, N«ng V¨n H¶i (2000). Ph©n tÝch tr×nh tù cña ®o¹n ®iÒu khiÓn cña gien tæng hîp ®­êng (RSUC1-PROMOTER) tõ gièng lóa C71. T¹p chÝ Sinh häc, tËp 23 (2), tr. 45-50. Le Thi Thu H., Dinh Duy K., Nong Van H., Le Tran B (2000). Nucleotide sequence of sucrose synthase promoter from rice C71 cultivar. EMBL Nucleotide Sequence Database, Accession Number AJ401233. Lª ThÞ Thu HiÒn, TrÞnh C«ng Sù, TrÇn ThÞ Ph­¬ng Liªn, Ph¹m BÝch Ngäc, §inh Duy Kh¸ng, N«ng V¨n H¶i, Lª TrÇn B×nh (2002). ThiÕt kÕ vect¬ vµ biÓu hiÖn gen m· ho¸ protein ®éc tè cryIA(c) cã nguån gèc tõ vi khuÈn Bacillus thuringiensis b»ng vi khuÈn Escherichia coli. T¹p chÝ Sinh häc, tËp 24(3), tr. 39-46. Lª ThÞ Thu HiÒn, §inh Duy Kh¸ng, Lª TrÇn B×nh, N«ng V¨n H¶i (2002). ThiÕt kÕ vect¬ mang gien ®éc tè cryIA(c) d­íi sù ®iÒu khiÓn cña ®o¹n khëi ®éng cña gien tæng hîp ®­êng (Rsuc1 promoter) ph©n lËp tõ gièng lóa C71. T¹p chÝ Khoa häc vµ C«ng nghÖ, tËp 40, sè §B, tr. 135-141. Le Thi Thu Hien, Lam Dai Nhan, Kieu Huu Anh, Nong Van Hai, Le Tran Binh (2002). Construction of Agrobacterium tumefaciens strains carrying Bt pesticidal genes. Advances in Natural Sciences 3(2), pp. 175-180. Tµi liÖu tham kh¶o Tµi liÖu tiÕng ViÖt Lª TrÇn B×nh, Lª ThÞ Muéi (1998), Ph©n lËp gen vµ chän dßng chèng chÞu ngo¹i c¶nh bÊt lîi ë c©y lóa, Nhµ XuÊt B¶n §¹i häc Quèc gia Hµ Néi. Lª TrÇn B×nh (1999), “T×nh h×nh ph¸t triÓn c«ng nghÖ gene ë ViÖt Nam”, B¸o c¸o khoa häc, Héi nghÞ C«ng nghÖ Sinh häc toµn quèc, Hµ Néi, tr. 71-75. NguyÔn ThÞ Liªn Chi, NguyÔn H÷u Hæ, NguyÔn V¨n UyÓn (1994), “ChuyÓn gen kh¸ng kanamycin vµo c©y thuèc l¸ Nicotiana Tabacum vµ m« l¸ c©y cµ óc S. laciniatum b»ng vi khuÈn Agrobacterium tumefaciens”, Di truyÒn häc vµ øng dông 1, tr. 23-28. T¹ Kim ChØnh (1996), TuyÓn chän mét sè chñng vi nÊm diÖt c«n trïng g©y h¹i ë ViÖt Nam vµ kh¶ n¨ng sö dông, LuËn ¸n Phã TiÕn sÜ Khoa häc Sinh häc, ViÖn C«ng nghÖ Sinh häc, Hµ Néi. NguyÔn L©n Dòng (1981), Sö dông vi sinh vËt ®Ó phßng trõ s©u h¹i c©y trång, Nhµ XuÊt b¶n Khoa häc Kü thuËt Hµ Néi. NguyÔn V¨n §¹t, NguyÔn Thanh Thuû, NguyÔn Huy Hoµng, N«ng V¨n H¶i, Lª TrÇn B×nh, Tr­¬ng Nam H¶i (2000), “BiÓu hiÖn gen m· ho¸ cho protein bÊt ho¹t ribosome cña m­íp ®¾ng trong nÊm men Pichia pastoris”, Nh÷ng vÊn ®Ò Nghiªn cøu C¬ b¶n trong C«ng nghÖ Sinh häc, B¸o c¸o Khoa häc Héi nghÞ Sinh häc Quèc gia, Nhµ XuÊt B¶n §¹i häc Quèc gia Hµ Néi, tr. 44-47. NguyÔn Thuý Hµ, Hoµng Quèc Tr­êng, Tèng Quúnh Mai, NguyÔn ThÞ Tþ, NguyÔn H÷u HiÕn, Phan Quèc Kinh, Lª TrÇn B×nh, Phan V¨n Chi (1999), “T¸ch dßng vµ biÓu hiÖn gen m· ho¸ cho protein bÊt ho¹t ribosome nhãm 1 ë E. coli”, B¸o c¸o Khoa häc Héi nghÞ C«ng nghÖ Sinh häc Toµn quèc, Hµ Néi, tr. 1121-1128. Lª ThÞ Thu HiÒn, §µo ThÞ Hång V©n, §Æng ThÞ Thu, N«ng V¨n H¶i (1999), “Ph©n lËp gen m· ho¸ lectin vµ protein k×m h·m α-amylase ë h¹t ®Ëu c« ve (Phaseolus vulgaris)”, Kû yÕu ViÖn C«ng nghÖ Sinh häc, Nhµ XuÊt B¶n Khoa häc vµ Kü thuËt, tr. 116-120. Lª ThÞ Thu HiÒn, §inh Duy Kh¸ng, Lª TrÇn B×nh, N«ng V¨n H¶i (2000), “Gen kh¸ng c«n trïng vµ øng dông trong c«ng nghÖ chuyÓn gen thùc vËt”, Kû yÕu ViÖn C«ng nghÖ Sinh häc, Nhµ XuÊt B¶n Khoa häc vµ Kü thuËt, tr. 31-39. NguyÔn Huy Hoµng, §µo Duy Phong, Nghiªm Ngäc Minh, Lª TrÇn B×nh, N«ng V¨n H¶i (2000), “Ph©n lËp vµ ®äc tr×nh tù gen m· ho¸ protein bÊt ho¹t ribosome tõ c©y m­íp ®¾ng (Momordica charantia)”, Nh÷ng vÊn ®Ò Nghiªn cøu C¬ b¶n trong C«ng nghÖ Sinh häc, B¸o c¸o Khoa häc Héi nghÞ Sinh häc Quèc gia, Nhµ XuÊt B¶n §¹i häc Quèc gia Hµ Néi, tr. 84-88. TrÇn BÝch Lan, NguyÔn Lan Hoa, NguyÔn §øc Doanh, TrÇn Duy Quý (1998), “KÕt qu¶ b­íc ®Çu sö dông Agrobacterium tumefaciens trong nghiªn cøu chuyÓn gen vµo lóa”, Héi nghÞ Toµn quèc lÇn thø nhÊt vÒ C«ng nghÖ Sinh häc c©y lóa, HuÕ, tr. 98-101. TrÇn ThÞ Ph­¬ng Liªn (1999), Nghiªn cøu ®Æc tÝnh ho¸ sinh vµ sinh häc ph©n tö cña mét sè gièng ®Ëu t­¬ng cã kh¶ n¨ng chÞu nãng, chÞu h¹n ë ViÖt Nam, LuËn ¸n TiÕn sÜ Sinh häc, ViÖn C«ng nghÖ Sinh häc, Hµ Néi. TrÇn ThÞ Ph­¬ng Liªn, N«ng V¨n H¶i (1997), “ChuyÓn tæ hîp gen GUS-BNG vµo c©y thuèc l¸”, T¹p chÝ Khoa häc vµ C«ng nghÖ, 2, tr. 23-27. §Æng Träng L­¬ng, R. Offringa, Vò §øc Quang, NguyÔn H÷u §èng, TrÇn Duy Quý, W. Dolf, Pau J.J. Hooykaas (1999), “ThiÕt kÕ l¹i cÊu tróc gen Bt (Bacillus thuringensis) ®Ó chuyÓn vµo c©y hai l¸ mÇm”, B¸o c¸o Khoa häc Héi nghÞ Sinh häc Toµn quèc, Nhµ XuÊt b¶n Khoa häc kü thuËt Hµ Néi, tr. 1371-1376. §Æng Träng L­¬ng (2001), Nghiªn cøu ¸p dông kü thuËt chuyÓn gen nhê vi khuÈn Agrobacterium tumefaciens nh»m gãp phÇn t¹o vËt liÖu chän gièng c¶i b¾p kh¸ng s©u ë ViÖt Nam, LuËn ¸n TiÕn sÜ N«ng nghiÖp, ViÖn Khoa häc Kü thuËt N«ng nghiÖp ViÖt Nam, Hµ Néi. §Æng Träng L­¬ng, NguyÔn §øc Doanh, Vò §øc Quang, NguyÔn H÷u §èng, TrÇn Duy Quý (2001), “Nghiªn cøu chuyÓn gen cryIA(c) kh¸ng s©u vµo mét sè gièng c¶i b¾p ( Brassica oleracea var capitata) qua Agrobacterium”, KÕt qu¶ nghiªn cøu khoa häc 1999- 2000 ViÖn Di truyÒn N«ng nghiÖp, Nhµ XuÊt b¶n N«ng nghiÖp Hµ Néi, tr 120- 128. Hoµng Kim Oanh, Lª V¨n S¬n, L©m §¹i Nh©n, §inh ThÞ Phßng, Lª ThÞ Muéi, Lª TrÇn B×nh (1998), “KÕt qu¶ nghiªn cøu t¹o c©y chuyÓn gen”, Tãm t¾t B¸o c¸o Héi nghÞ toµn quèc lÇn thø nhÊt vÒ C«ng nghÖ Sinh häc c©y lóa, tr. 44-45. Phan Tè Ph­îng, Ph¹m Thu H»ng, Vò §øc Quang, TrÇn Duy Quý, Lili C., Zhang S., Fauquet C.M., Beachy R.N. (1998), “ChuyÓn gen kh¸ng hygromycin, gen gus vµ gen kh¸ng bÖnh b¹c l¸ vµo lóa b»ng sóng b¾n gen”, T¹p chÝ N«ng nghiÖp vµ C«ng nghiÖp thùc phÈm 2, tr. 56-57. NguyÔn H÷u Phæ, Lª TÊn §øc, NguyÔn ThÞ Thanh, NguyÔnV¨n UyÓn (1999), “T¹o c©y thuèc l¸ (Nicotiana tabacum L.) kh¸ng thuèc trõ cá basta b»ng chuyÓn gen th«ng qua vi khuÈn Agrobacterium tumefaciens”, B¸o c¸o Khoa häc Héi nghÞ Sinh häc Toµn quèc, Nhµ XuÊt b¶n Khoa häc kü thuËt Hµ Néi, tr. 1297-1304. NguyÔn H÷u T©m, Lª TÊn §øc, NguyÔn ThÞ Thanh, NguyÔn V¨n UyÓn (1998), “Sö dông vi khuÈn Agrobacterium tumefaciens ®Ó chuyÓn gen kh¸ng hygromycin vµ gen gusA vµo lóa indica (Ozyra sativa L.)”, Tãm t¾t B¸o c¸o Héi nghÞ toµn quèc lÇn thø nhÊt vÒ C«ng nghÖ Sinh häc c©y lóa, tr. 48-49. NguyÔn ThÞ Thanh, Lª TÊn §øc, NguyÔn H÷u Phæ, NguyÔn V¨n UyÓn (1998), “T¹o c©y cµ tÝm (Solanum melongena L.) mang gen Bt, gen bar vµ gen gusA dïng ph­¬ng ph¸p chuyÓn gen gi¸n tiÕp nhê vi khuÈn Agrobacterium tumefaciens”, Tãm t¾t B¸o c¸o Héi nghÞ toµn quèc lÇn thø nhÊt vÒ C«ng nghÖ Sinh häc c©y lóa, tr. 82-83. NguyÔn ThÞ Thanh, Lª TÊn §øc, NguyÔn V¨n UyÓn (1999), “T¹o c©y c¶i b«ng (Brassica oleracea var botrycis) vµ c©y c¶i xanh (Brassica juncea) mang gen kh¸ng s©u cryIA(c) vµ gen bar kh¸ng thuèc cá d¹i th«ng qua Agrobacterium tumefaciens”, B¸o c¸o Khoa häc Héi nghÞ C«ng nghÖ Sinh häc Toµn quèc, Hµ Néi, tr. 1287-1295. Vâ ThÞ Thø (1996), Nghiªn cøu ®Æc ®iÓm sinh häc vµ kh¶ n¨ng øng dông cña mét sè chñng vi khuÈn thuéc chi Bacillus, LuËn ¸n Phã TiÕn sÜ Khoa häc Sinh häc, ViÖn C«ng nghÖ Sinh häc, Hµ Néi. Mai Tr­êng, NguyÔn H÷u Hæ, Lª TÊn §øc, NguyÔn ThÞ Thanh, NguyÔn V¨n UyÓn (1998), “B­íc ®Çu chuyÓn gen bar, gen gusA vµ gen cryIA(c) vµo c©y ®Ëu xanh (Vigna radiata L.) nhê vi khuÈn Agrobacterium tumefaciens”, Héi nghÞ Toµn quèc lÇn thø nhÊt vÒ C«ng nghÖ Sinh häc c©y lóa, HuÕ, tr. 86-87. Tµi liÖu tiÕng Anh Adang M.J., Brody M.S., Cardineau G., Eagan N., Roush R.T., Shewmaker C.K., Jones A., Oakes J.V., McBride K.E. (1993), “The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants”, Plant Molecular Biology 21, pp. 1131-1145. Alam M.F., Datta K., Abrigo E., Oliva N., Tu J., Virmani S.S., Datta S.K. (1999), “Transgenic insect-resistant maintainer line (IR68899B) for improvement of hybrid line”, Plant Cell Reports 18, pp. 572-575. Alasdair D.I., Bruun R.T. (2000), “Promoter”, Patent WO0078975. Altosaar I., Sardana R., Dukiandjiev S., Giband M., Cheng X. (1997), “Synthetic Bacillus thuringiensis (Bt) toxin genes for insect resistance and their rapid bioassay using maize endosperm suspension cultures”, Rice Genetics III, pp. 737-742. Alves A.C., Quecini V.M., Vieira M.L.C. (1999), “Plant transformation: Advances and perspectives”, Scientia Agricola 56(1), pp. 1-12. An G. (1986), “Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells”, Plant Physiology 81, pp.86-91. An G., Ebert P.R., Mitra A., Ha S.B. (1988), “Binary vectors”, In: Plant molecular biology manual A3, Gelvin S.B., Schilperoort R., and Verma D. P. (Eds.), pp. 1-19. Angenon G., Van Montagu M. (1992), “Transgenic plants: Agrobacterium-mediated transformation and its application in plant molecular biology research and biotechnology”. In: Biotechnology and crop improvement in Asia, pp. 181-199. Anon (2000), “Novartis pins hopes for GM seeds on new marker system”, Nature 406, pp. 924. Armitage P., Walden R., Draper J. (1988), “Vectors for the transformation of plant cells using Agrobacterium”, In: Plant genetic transformation and gene expression, A laboratory manual, Draper J., Scott R., Armitage P., (Eds.), Blackwell Scientific Publications, pp. 3-64. Barbieri L., Battelli M.G., Stirpe F. (1993), “Ribosome-inactivating proteins from plants”, Biochimica et Biophysica Acta 1154, pp. 237-282. Barton K.A., Miller M.J. (1991), “Insecticidal toxins in plants”, EPA 0431829 A1 910612. Becker D., Kemper E., Schell J., Masterson R. (1992), “New plant binary vectors with selectable markers located proximal to the left T-DNA border”, Plant Molecular Biology 20, pp. 1195-1197. Becker C., Shutov A.D., Nong V.H., Senyuk .L., Jung R., Horstmann C., Fircher J., Nielsen N.C., Muntz K. (1995), “Purification, cDNA cloning and characterization of proteinase B, an asparagine – specific endopeptidase”, European Journal of Biochemistry 228, pp. 456-462. Bevan M. (1984), “Binary Agrobacterium vectors for plant transformation”, Nucleic Acids Research 12, pp. 8711-8721. Boulter D., Edwards G.A., Gatehouse A.M.R., Gatehouse J.A., Hilder V.A. (1990), “Additive protective effects of incorporating two different higher plant derived insect resistance genes in transgenic tobaco plants”, Journal of Crop Protection 9, pp. 351-354. Boulter D. (1993), “Insect pest control by copying nature using genetically engineered crops”, Phytochemistry 34, pp. 1453-1466. Bourdon V., Harvey A., Lonsdale D.M. (2001), “ Introns and their positions affect the translational activity of mRNA in plant cells”, EMBO Reports 2, pp. 394-398. Brears T., Walker E.L., Coruzzi G.M. (1991), “A promoter sequence involved in cell-specific expression of the pea glutamine synthetase GS3A gene in organs of transgenic tobacco and alfalfa”, The Plant Journal 1, pp. 235-244. Brock T.D., Madigan M.T. (1991), Biology of microorganisms, Prentice-Hall International Editions, pp. 301-302, 663-666. Burkness E.C., Hutchison W.D., Bolin P.C., Bartels D.W., Warnock D.F., Davis D.W. (2001), “Field efficacy of sweet corn hybrids expressing a Bacillus thuringiensis toxin for management of Octrinia nubilalis (Lepidoptera: Crambidae) and Helicoverpa zea (Lepidoptera: Noctuidae)”, Journal of Economic Entomology 94(1), pp. 197-203. Callis J., Fromm M., Walbot V. (1987), “Introns increase gene expression in cultured maize cells”, Gene and Development 1(10), pp. 1183-1200. Carozzi N.B., Warren G.W., Desai N., Jayne S.M., Lotstein R., Rice D.A., Evola S., Koziel M.G. (1992), “Expression of a chimeric CaMV35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco”, Plant Molecular Biology 20, pp. 539-548. Chakrabarti S.K., Mandaokar A.D., Kumar A.P., Sharma R.P. (1998), “Synergistic effect of CryIAc and CryIF δ-endotoxons of Bacillus thuringiensis on cotton bollworm, Helicoverpa armigera”, Current Science 75 (7), pp. 663-664. Chan M.T., Lee T.M., Chang H.H. (1992), “Transformation of indica rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens”, Plant Cell Physiology 33, pp. 577-583. Chaubet-Gigot N., Kapros T., Flenet M., Kahn K., Gigot C., Waterborg J.H. (2001), “Tissue-specific enhancement of transgene expression by introns of replacement histone H3 genes of Arabidopsis”, Plant Molecular Biology 45, pp. 17-30. Chen L., Marmey P., Taylor N.J., Brizard J., Espinoza C., D’Cruz P., Huet H., Zhang S., de Kocho A., Beachy R.N., Fauquet C.M. (1998), “Expression and inheritance of multiple transgenes in rice plants”, Nature Biotechnology 16, pp. 1060-1064. Cheng X., Sardana R., Kaplan H., Altosaar I. (1998), “Agrobacterium - transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer”, Proceedings of the National Academy of Sciences of the USA 95, pp. 2767-2772. Cheong H., Gill S.S. (1997), “Cloning and characterization of a cytolytic and mosquitocidal δ-endotoxin from Bacillus thuringiensis subsp. jegathesan”, Applied and Environmental Microbiology 63 (8), pp. 3254-3260. Chilton M.D. (2001), “Agrobacterium. A Memoir”, Plant Physiology 125, pp. 9-14. Christensen A.H., Sharrock R.A., Quail P.H. (1992), “Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation”, Plant Molecular Biology 18, pp. 675-689. Christensen A.H., Quail P.H. (1996), “Ubiquitin promoter - based vectors for high-level expression of selectable and/ or screenable marker genes in monocotyledonous plants”, Transgenic Research 5, pp. 213-218. Christou P. (1996), “Transformation technology”, Trends in Plant Science 1 (12), pp.423-431. Clancy M., Hannah L.C. (2002), “Splicing of the maize Sh1 first intron is essential for enhancement of gene expression, and a T-rich motif increases expression without affecting splicing”, Plant Physiology 130, pp. 918-929. Cornejo M.J., Luth D., Blankenship K.M., Anderson O.D., Blechl A.E. (1993), “Activity of a maize ubiquitin promoter in transgenic rice”, Plant Molecular Biology 23 (3), pp. 567-581. Dale E.C., Ow D.W. (1991), “Gene transfer with subsequent removal of the selection gene from the host genome”, Proceedings of the National Academy of Sciences of the USA 88, pp. 10558-10562. Daniel M. B., Michael D. R., Edelstein S. J. (1996), Protein methods, 2d Ed Wiley-Liss, Inc. Datta K., Vasquez A., Tu J., Torrizo L., Alam M.F., Oliva N., Abrigo E., Khush G.S., Datta S.K. (1998), “’Constitutive and tissue-specific differential expression of the cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest”, Theoretical Applied Genetics 97, pp.20-30. De Cosa B., Moar W., Lee S-B., Miller M., Daniell H. (2000), “Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals”, Nature Biotechnology 19, pp. 71-74. De la Riva G.A., Adang M.J. (1996), “Expression of Bacillus thuringiensis delta-endotoxin genes in transgenic plants”, Biotechnologia Aplicada 13(4), pp. 169-174. Dean D.H., Adang M.J. (1992), “Protein engineering of Bacillus thuringiensis δ-endotoxins and genetic manipulation for plant protection”, In: Plant Protein Engineering, Shewry P.R., Gutteridge S. (Eds.), Cambridge University Press, Cambridge, UK, pp. 293-311. Deblaere R., Reynaerts A., Hofte H., Hernalsteens J.D., Leemans J., Van Montagu M. (1987), “Vector for cloning in plant cells”, Methods in Enzymology 153, pp. 277-292. Diehn S.H., Chiu W.L., De Rocher E.J., Green P.J. (1998), “Premature polyadenylation at multiple sites within a Bt toxin gene-coding region”, Plant Physiology 117, pp. 1433-1443. Dina K.J., Thyge O.F., Guldager p.S., Kirsten B., Anna H., Morten J., John N., Iain D. (1998), “Mannozase or xylose based positive selection”, Patent US5767378. Ding X., Gopalakrishnan B., Johnson L.B., White F.F., Wang X., Morgan T.D., Kramer K.J., Muthucrishnan S. (1998), “Insect resistance of transgenic tobacco expressing an insect chitinase gene”, Transgenic Research 7, pp. 77-84. Douglas C.J., Hauffe K.D., Ites-Morales M.E., Ellard M., Paszkowski U., Hahlbrock K., Dangl J.L. (1991), “Exonic sequences are required for elicitor and light activation of a plant defense gene, but promoter sequences are sufficient for tissue specific expression”, The EMBO Journal 10(7), pp. 1767-1775. Draper J., Scott R., Armitage P. (1988), Plant genetic transformation and gene expression: A laboratory manual, Blackwell Scientific Publications, pp. 3-160. Duan X., Li X., Xue Q.Z., Abo-El-Saad M., Xu D., Wu R. (1996), “Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant”, Nature Biotechnology 14, pp. 494-498. Edmonds H.S., Gatehouse L.N., Hilder V.A., Gatehouse J.A. (1996), “The inhibitory effects of the cysteine protease inhibitors, oryzacystatin, on digestive proteases and on larval survival and development of the southern corn rootworm (Diabrotica undecimpunctata howard)”, Entomologia Experimentalis et Applicata 78, pp. 83-94. Edwards K., Johnstone C. (1991), “A simple rapid method for the preparation of plant genomic DNA for PCR”, Nucleic Acids Research 19, pp. 1349. Estruch J.J., Carozzi N.B., Desai N., Duck N.B., Warren G.W., Koziel M.G. (1997), “Transgenic plants: an emerging approach to pest control”, Nature Biotechnology 15, pp. 137-141. Fedoroff N.V., Cohen J.E. (1999), “Plants and population: Is there time?”, Proceedings of the National Academy Sciences of the USA 96, pp. 5903-5907. Finardi-Filho F., Mirkov T.E., Chrispeels M.J. (1996), “A putative precursor protein in the evolution of the bean α-amylase inhibitor”, Phytochemistry 43(1), pp. 57-62. Firoozabady E., DeBoer D.L., Merlo D.J., Halk E.L. (1987), “Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants”, Plant Molecular Biology 10, pp. 105-116. Fischoff D.A., Bowdis K.S. (1987), “Insect tolerant transgenic tomato plant”, Biotechnology 5, pp. 807. Frame B.R., Shou H., Chikwamba R.K., Zhang Z., Xiang C., Fonger T.M., Pegg S.E.K., Li B., Nettleton D.S., Pei D., Wang K. (2002), “Agrobacterium tumefaciens - mediated transformation of maize embryos using a standart binary vector system”, Plant Physiology 129, pp. 13-22. Gallie D.R., Young T.E. (1994), “The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression”, Plant Physiology 106(3), pp. 929-939. Gasson M.J. (2000), “Gene transfer from genetically modified food”, Current Opinion in Biotechnology 11, pp. 505-508. Gasson M., Burke D. (2001), “Scientific perspectives on regulating the safety of genetically modified foods”, Nature Reviews Genetics, pp. 217-222. Gatehouse A.M.R., Hilder V.A., Powell K.S., Wang M., Davison G.M., Gatehouse L.N., Down R.E., Edmonds H.S., Boulter D., Newell C.A., Merryweather A., Hamilton W.D.O, Gatehouse J.A. (1994), “Insect-resistance transgenic plants: Choosing the gene to do the ‘job’ ”, Biochemical Society Transactions 22, pp. 944-949. Gatehouse A.M.R., Bown D.P., Wilkinson H.S., Down R.E., Ford L., Gatehouse J.A. (1998), “The use of transgenic plants for the control of insect pests”, BCPC Symposium Proceedings 71: Biotechnology in crop protection: Facts and fallacies, pp. 25-32. Gill S.S., Cowles E.A., Pietrantonio P.V. (1992), “The mode of action of Bacillus thuringiensis endotoxins”, Annual Review of Entomology 37, pp. 615-636. Gordon M.P. (1998), “Discovery of the T-DNA of Agrobacterium tumefaciens”, In: Discoveries in plant biology I, Kung S-D., Yang S-F. (Eds.), World Scientific Publishing, Singapore, New Yersey, London, Hong Kong, pp. 111-113. Graham J., Gordon S.C., McNicol R.J. (1997), “The effect of the CpTI gene in strawberry against attack by vine weevil (Otiorhynchus sulcatus F. Coleoptera: Curculionidae)”, Annals Applied Biology 131, pp. 133-139. Grochulski P., Masson L., Borisova S., Pusztai - Carey M., Schwartz J.L., Brousseau R., Cygler M. (1995), “Bt CryIA(a) insecticidal toxin: crystal structure and channel formation”, Journal of Molecular Biology 254, pp. 447-464. Hajdukiewicz P., Svab Z., Maliga P. (1994), “The small, versatile pPZP familly of Agrobacterium binary vectors for plant transformation”, Plant Molecular Biology 25, pp 989-994. Hallahan D.L., Pickett J.A., Wadham L.J., Wallsgrove R.M., Woodcock C.M. (1992), “Potential of secondary metabolites in genetic engineering of crops for resistance”, In: Plant Genetic Manipulation for Crop Protection, Gatehouse A.M.R., Hilder V.A., boulter D. (Eds.), CAB International, Wallingford, pp. 215-248. Hames B.D., Rickwood D. (1989), Gel electrophoresis of protein: A practical apporoach, 2 end IRL, Press, Oxford. Hamilton C.M., Frary A., Lewis C., Tanksley S.D. (1996), “Stable transfer of intact high molecular weight DNA into plant chromosomes”, Proceedings of the National Academy of Sciences of the USA 93, pp. 9975-9979. Hamilton C.M. (1997), “A binary - BAC system for plant transformation with high-molecular - weight DNA”, Gene 200, pp. 107-116. Hedley P.E., Maddison A.L., Davidson D., Machray G.C. (2000), “Differential expression of invertase genes in internal and external phloem tissues of potato (Solanum tuberosum L.), Journal of Experimental Botany 51(345), pp. 817-821. Hellens R. (2000), “pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation”, Plant Molecular Biology 42, pp. 819-832. Hellens R., Mullineaux P. (2000), “A guide to Agrobacterium binary Ti-vectors”, Trends in Plant Science 5, pp. 446-451. Hemmer W. (1997), “Foods derived from genetically modified organisms and detection methods”, Http:/www.bats.ch/abstr/297intro.htm. Herrera-Estrella L., Depicker A., Van Montagu M., Schell J. (1983), “Expression of chimeric genes transferred into plant cells using a Ti plasmid-derived vector”, Nature 303, pp. 209-213. Herrera-Estrella L. (2000), “Genetic modified crops and developing countries”, Plant Physiology 124, pp. 923-925. Herrera-Estrella L., Alvarez-Morales A. (2001), “Genetically modified crops: hope for developing countries?”, EMBO Reports 2(4), pp. 256-258. Hiei Y., Ohta S., Komari T., Kumasho T. (1994), “Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA”, The Plant Journal 6, pp. 271-282. Hilder V.A., Boulter D. (1999), “Genetic engineering of crop plants for insect resistance - a critical review”, Crop Protection 18, pp. 177-191. Hoekema A., Hirsch P.R., Hooykaas P.J.J., Schilperoort R.A. (1983), “A binary plant vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid”, Nature 303, pp. 179-180. Hoekema A., Hooykaas P.J.J., Schilperoort R.A. (1984), Transfer or the octopine T-DNA segment to plant cells mediated by different types of Agrobacterium tumor- or root-inducing plasmids: Generality of virulence systems, Journal of Bacteriology 158, pp. 383-385. Hoffmann M.P., Zalom F.G., Wilson L.T., Smilanick J.M., Malyj L.D., Kiser J., Hilder J.A., Barnes W.M. (1992), “Field evaluation of transgenic tobacco containing genes encoding Bacillus thuringiensis δ-endotoxin or cowpea trypsin inhibitor: Efficacy against Heliocoverpa zea (Lepidopteran: Noctuidae)”, Journal of Economic Entomology 6, pp. 2516-2522. Hofte H., Whiteley H.R. (1989), “Insecticidal proteins of Bacillus thuringiensis”, Microbiology Review 53 (2), pp. 242-255. Holster M., De Waele D., Depicker A., Messens E., Van Montagu M., Schell J. (1987), “Transfection and transformation of A. tumefaciens”, Molecular and General Genetics 163, pp. 181-187. Hooykaas P.J.J., Schilperoort R.A. (1992), “Agrobacterium and plant genetic engineering”, Plant Molecular Biology 19, pp. 15-38. Horsch R.B., Fraley R.T., Rogers S.G., Sanders P.R., Lloyd A., Hoffmann N. (1984), “Inheritance of functional foreign genes in plants”, Science 223, pp. 496-498. Huang J.W., Chen J.T., Yu W.P., Shyur L.F., Wang A.Y., Sung H.Y., Lee P.D., Su J.C. (1996), “Complete structures of three rice sucrose synthase isogenes and differential regulation of their expressions”, Bioscience, Biotechnology, and Biochemistry 60, pp. 233-239. Ishida Y., Saito H., Ohta S., Hiei Y., Komari T., Kumashiro T. (1996), “High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens”, Nature Biotechnology 14, pp. 745-750. Ishimoto M., Chrispeels M.J. (1996), “Protective mechanism of the Mexican bean weevil against high levels of α-amylase inhibitor in the common bean”, Plant Physiology 111, pp. 393-401. James C., Krattiger A.F. (1996), “Global review of the field testing and commercialization of transgenic plants, 1986 to 1995: The first decade of crop biotechnology”, ISAAA Briefs 1, pp. 1-31. James C. (1997), “Global status of transgenic crops in 1997”, ISAAA Briefs 5, pp. 1-27. James C. (2000), “Preview: Global review of commercialized transgenic crops: 2000”, ISAAA Briefs 21, pp. 1-15. James C. (2001), “Preview: Global review of commercialized transgenic crops: 2001”, ISAAA Briefs 24, pp. 1-20. Jang I.C., Choi W.B., Lee K.H., Song S.I., Nahm B.H., Kim J.K. (2002), “High level and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots”, Plant Physiology 129, pp. 1473-1481. Jefferson R.A., Kavanagh T.A., Bevan M.W. (1987), “GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants”, The EMBO Journal 6, pp. 3901-3907. Jouanin L., BonadÐ-Bottino M., Giard C., Morrot G., Giband M. (1998), “Transgenic plant for insect resistance”, Plant Science 131(1), pp. 1-11. Kaur S. (2000), “Molecular approaches towards development of novel Bacillus thuringiensis biopesticides”, World Journal of Microbiology and Biotechnology 16, pp. 781-793. Kay R., Chan A., Daly M., McPherson J. (1987), “Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes”, Science 236, pp.1299-1302. Knowles B.H., Dow J.A.T. (1993), “The crystal δ-endotoxin gene of Bacillus thuringiensis: Modes of action on the insect gut”, BioEssays 15, pp. 469-476. Komari T., Hiei Y., Saito Y., Murai N., Kumashiro T. (1996), ”Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers”, The Plant Journal 10(1), pp.165-174. Koziel M.G., Beland G.L., Bowman C., Carozzi N.B., Crenshaw R., Crossland L., Dawson J., Desai N., Hill M., Dadwell S., Lewis K., Maddox D., McPherson K., Meghji R., Merlin E., Rhodes R., Warren G.W., Wright M., Evola S.V. (1993a), “Field performance of elite transgenic maize plant expressing an insecticidal protein derived from Bacillus thuringiensis”, Bio/Technology 11, pp. 194-200. Koziel M.G., Caozi N.B., Curier T.C., Warren G.W., Evola S.V. (1993b), “The insecticidal crystal proteins of Bacillus thuringiensis: Past, present and future use”, Biotechnology and Genetic Engineering Reviews 11, pp. 171-228. Krattiger A.F. (1997), “Insect resistance in crops: A case study of Bacillus thuringiensis and its transfer to developing countries”, ISAAA Briefs 2, pp. 1-42. Laemmli U.K. (1970), “Cleavage of structural proteins during the assembly of the head of bacteriophage T4”, Nature 227, pp. 680-685. Laporte M.M., Galagan J.A., Prasch A.L., Vanderveer P.J., Hanson D.T., Shewmaker C.K., Sharkey T.D. (2001), “Promoter strength and tissue specificity effects on growth of tomato plants transformed with maize sucrose-phosphate synthase”, Planta 212(5-6), pp. 817-822. Larkin J.C., Oppenheimer D.G., Pollock S., Marks M.D. (1993), “Arabidopsis Glabrous1 gene requires downstream sequences for function”, The Plant Cell 5, pp. 1739-1748. Last R.L., Rose A.B. (1999), “Methods & compositions for enhancing the expression of genes in plants”, Patent US5861277. Lazzeri P.A. (1998), “Techniques for the development of transgenic crops in crop protection”, BCPC Symposium proceedings 71: Biotechnology in crop protection: Facts and fallacies, pp. 3-9. Lewin B. (1997), Genes VI, Oxford University Press, Oxford-New York- Tokyo. Li J., Caroll J., Ellar D.J. (1991), “Crystal structure of insecticidal d-endotoxin from Bacillus thuringiensis at 2.5∑ resolution”, Nature 353, pp. 815-821. Luehrsen K.R., Walbot V., (1991), “Intron enhancement of gene expression and the splicing efficiency of introns in maize cells”, Molecular and General Genetics 225, pp, 81-93. Luehrsen K.R., Walbot V., (1994), “Addition of A-and U-rich sequence increases the splicing efficiency of a deleted form of a maize intron”, Plant Molecular Biology 24, pp. 449-463. Ma H., Yanofsky M.F., Klee H.J., Bowman J.L., Meyerowitz E.M. (1992), “Vectors for plant transformation and cosmid libraries”, Gene 117, pp. 161-167. Malone L.A., Giacon H.A., Burgess E.P.J., Maxwell J.Z., Christeller J.T., Laing W.A. (1995), “Toxicity of trypsin endopeptidase inhibitors to honey bees (Hymenoptera: Apodae)”, Journal of Economic Entomology 88, pp. 46-50. Mascarenhas J.P., Hamilton D.A. (1992), “Artifacts in the localization of GUS activitiy in anthers of petunia transformed with CaMV 35S- GUS construct”, The Plant Journal 2(3), pp 405-408. McBride K.E., Summerfelt K.R. (1990), “Improved binary vectors for Agrobacterium - mediated plant transformation”, Plant Molecular Biology 14, pp. 269-276. McCormac A.C., Elliott M.C., Chen D.F. (1998), “A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation”, Molecular Biotechnology 9 (2), pp. 155-159. McCormac A.C., Elliot M.C., Chen D.F. (1999), “pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities”, Molecular and General Genetics 261, pp. 226-235. McElroy D., Zhang W., Cao J., Wu R. (1990), “Isolation of an efficient actin promoter for use in rice transformation”, The Plant Cell 2, pp. 163-171. McLean B.G., Greene E.A., Zambryski P.C. (1994), “Mutants of Agrobacterium VirA that active vir gene expression in the absence of the inducer acetosyringone”, The Journal of Biological Chemistry 269 (4), pp. 2645-2651. Merritt C.R. (1998), “The commercialization of transgenic crops-the Bt experience”, BCPC Symposium Proceedings 71: Biotechnology in crop protection: Facts and fallacies, pp. 79-86. Michaud D. (1997), “Avoiding protease-mediated resistance in herbivorous pests”, Trends in Biotechnology 15, pp. 4-6. Mooney P.A., Goodwin P.B., Dennis E.S., Liewellyn D.J. (1991), “Agrobacterium tumefaciens-gene transfer into wheat tissues”, The Journal of Plant Cell Tissue and Organ Culture 25, pp.209-218. Moreno J., Altabella T., Chrispeels M.J. (1990), “Characterization of α-amylase- inhibitor, a lectin-like protein in the seeds of Phaseolus vulgaris”, Plant Physiology 92, pp. 703-709. Muramatsu M., Fukazawa C. (1993), “A high-order structure of plant storage proprotein allows its second conversion by an asparagine-specific cysteine protease, a novel proteolytic enzyme”, European Journal of Biochemistry 215, pp. 123-132. Murray M.G., Thompson W.F. (1980), “Rapid isolation of high molecular weight plant DNA”, Nucleic Acids Research 8, pp. 4321-4325. Nayak P., Basu D., Das S., Basu A., Ghosh D., Ramakrishnan N.A., Ghosh M., Sen S. K. (1997), “Transgenic elite indica rice plants expressing CryIAc d-endotoxin of Bacillus thurigiensis are resistant against yellow stem borer (Scirpophaga incertular)”, Agricultural Sciences 94, pp. 2111-2116. Negrotto D., Jolley M., Beer S., Wench A.R., Hansen G. (2000), “The use of phosphomannozase-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation”, Plant Cell Reports 19, pp. 798-803. Odell J.T., Knowlton S., Lin W., Mauvais C.J. (1988), “Properties of an isolated transcription stimulating sequence derived from the cauliflower mosaic virus 35S promoter”, Plant Molecular Biology 10, pp. 263-272. Oerke E.C., Dehne H.W., Schonbeck F., Weber A. (1994), Crop production and crop protection: Estimated losses in major food and cash crops, Elsevier, Amsterdam. Olhoft P.M., Somers D.A. (2001), “L-cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells”, Plant Cell Reports 20, pp. 706-711. Panbangred W., Panjaisee S., Tantimavanich S. (2000), “Expression of the mosquitocidal cryIVB gene under the control of different promoters in Bacillus sphaericus 2362 and acrystalliferous Bacillus thuringiensis subsp. israelensis c4Q2-72”, World Journal of Microbiology and Biotechnology 16, pp. 163-169. Perlak F.J., Deaton R.W., Amstrong T.A., Fuchs R.L., Sims S.R., Greenplate J.T., Fischhoff D.A. (1990), “Insect resistant cotton plants”, Bio/Technology 8, pp. 939-943. Perlak F.J., Fuchs R.L., Dean D.A., McPherson S.L., Fischhoff D.A. (1991), “Modification of the coding sequence enhances plant expression of insect control protein genes”, Proceedings of the National Academy of Sciences of the USA 88, pp. 3324-3328. Plesse B., Criqui M-C., Durr A., Parmentier Y., Fleck J., Genschik P. (2001), “Effects of the polyubiquitin gene Ubi.U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum”, Plant Molecular Biology 45, pp. 655-667. Purcell J.P., Greenplate J.T., Jennings M.G., Ryers J.S., Pershing J.C., Sims S.R., Prinsen M.J., Corbin D.R., Tran M., Sammons R.D., Stonard R.J. (1993), “Cholesterol oxidase: A potent insecticidal protein active against bollweevil larvae, Biochemical and Biophysical Research Communications 196, pp. 1406-1413”. Rajamohan F., Cotrill J.A., Gould F., Dean D.H. (1996), “Role of domain II, loop 2 residues of Bt CryIAb δ-endotoxin in reversible and irreversible binding to Manduca sexta and Heliothis virescens”, Journal of Biological Chemistry 271, pp. 2390-2397. Rajamohan F., Dean D.H. (1996), “Chapter 7. Molecular biology of bacteria on biologicial control of insects”, In: Molecular biology of the biological control of pests and diseases of plants, Gunasekaran M., Weber D.J. (Eds.),CRC Press, Boca Raton, Florida, pp. 105-122. Rao K.V., Rathore K.S., Hodges T.K., Fu X., Stoger E., Sudhakar D., Williams S., Christou P., Bharathi M., Bown D.P., Powell K.S., Spence J., Gatehouse A.M., Gatehouse J.A. (1998), “Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper”, Plant Jounal 15(4), pp. 469-477. Rasul N.M., Mohammad K., Islam M.R., Seraj Z.I. (1997), “Tranformation of an indica rice cultivar Binnatoa with Agrobacterium tumefaciens”, Plant Tissue Culture 7(2), pp. 71-80. Reeck G.R., Kramer K.J., Baker J.E., Kanost M.R., Fabrick J.A., Behnke C.A. (1997), “Proteinase inhibitors and resistance of transgenic plants to insect. In: Advance in insect control: The role of transgenic plant, Carozzi N., Koziel M. (Eds.), Taylor and Francis, London, pp. 157-183. Riazuddin S., Husnain T., Khan E., Karim S., Khanum M., Altosaar I. (1996), “Transformation of indica rice with Bt pesticidal genes”, In: Rice Gentics III, pp. 730-734. Robert L. (1998), “Flower-specific promoters”, PBI Bulletin, Rose A.B., Beliakoff J.A. (2000), “Intron-mediated enhancement of gene expression independent of unique intron sequence and splicing”, Plant Physiology 122, pp. 535-542. Roush R.T. (1994), “Managing pests and their resistance to Bacillus thuringiensis: Can transgenic crops be better than sprays?” Biocontrol Science and Technology 4, pp. 501-516. Sambrook J., Fritsch E.F., Maniatis T. (1989), Molecular cloning I, III: A laboratory manual, Cold Spring Harbor Laboratory Press, 1989, I (1.21-1.67), III (Appendix A, B). Sanchis V.D., Lereclus G., Menou G., Chaufaux J., Lecadeet M.-M. (1988), “Multiplicity of delta-endotoxin genes with different insecticidal specificities in Bacillus thuringiensis aizawai 7.29”, Molecular Microbiology 2, pp. 393-404. Sanger F., Nicklen S., Coulson A.R. (1977), “DNA sequencing with chain termination inhibitors”, Proceedings of the National Academy of Sciences of the USA 74, pp. 5463-5467. Sardana R., Altosaar I. (1996), “Identification and use of specific promoters and synthetic Bacillus thuringiensis toxin genes in rice biotechnology”, In: Rice Genetics III, pp. 723-729. Sardana R., Dukiandjiev S., Giband M., Cheng X., Cowan K., Sauder C., Altosaar I. (1996), “Construction and rapid testing of synthetic and modified toxin gene sequences CryIA (b&c) by expression in maize endosperm culture”, Plant Cell Reports 15, pp. 677-681. Sarma K.S., Sunilkumar G., Balamani V., Veluthambi K. (1995), “GUS activity and generation of transformed shoot buds are highly correlated in Agrobacterium- transformed tobacco”, Plant Molecular Biology Reporter 13(3), pp. 377-382. Schledzewski K., Mendel R. (1994), “Quantitative transient gene expression: Comparison of the promoters for maize polyubiquitin 1, rice actin 1, maize derived Emu and CaMV 35S in cells of barley, maize and tobacco”, Transgenic Research 3, pp. 249-255. Schnepf H.E., Whitely H.R (1981), “Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli”, Proceedings of the National Academy of Sciences of the USA 78, pp. 2893-2897. Shewry P.S., Lucas J.A. (1997), “Plant proteins that confer resistance to pests and pathogens”, Advances in Botanical Research 26, pp. 1351-1392. Shimamoto K. (1991), “Transgenic rice plants”, In: Molecular approachs to crop improvement, Dennis E.S., Llewellyn D.J. (Eds.), Springer- Verlag, Wien, NewYork, pp. 1-15. Smith R.H., Hood E.E.(1995), “Agrobacterium tumefaciens transformation of monocotyledons”, Crop Science 35(2), pp. 301-309. Stewart C.N., Adang M.J., All J.N., Boerma H.R., Cardineau G., Tucker D., Parrott W.A. (1996), “Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis cryIA(c) gene”, Plant Physiology 112, pp. 121-129. Stewart S.D., Adamczyk J.J., Knighten K.S., Davis F.M. (2001), “Impact of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringensis Berliner on growth and survival of noctuid (Lepidoptera) larvae”, Journal of Economic Entomology 94(3), pp. 752- 760. Studier F.W., Rosenberg A.H., Dunn J.J., Dubendorff J.W. (1990), “Use of T7 RNA polymerase to direct expression of cloned genes”, Methods in Enzymology 185, pp. 60-89. Sudhakar D., Fu X., Stoger E., Williams S., Spence J., Brown D.P., Bharathi M., Gatehouse J.A., Christou P. (1998), “Expression and immunolocalisation of the snowdrop lectin, GNA in transgenic rice plants”, Transgenic Research 7, pp. 371-378. Sutton D.W., Havstad P.K., Kemp J.D. (1992), “Synthetic cryIIIA gene from Bacillus thurigiensis improved for high expression in plants”, Transgenic Research 1, pp. 228-236. Tabashnik B.E. (1994), “Evolution of resistance to Bacillus thuringiensis”, Annual Review of Entomology 39, pp. 47-79. TempÐ J., Goldmann A. (1992), “Occurrence and biosynthesis of opine”, Molecular Biology of Plant Tumors, pp. 427-449. Udayasuriyan Y., Nakamura A., Mori H., Masaki H., Uozumi T. (1994), “Cloning of a new cryIA(a) gene from Bacillus thuringiensis strain FU-2-7 and analysis of chimaric CryIA(a) protein for toxicity”, Bioscience, Biotechnology, and Biochemistry 58(5), pp. 830-835. Ulmasov B., Folk W. (1995), “Analysis of the role of 5’ and 3’ flanking sequence elements upon in vivo expression of the plant tRNATrp genes”, The Plant Cell 7(10), pp. 1723-1734. Van Haute E., Joos H., Maes M., Warren G., Van Montagu M., Schell J. (1983), “Intergenic transfer and exchange recombination of restriction fragments cloned in pBR322: A novel strategy for the reversed genetics of Ti-plasmids of Agrobacterium tumefaciens”, The EMBO Journal 2, pp. 411-417. Velten J., Schell J. (1985), “Selection-expression plasmid vectors for use in genetic transformation of higher plants”, Nucleic Acids Research 13 (19), pp. 6981-6999. Vijayachandra K., Palanichelvam K., Veluthambi K. (1995), “Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation”, Plant Molecular Biology 29, pp. 125-133. Voisey C.R., White D.W., Wigley P.J., Chilcott C.N., McGregor P.G., Woodfield D.R. (1994), “Release of transgenic white clover plants expressing Bacillus thuringiensis genes: An ecological perspective”, Biocontrol Science and Technology 4, pp. 475-481. Walden R., Koncz C., Schell J. (1990), “The use of gene vectors in plant molecular biology”, Methods in Molecular and Cellular Biology 1(516), pp. 175-194. Walden R. (1993), “Cell culture, transformation and gene technology”, In: Plant Biochemistry and Molecular Biology, Lea P.J., Leegood R.C. (Eds.), pp. 276-295. Walden R., Wingender R. (1995), “Gene - transfer and plant - regeneration techniques”, Trends in Biotechnology 13, pp. 324-331. Walkerpeach C.R., Velten J. (1994), “Agrobacterium - mediated gene transfer to plant cells: cointegrate and binary vector systems”, Plant Molecular Biology, Manual B1, pp. 1-19. Walter F.S., Slatin S.L., Kulesza C.A., English L.H. (1993), “Ion channel activity of N-terminal fragments from CryIA(c) delta-endotoxin”, Biochemical and Biophysical Research Communications 196 (2), pp. 921-926. Wang M.B., Boulter D., Gatehouse J.A. (1992), “A complete sequence of the rice sucrose synthase-1 (RSs1) gene”, Plant Molecular Biology 19(5), pp. 881-885. Wolfersberger. (1996), “Site-directed mutations in the third domain of Bacillus thuringiensis delta-endotoxin CryIAa affect its ability to increase the permeability of Bombyx mori midgut brush border membrane vesicles”, Applied and Environmental Microbiology 62 (1), pp. 279-282. Wong E.Y., Hironaka C.M., Fischhoff D.A. (1992), “Arabidopsis thaliana small subunit leader and transit peptide enhance expression of Bacillus thuringiensis proteins in transgenic plants”, Plant Molecular Biology 20, pp. 81-93. Wu R. (1998), “Chapter 9. Discovery of transgenic plants”, In: Discoveries in plant biology, Kung S.D., Yang S.F. (Eds.), World Scientific Publishing Co., Singapore, New Yersey, London, Hong Kong, pp. 115-129. Xu D.P., Xue Q.Z., McElroy D., Mawal Y., Hilder V.A., Wu R. (1996), “Constitutive expression of a cowpea trypsin-inhibitor gene, CpTI, in transgenic rice plants confers resistance to 2 major rice insect pests”, Molecular Breeding 2, pp. 167-173. Yang N.S., Russell D. (1990), “Maize sucrose synthase-1 promoter directs phloem cell-specific expression of GUS gene in transgenic tobacco plants”, Proceedings of the National Academy of Sciences of the USA 87, pp. 4144-4148. Yin Y., Chen L., Beachy R. (1997), “Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice”, The Plant Journal 12, pp. 1179-1188. Yu W.P., Wang A.Y., Yuang R.H., Sung H.Y., Su J.C. (1992), “ Isolation and sequences of rice sucrose synthase cDNA and genomic DNA”, Plant Molecular Biology 18(1), pp. 139-142. Zambryski P., Joos H., Genetello C., Leemans J., Van Montagu M., Schell J. (1983), “Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity”, The EMBO Journal 2, pp. 2143-2150. Zhu J., Oger P.M., Schrammeijer B., Hooykaas P.J., Farrand S.K., Winans S.C. (2000), “The bases of crown gall tumorigenesis”, Journal of Bacteriology 182 (14), pp. 3885-3895. Zupan J., Zambryski P. (1995), “Transfer of T-DNA from Agrobacterium to the plant cell”, Plant Physiology 107, pp. 1041-1047. Zupan J., Muth T.R., Draper O., Zambryski P. (2000), “The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights”, The Plant Journal 23, pp. 11-28.