Luận án Nghiên cứu biểu hiện protein tái tổ hợp miraculin trong dòng tế bào BY2, rễ tơ thuốc lá và cây cà chua chuyển gen

Về lý thuyết, hàm lƣợng protein tái tổ hợp có thể tăng khi số bản copy trong cây chủ tăng. Tuy nhiên, tăng số bản copy của gen chuyển bằng cách biến nạp cùng lúc nhiều bản copy không phải luôn đem lại kết quả tốt cho sản lƣợng protein tái tổ hợp bởi vì các thể biến nạp nhiều bản copy thƣờng có mức độ biểu hiện của gen thấp hoặc “gen câm” (Jorgensen et al., 1996). Trong nghiên cứu này, gen miraculin đã đƣợc thay đổi mã di truyền đã đƣợc chuyển vào cây cà chua với tỷ lệ cao đạt lần lƣợt 42,11% và 52,38% tƣơng ứng với các dòng cà chua đƣợc chuyển cấu trúc E8-pro/Mir/HSP-ter và 35S-pro/Mir/NOSter. Bằng phân tích PCR và Southern blot, bƣớc đầu đã xác định đƣợc 27 dòng cây cà chua chuyển gen trong đó 16 dòng đƣợc chuyển cấu trúc E8-pro/Mir/HSP-ter và 11 dòng đƣợc chuyển cấu trúc 35S-pro/Mir/NOS-ter; 5 dòng mang 1 bản copy (T.E8.4, T.E8.7, T.E8.8, T.35S.2, T.35S8). Đối với 11 dòng mang cấu trúc 35Spro/Mir/NOS-ter, mức độ biểu hiện mRNA của gen miraculin cũng đƣợc xác nhận bằng kỹ thuật RT-PCR. Năm dòng cà chua mang bản đơn copy của gen chuyển đƣợc trồng và chăm sóc trong điều kiện phòng thí nghiệm, các dòng này sinh trƣởng bình thƣờng tƣơng tự cây cà chua PT18 không chuyển gen. Sau 80-90 ngày cây ra hoa, hàm lƣợng miraculin tái tổ hợp trong vỏ ngoài quả cao nhất ở T.E8.8 (đạt 118,0 ng/µg protein tan tổng số) và gấp khoảng hơn 4 lần so với hàm lƣợng miraculin tái tổ hợp ở dòng T35S.2 và T35S.8.

pdf147 trang | Chia sẻ: tueminh09 | Ngày: 24/01/2022 | Lượt xem: 83 | Lượt tải: 0download
Bạn đang xem trước 20 trang tài liệu Luận án Nghiên cứu biểu hiện protein tái tổ hợp miraculin trong dòng tế bào BY2, rễ tơ thuốc lá và cây cà chua chuyển gen, để xem tài liệu hoàn chỉnh bạn click vào nút DOWNLOAD ở trên
ell and hairy root was observed in transformants with HSP-pro/Mir/HSP-ter cassette. Modified miraculin gene was has been also transferred into tomato plants with transformation high rate reached from 42.11% to 52.38 %. Trasgenic tomato lines were comfirmed by PCR and Southern blot analysis for 27 lines transgenic tomato plants in which 16 lines are transferred E8-pro/Mir/HSP-ter and 11 35S- pro/Mir/NOS-ter casstte, there are 5 transgenic tomato lines carrying single copy (TE8.2, TE8.7, TE8.8, T35S.2, T35S8). For 11 transgenic tomato lines carrying 35S-pro/Mir/NOS-ter cassette, expression genes miraculin in mRNA levels were also confirmed by RT-PCR analysis. Recombinant miraculin content in the red fruit exocarp of E8-pro/Mir/HSP-ter (T.E8.8) reached 118,0 ng/µg in total solute protein and was approximately 4,0 times higher than that in the 35S-pro/Mir/NOS-ter transgenic line. Furthermore, these transgenic tomato are expressed system to produce recombinant miraculin in the agriculture. Recombinant miraculin in transgenic tomato is the same taste-modifying as the natural miracluin in miracle fruit. From these results, all transgenic lines are potential for production of recombinant miraculin protein in the future. 112 TÀI LIỆU THAM KHẢO 1. Almeida D, Huber D (1999) Apoplastic pH and inorganic ion levels in tomato fruit: a potential means for regulation of cell wall metabolism during ripening. Physiol Plant 105(3):506-512. 2. Albenne C, Canut H, Jamet E (2013) Plant cell wall proteomics: the leadership of Arabidopsis thaliana. Front. Plant Sci 4:111. doi: 10.3389/fpls.2013.00111 3. Aluru M, Xu Y, Guo R, Wang Z, Li S, White W, et al (2008) Generation of transgenic maize with enhanced provitamin A content. J Exp Bot 59:3551- 3562. 4. Alvarez M, Pinyerd H, Crisantes J, Rigano M, Pinkhasov J, Walmsley A, Mason H, Cardineau G (2006) Plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice. Vaccine 24(14):2477- 2490. 5. Amin J, Anathan J, Voellmy R (1988) Key features of heat shock regulatory elements. Mol Cell Biol 8:3761-3769. 6. Aviezer D, Almon-Brill E, Shaaltiel Y, Galili G, Chertkoff R, Hashmueli S, Galun E, Zimran A (2008) Novel enzyme replacement therapy for Gaucher disease: On-going phase III clinical trial with recombinant human glucocerebrosidase expressed in plant cells. 7. Azlan G, Marziah M, Radzali M, Johari R (2002) Establishment of Physalis minimahairy roots culture for the production of physalins. Plant Cell Tiss Org Cult 69:271-278. 8. Berberich T, Takagi T, Miyazaki A, Otani M, Shimada T, Kusano T (2005) Production of mouse adiponectin, an anti-diabetic protein, in transgenic sweet potato plants. J Plant Physiol 162:1169-1176. 9. Chen G, Inouye M (1990) Suppression of the negative effect of minor arginine codons on gene expression; preferential usage of minor codons within thefirst 25 codons of the Escherichia coli genes. Nucl Acid Res 181465–1473. 113 10. Chen T, Lin Y, Lee Y, Yang N, Chan M (2004) Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures. Transgenic Res 13:499-510. 11. Chilton M, Tepfer D, Petit A, David C, Casse-Delbart F, Tempe J (1982) Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295:432-434. 12. Christey M, Sinclair B (1992) Regeneration of transgenic kale (Brassica oleracea var. acephala), rape (B. napus) and turnip (B. campestris var.rapifera) plants via Agrobacterium rhizogenes mediated transformation. Plant Sci 87:161-169. 13. Chung H, Park J-H, Lee H, Kim K, Sunter G, Kim J, Kim W, Chung I (2014) Expression of a functional recombinant chimeric protein of human hepatis A virus VP1 and an Fc antibody fagment in transgenic tomato plants. Plant Biotechnol Rep 8:243-249. 14. Comai L, Moran P, Maslyar D (1990) Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements. Plant Mol Biol 15(3):373-381. 15. Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, et al (2001) Comparative analysis of transgenic rice plants obtained by Agrobactrium- mediated transformation and particle bombardment. Mol Breed 7(1):25-33. 16. Daniell H, Chebolu S, Kumar S, Singleton M, Falconer R (2005) Chloroplast derived vaccine antigens and other therapeutic proteins. Vaccine 23(15):1779- 1783. 17. Daniell H, Ruiz G, Denes B, Sandberg L, Langridge W (2009) Optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function. BMC Biotechnol 933. 114 18. Daniell H, Streatfield S, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6(5):219-226. 19. Davidovich-Rikanati R, Sitrit Y, Tadmor Y, Iijima Y, Bilenko N, Bar E, Carmona B, Fallik E, Dudai N, Simon J, Pichersky E, Lewinsohn E (2007) Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway. Nat Biotechnol 25:899-901. 20. Deikman J, Xu R, Kneissl M, Ciardi J, Kim K, Pelah D (1998) Separation of cis elements responsive to ethylene, fruit development, and ripening in the 5'- flanking region of the ripening-related E8 gene. Plant Mol Biol 371001–1011. 21. DeMuynck B, Navarre C, Nizet Y, Stadlmann J, Boutry M (2009) Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells. Transgenic Res 18:467-482. doi:10.1007/s11248-008-9240-1. 22. Depicker A, Van Montagu M (1997) Post-transcriptional gene silencing in plants. Curr Opin Cell Biol 9(3):373-382. 23. Desai PN SN, Padh H (2010) Production of heterologous proteins in plants: strategies for optimal expression. Biotechnol Adv 28:427-435. 24. Đỗ Xuân Đồng, Chu Hoàng Hà, Lê Trần Bình (2007) Nghiên cứu quy trình tái sinh và hệ thống chuyển gen cho một số giống cà chua (Lycopersicon esculentum L.) của Việt Nam. Tạp chí Công nghệ Sinh học 5(5):217-223. 25. Drake PMW, Barbi T, Sexton A, McGowan E, Stadlmann J, Navarre C, et al (2009) Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco. FASEB J .23:3581-3589. 26. Drake P, Chargelegue D, Vine N, van Dolleweerd C, Obregon P, Ma J (2003) Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots. Plant Molecular Biology 52(1):233-241. 115 27. Duhita N, Hiwasa-Tanase K, Yoshida S, Ezura H (2009) Single-step purification of native miraculin using immobilized metal affinity chromatography. J Agric Food Chem 57:5148-5151. 28. Eibl R, Eibl D (2008) Design of bioreactors suitable for plant cell and tissue cultures. Phytochemistry Reviews 7593-598. 29. Firsov A, Pushin A, Korneeva I, Dolgov S (2012) Transgenic Tomato Plants as Supersweet Protein Thaumatin II Producers. Applied Biochemistry and Microbiology 48(9):746-751. 30. Fischer R, Schumann D, Zimmermann S, Drossard J, Sack M, Schillberg S (1999) Expression and characterization of bispecific single-chain Fv fragments produced in transgenic plants. Eur. J. Biochem 262:810-816. 31. Gallie D (1993) Posttranscriptional regulation of gene expression in plants. Annu Rev Plant Physiol Plant Mol Biol 44:77-105. 32. Gaume A, Komarnytsky S, Borisjuk N, Raskin I ( 2003 ) Rhizosecretion of recombinant proteins from plant hairy roots. Plant Cell Rep 211188-1193. 33. Goulet C, Khalf M, Sainsbury F, D‟Aoust M-A, Michaud D (2012) A protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast. Plant Biotechnol. J 10:83-94. doi: 10.1111/j.1467- 7652.2011.00643.x 34. Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18(11):1151-1155. 35. Golovkin M, Spitsin S, Andrianov V, Smirnov Y, Xiao Y, Pogrebnyak N, et al (2007) Smallpox subunit vaccine produced in planta confers protection in mice. Proc Natl Acad Sci USA 104:6864-6869. 36. Guerineau F, Lucy A, Mullineaux P (1992) Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts. Plant Mol Biol 18:815-818. 37. Gurr S, Rushton P (2005) Engineering plants with increased disease resistance: how are we going to express it? Trends Biotechnol 23:283-290. 116 38. Gustafssion C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22(7):346-353. 39. Gutierrez-Ortega A, Sandoval-Montes C, de Olivera-Flores T, Santos- Argumedo L, Gomez-Lim M (2005) Expression of functional interleukin-12 from mouse in transgenic tomato plants. Transgenic Res 14:877-885. 40. Gutierrez R, Macintosh G, Green P (1999) Current perspectives on mRNA stability in plants: multiple levels and mechanisms of control. Trends Plant Sci 4(11):429-438. 41. Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp 41:95-98. 42. Häkkinen ST, Raven N, Henquet M, Laukkanen M-L, Anderlei T, Pitkänen J- P, et al (2014) Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody. Biotechnol.Bioeng 111:336-346. doi: 10.1002/bit.25113 43. He D, Qian K, Shen G, Li Y, Zhang Z, Su Z, et al (2007) Stable expression of foot-and-mouth disease virus protein VP1 fused with cholera toxin B subunit in the potato (Solanum tuberosum). Colloids Surf B Biointerfaces 55159–163. 44. Hernandez-Garcia C, Martinelli A, Bouchard R, Finer J (2009) A soybean (Glycine max) polyubiquitin promoter gives strong constitutive expression in transgenic soybean. Plant Cell Rep 28:837-849. 45. Hirai T, Fukukawa G, Kakuta H, Fukuda N, Ezura H (2010a) Production of recombinant miraculin using transgenic tomato in a closed-cultivation system. J Agric Food Chem 58:6096-6101. 46. Hirai T, Kim Y, Kato K, Hiwasa-Tanase K, Ezura H (2011a) Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter. Transgenic Res 20(6):1285-1292. 47. Hirai T, Kurokawa N, Duhita N, Hiwasa-Tanase K, Kato K, Ezura H (2011b) The HSP terminator of Arabidopsis thaliana induces extremely high-level 117 accumulation of miraculin protein in transgenic tomato. J Agric Food Chem 59:9942-9949. 48. Hirai T, Sato M, Toyooka K, Sun H-J, Yano M, Ezura H (2010b) Miraculin, a taste-modifying protein is secreted into intercellular spaces in plant cells. J Plant Physiol 167:209-215. 49. Hiwasa-Tanase K, Nyarubona M, Hirai T, Kato K, Ichikawa T, Ezura H (2011) High-level accumulation of recombinant miraculin protein in transgenic tomatoes expressing a synthetic miraculin gene with optimized codon usage terminated by the native miraculin terminator. Plant Cell Rep 30:113-124. 50. Ho M-W, Ryan A, Cummins J (1999) Cauliflower mosaic viral promoter-A recipe for disaster? Microb Ecol Health Dis 11(194197): 51. Howard J, Hood E (2005) Bioindustrial and biopharmaceutical products produced in plants. Advances in Agronomy 8591-124. 52. 53. 54. Igeta H, Tamura Y, Nakaya K, Nakamura Y, Kurihara Y (1991) Determination of disulfide array and subunit structure of taste- modifying protein, miraculin. Biochim Biophys Acta 1079:303-307. 55. Ingelbrecht I, Herman L, Dekeyser R, Van Montagu M, Depicker A (1989) Different 3‟ end regions strongly influence the level of gene expression in plant cells. Plant Cell Physiol 1:671-680. 56. Ito K, Asakura T, Morita Y, Nakajima K, Koizumi A, Shimizu-Ibuka A, Masuda K, Ishiguro M, Terada T, Maruyama J, Kitamoto K, Misaka T, Abe K (2007) Microbial production of sensory-active miraculin. Biochem Biophys Res Commun 360:407-411. 57. Ito K, Sugawara T, Koizumi A, Nakajima K, Shimizu-Ibuka A, Shiroishi M, Asada H, Yurugi-Kobayashi T, Shimamura T, Asakura T, Masuda K, Ishiguro M, Misaka T, Iwata S, Kobayashi T, Abe K (2010) Bulky high-mannose-type 118 N-glycan blocks the taste-modifying activity of miraculin. Biochim Biophys Acta 1800:986-992. 58. Izawa H, Ota M, Kohmura M, Ariyoshi Y (1996) Synthesis and character- ization of the sweet protein brazzein. Biopolymers 39(1): 95-101. 59. Jha S, Sanyal I, Amla D (2015) High-level expression and purification of a therapeutic recombinant serine protease inhibitor from transgenic tomato plants. International Journal of Advance Research In Science And Engineering 4(4):1158-1176. 60. Jiang X, He Z, Peng Z, Qi Y, Chen Q, Yu S (2007) Cholera toxinB protein in transgenic tomato fruit induces systemic immune response in mice. Transgenic Res 16:169-175. 61. Jin S, Sun H-J, Al Bachchu M, Chung S, Lee J, Han S-I, Yun J, Boo K, Lee D, Riu K, Kim J-H (2013) Production of recombinant miraculin protein using transgenic citrus cell suspension culture system. Journal of the Korean Society for Applied Biological Chemistry 56(3):271-274. 62. Jones H, Doherty A, Wu H (2005) Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 15. 63. Jorgensen R, Cluste P, English J, Que Q, Napoli C (1996) Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single copy vs. complex T-DNA sequences. Plant Mol Biol 31:957-973. 64. Kaneko R, Kitabatake N (1999) J Agric Food Chem. Heat-induced formation of intermolecular disulfide linkages between thaumatin molecules that do not contain cysteine residues 47(12):4950-4955. 65. Kang T, Kim B, Yang J, Yang M (2006) Expression of a synthetic cholera toxin B subunit in tobacco using ubiquitin promoter and bar gene as a selectable marker. Mol Biotechnol 32:93-100. 119 66. Kang T, Loc N, Jang M, Yang M (2004c) Modification of the cholera toxin B subunit coding sequence to enhance expression in plants. Mol Breeding 13:143-153. 67. Kant R (2005) Sweet proteins-potential replacement for artificial low calorie sweeteners. Nutr J 45. 68. Kantor M, Sestras R, K C (2013) Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum. Pesq.aropec.bras (Brasília) 48(1):73-79. 69. Karnoup A, Turkelson V, Anderson W (2005) O-linked glycosylation in maize-expressed human IgA1. Glycobiology 15:965-981. 70. Kato K, Maruyama S, Hirai T, Hiwasa-Tanase K, Mizoguchi T, Goto E, Ezura H (2011) A trial of production of the plant-derived high-value recombinant protein in a plant factory: photosynthetic photon fluxes affect the accumulation of recombinant miraculin in transgenic tomato fruits. Plant Signal Behav 8:1172-1179. 71. Kato K, Yoshida R, Kikuzaki A, Hirai T, Kuroda H, Hiwasa-Tanase K, Takane K, Ezura H, Mizoguchi T (2010) Molecular breeding of tomato lines for mass production of miraculin in a plant factory. J Agric Food Chem 58:9505-9510. 72. Kim T, Baek M, Lee E, Kwon T, Yang M (2008) Expression of human growth hormone in transgenic rice cell suspension culture. Plant Cell Rep 27:885-891. 73. Kim Y, Hirai T, Kato K, Hiwasa-Tanase K, Ezura H (2010a) Gene dosage and genetic background affect miraculin accumulation in transgenic tomato fruits. Plant Biotechnol 27:333-338. 74. Kim Y, Kato K, Hirai T, Hiwasa-Tanase K, Ezura H (2010b) Spatial and developmental profiling of miraculin accumulation in transgenic tomato fruits expressing the miraculin gene constitutively. J Agric Food Chem 58:282-286. 120 75. Kittipongpatana N, Hock R, Porter J (1998) Production of solasodine by hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. Plant Cell Tiss Org Cult 52:133-143. 76. Kohmura M, Nio N, Ariyoshi Y (1992) Solid-phase synthesis of [AsnA16]-, [AsnA22]-, [GlnA25]-, and [AsnA26] monellin, analogues of the sweet protein monellin. Biosci Biotechnol Biochem 56(3):472-476. 77. Koizumi A, Tsuchiya A, Nakajima K, Ito K, Terada T, Shimizu-Ibuka A, Briand L, Asakura T, Misaka T, Abe K (2011) Human sweet taste receptor mediates acid-induced sweetness of miraculin. Proc Natl Acad Sci USA 108(40):16819-16824. 78. Krolicka A, Staniszewska I, Bielawski K, Malinski E, Szafranek J, Lojkowska E (2001) Establishment of hairy root cultures of Ammi majus. Plant Sci 160:259-264. 79. Kurihara K, Beidler L (1968) Taste-modifying protein from miracle fruit. Science 161:1241-1243. 80. Kurihara K, Beidler L (1969) Mechanism of the action of taste-modifying protein. Nature 222:1176-1178. 81. Kurihara Y (1992) Characteristics of antisweet substances, sweet proteins, and sweetness-inducing proteins. Crit. Rev. Food Sci. Nutr 32:231-252. 82. Kurihara Y, Nirasawa S (1997) Structures and activities of sweetness- inducing substances (miraculin, curculin, strogin) and the heat- stable sweet protein, mabinlin. FFI J Jpn 174:67-74. 83. Kurokawa N, Hirai T, Takayama M, Hiwasa-Tanase K, Ezura H (2013) An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin. Plant Cell Rep 32:529-536. 84. Kwon T, Kim Y, Lee J, Yang M (2003) Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor 121 in transgenic tomato suspension cultures. Biotechnology Letters 25:1571- 1574. 85. Lal P, Ramachandran V, Goyal R, Sharma R (2007) Edible vaccines: current status and future. Indian J Med Microbiol 25(2):93-102. 86. Lê Trần Bình, Phan Văn Chi, Nông Văn Hải, Trƣơng Nam Hải, Lê Quang Huấn (2003) Áp dụng các kỹ thuật phân tử trong nghiên cứu tài nguyên sinh vật Việt Nam. Nxb Khoa học Kỹ thuật, Hà Nội. 87. Lee S, Lee J, Chang H, Cho J, Jung J, Lee W (1999) Solution structure of a sweet protein single-chain monellin determined by nuclear magnetic resonance and dynamical simulated annealing calculations. Biochemistry 38(8):2340-2346. 88. Li H, Ramalingam S, Chye M (2006b) Accumulation of recombinant SARS- CoV spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines. Exp Biol Med 231:1346-1352. 89. Liu Q, Xue Q (2005) Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species. J Genet 84(1):55-62. 90. Lohmann C, Egger-Schumacher G, Wunderlich M, Schoffl F (2004) Two different heat shock transcription factors regulate immediate early expression of stress genes in Arabidopsis. Mol Gennet Genomics 271(1):11-21. 91. Lutcke H, Chow K, Mickel F, Moss K, Kern H, Scheele G (1987) Selection of AUG initiation codon differs in plants and animals. EMBO J 6(1):43-48. 92. Mandal MK, Fischer R, Schillberg S, Schiermeyer A (2014) Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacumcv. Bright yellow 2 (BY-2) suspension cells. Biotechnol. J 9:1065-1073. doi: 10.1002/biot.201300424. 93. Mason H, Warzecha H, Mor T, Arntzen C (2002) Edible plant vaccines: application for prophylactic and molecular medicine. Trends Mol Med 8(7):324-329. 122 94. Masuda T, Kitabatake N (2006) Developments in biotechnological production of sweet proteins. J Biosci Bioeng 102:375-389. 95. Masuda Y, Nirasawa S, Nakaya K, Kurihara Y (1995) Cloning and sequencing of a cDNA encoding a taste-modifying protein, miraculin. Genes Dev 161:175-177. 96. Matsui T, Takita E, Sato T, Aizawa M, Ki M, Kadoyama Y, Hirano K, Kinjo S, Asao H, Kawamoto K, Kariya H, Makino S, Hamabata T, Sawada K, Kato K (2011) Production of double repeated B subunit of Shiga toxin 2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease. Transgenic Res 20:735-748. 97. Matsuyama T, Satoh M, Nakata R, Aoyama T, Inoue H (2009) Functional expression of miraculin, a taste-modifying protein in Escherichia coli. J Biochem 145:445-450. 98. Menzel G, Harloff H, Jung C (2003) Expression of bacterial poly (3- hydroxybutyrate) synthesis genes in hairy roots of sugar beet (Beta vulgaris L.). Appl Microbiol Biotechnol 60:571-576. 99. Meyer P (1996b) Repeat-induced gene silencing: common mechanisms in plants and fungi. Biol Chem Hoppe Seyler 37787-95. 100. Mor T, Sternfeld M, Soreq H, Arntzen C (2001) Expression of Recombinant Human Acetylcholinesterase in Transgenic Tomato Plants. Biotechnology and Bioengineering 75(3):259-266. 101. Muir S, Collins G, Robinson S, Hughes S, Bovy A, De Vos C, Van Tunen A, Verhoeyen M (2001) Overexpression of petunia chalcone isomerase in tomato results in fruit containing dramatically increased levels of flavonoids. Nat Biotechnol 19(5):470-474. 102. Nagata T, Nemoto Y, Seiichiro H (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. Int. Rev. Cytol 13:21-30. 123 103. Nagaya S, Kawamura K, Shinmyo A, Kato K (2010) The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol 51:328-332. 104. Chu Văn Mẫn (2010) Tin học trong công nghệ sinh học. Nhà xuất bản Giáo dục Việt Nam. 105. Nirasawa S, Nishino T, Katahira M, Uesugi S, Hu Z, Kurihara Y (1994) Structures of heat-stable and unstable homologues of the sweet protein mabinlin. The difference in the heat stability is due to replacement of a single amino acid residue. Eur J Biochem 223(3):989-995. 106. Niemer M, Mehofer U, Torres Acosta JA,Verdianz M, Henkel T, Loos A, et al. (2014) The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation:down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant- based expression platforms. Biotechnol. J 9:493-500. doi: 10.1002/biot.201300207. 107. Nocarova E, Fischer L (2009) Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression. BMC Plant Biol 9 108. Novina C, Roy A (1996) Core promoters and transcriptional control. Trends Genet 12:351-355. 109. Ota M, Sawa A, Nio N, Ariyoshi Y (1999) Enzymatic ligation for synthe-sis of single-chain analogue ofmonellin by transglutaminase. Biopolymers 50(2):193-200. 110. Peñarrubia L, Kim R, Giovannoni J, Kim S, Fischer R (1992) Production of the sweet protein monellin in transgenic plants. Nat Biotechnol 10:561-564. 111. Perlak F, Fuchs R, Dean D, McPherson S, Fischhoff D (1991) Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA 88(8):3324-3328. 124 112. Pham Bich Ngoc: Production and Secretion of Recombinant Sweet-Tasting Thaumatin from Suspension Cells and Hairy Roots of Nicotiana tabacum. University of Heidelberg, Germany (2009). 113. Pineda B, Gimenez-Caminero E, Garcia-Sogo B, Anton M, Atarés A, Capel J, Lozano R, Angosto T, Moreno V (2010) Genetic and physiological characterization of the arlequin insertional mutant reveals a key regulator of reproductive development in tomato. Plant Cell Physiol 51(3):435-447. 114. Plasson C, Michel R, Lienard D, Saint-Jore-Dupas C, Sourrouille C, de March G, Gomord V (2009) Production of recombinant proteins in suspension- cultured plant cells. Methods Mol Biol 483:145-161. 115. Pogrebnyak N, Golovkin M, Andrianov V, Spitsin S, Smirnov Y, Egolf R KH (2005) Severe acute respiratory syndrome (SARS) S protein production in plants: development of recombinant vaccine. Proc Natl Acad Scie USA 102(25):9062-9067. 116. Qiu D, Diretto G, Tavarza R, Giuliano G (2007) Improved protocol for Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene CsZCD. Sci Hortic 112(2):172-175. 117. Reddy C, Vijayalakshmi M, Kaul T, Islam T, Reddy M (2015) Improving Flavour and Quality of Tomatoes by Expression of Synthetic Gene Encoding Sweet Protein Monellin. Mol Biotechnol 57(5):448-453. 118. Reuter L, Bailey M, Joensuu J, Ritala A (2014) Scale-up of hydrophobin- assisted recombinant protein production in tobacco BY-2 suspension cells. Plant Biotechnology Journal 12(4):402-410. 119. Römer S, Fraser P, Kiano J, Shipton C, Misawa N, Schuch W, Bramley P (2000) Elevation of the provitamin a content of transgenic tomato plants. Nat Biotechnol 18(6):666-669. 125 120. Rouwendal G, Mendes O, Wolbert E, Boer A (1997) Enhanced expression in tobacco of the gene encoding green fluorescent protein by modification of its codon usage. Plant Mol Biol 33:989-999. 121. Ruhlman T, Ahangari R, Devine A, Samsam M, Daniell H (2007) Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts-oral administration protects against development of insulitis in non-obese diabetic mice. Plant Biotechnol J 5:495-510. 122. Saidi Y, Schaefer D, Goloubinoff P, Zrÿd J-P, Finka A (2009) The CaMV 35S promoter has a weak expression activity in dark grown tissues of moss Physcomitrella patens. Plant Signaling & Behavior 4(5):457-459. 123. Seo Y, Kim S, Harn C, Kim W (2011) Ectopic expression of apple fruit homogentisate phytyltransferase gene (MdHPT1) increases tocopherol in transgenic tomato (Solanum lycopersicumcv. Micro-Tom) leaves and fruits. Phytochemistry 72:321-329. 124. Sharma A, Sharma M (2009) Plants as bioreactors: Recent developments and emerging opportunities. Biotechnology Advances 27:811-832. 125. Sharp J, Doran P (2001) Strategies for enhancing mono-clonal antibody accumulation in plant cell and organ cultures. Biotechnol Prog 17:979-992. 126. Shin Y, Hong S, Kwon T, Jang Y, Yang M (2003) High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnol Bioeng 82:778-783. 127. Sivankalyani V, Takumi S, Thangasamy S, Ashakiran K, Girija S (2014) Punctured-hypocotyl method for high-efficient transformation and adventitious shoot regeneration of tomato. Scientia Horticulturae 165:357- 364. 128. Smith M, Mason H, Shuler M (2002) Hepatitis Bsurface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form. Biotechnology and Bioengineering 80:812- 822. 126 129. Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, et al (2000) Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42:583-590. 130. Streatfield S (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5(1):2-15. 131. Sugaya T, Yano M, Sun H-J, Hirai T, Ezura H (2008) Transgenic strawberry expressing a taste-modifying protein, miraculin. Plant Biotechnol J 25:329- 333. 132. Sun H-J, Cui M, Ma B, Ezura H (2006a) Functional expression of the taste- modifying protein, miraculin, in transgenic lettuce. FEBS Lett 580620-626. 133. Sun H-J, Kataoka H, Yano M, Ezura H (2007) Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants. Plant Biotech J 5:768-777. 134. Sun H-J, Uchii S, Watanabe S, Ezura H (2006b) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol 47:426-431. 135. Sun Q, Ding L, Lomonossoff G, Sun Y, Luo M, Li C, Jiang L, Xu Z (2011) Improved expression and purification of recombinant human serum albumin from transgenic tobacco suspension culture. J Biotechnol 155(2):164-172. 136. Theerasilp S, Hitotsuya H, Nakajo S, Nakaya K, Nakamura Y, Kurihara Y (1989) Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin. J Biol Chem 264:6655-6659. 137. Theerasilp S, Kurihara Y (1988) Complete purification and charac- terization of the taste-modifying protein, miraculin, from miracle fruit. J Biol Chem 263:11536-11539. 138. Torregrosa L, Bouquet A (1997) Agrobacterium rhizogenes and A. tumefaciens co-transformation to obtain grapevine hairy roots producing the coat protein of grapevine chrome mosaic nepovirus. Plant Cell Tiss Org Cult 49:53-62. 127 139. Trần Danh Thế, Vũ Văn Độ, Ngô Kế Sƣơng (2009) Bƣớc đầu trồng thử nghiệm và tách chiết hoạt chất miraculin trong trái cây thần kỳ (Synsepalum Dulcificum Daniell). Tạp chí Phát triển Khoa học và Công nghệ 13(1):54-61. 140. Twyman R, Schillberg S, Fischer R (2013) Optimizing the yield of recombinant pharmaceutical proteins in plants. Curr. Pharm. Des 19: 5486- 5494. doi: 10.2174/1381612811319310004. 141. van der Wel H, Larson G, Hladik A, Hladik C, Hellekant G, Glaser D, Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, et al (1989) Isolation and characterisation of pentadin, the sweet principle of Pentadiplandra brazzeana Baillon. Chem Senses 14:75-79. 142. Wakasa Y, Ozawa K, Takaiwa F (2009) Higher-level accumulation of foreign gene products in transgenic rice seeds by the callus-specific selection system. J Biosci Bioeng 107:78-83. 143. Wang J, Oard J (2003) Rice ubiquitin promoters: deletion analysis and potential usefulness in plant transformation systems. Plant Cell Rep 22:129- 134. 144. Wolin S, Walter P (1988) Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J 7:3559–3569. 145. Wongsamuth R, Doran P (1997a) Hairy roots as an expression system for production of antibodies. In: In: Hairy Roots. Culture and Applications, 89- 97, Harwood, Australia. 146. Xin Z, Chen J (2012) A high throughput DNA extraction method with high yield and quality. Plant Methods 826. 147. Yano A, Maeda F, Takekoshi M (2004) Transgenic tobacco cells producing the human monoclonal antibody to hepatitis B virus surface antigen. J. Med. Virol 73:208-215. 148. Yano M, Hirai T, Kato K, Hiwasa-Tanase K, Fukuda N, Ezura H (2010) Tomato is a suitable material for producing recombinant miraculin protein in genetically stable manner. Plant Sci 178:469-473. 128 149. Yasmeen A, Mirza B, Inayatullah S, Safdar N, Jamil M, Ali S, Choudhry M (2009) In planta transformation of tomato. Plant Mol Biol Rep 27(1):20-28. 150. Zemanek E, Wasserman B (1995) Issues and advances in the use of transgenic organisms for the production of thaumatin, the intensely sweet protein fromThaumatococcus danielli. . Crit Rev Food Sci Nutr 35(5):455-466. 151. Zhao G, Zhang Y, Hoon M, Chandrashekar J, Erlenbach I, Ryba N, Zuker C (2003) The receptors for mammalian sweet and umami taste. Cell 115(3):255- 266. 152. Zuo J, Chua N (2000) Chemical-inducible systems for regulated expression of plant genes. Curr Opin Biotechnol 11:146-151. PHỤ LỤC 1. So sánh trình tự nucleotide của promoter E8 thu đƣợc với trình tự AF515784.1 và DQ317599.1 10 20 30 40 50 60 70 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ---------- AAGCTTTCCC TAATGATATT GTTCATGTAA TTAAGTTTTG TGGAAGTGAG AGAGTCCAAT AF515784.1 ---------- ------.... .......... .......... .......... .......... .......... DQ317599.1 GAATTCATTT TT.ACA.... .......... .....C.... .......... .......... .......... 80 90 100 110 120 130 140 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTTGATAAGA AAAGAGTCAG AAAACGTAAT ATTTTAAAAG TCTAAATCTT TCTACAAATA AGAGCAAATT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 150 160 170 180 190 200 210 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TATTTATTTT TTAATCCAAT AAATATTAAT GGAGGACAAA TTCAATTCAC TT-GGTTGTA AAATAAACTT AF515784.1 .......... .......... .......... .......... .......... ..T....... .......... DQ317599.1 .......... .......... .......... .......... .......... ..-....... .......... 220 230 240 250 260 270 280 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter AAACCAATAA CCAAAGAACT AATAAATCCT GAAGTGGAAT TATTAAGGAT AAATGTACAT AGACAATGAA AF515784.1 .......... .......... .......... .......... .......... .......... ....G..... DQ317599.1 .......... .......N.. ........-. .......... .......... ..-....... .......... 290 300 310 320 330 340 350 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter GAAATAATAG GTTCGATGAA TTAATAATAA TTAAGGATGT TACAATCATC ATGTGCCAAG TATATACACA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 360 370 380 390 400 410 420 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ATATTCTATG GGATTTATAA TTTCGTTACT TCACTTAACT TTTGCGTAAA TAAAACGAAT TATCTGATAT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 430 440 450 460 470 480 490 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTTATAATAA AACAGTTAAT TAAGAACCAT CATTTTTAAC AACATAGATA TATTATTTCT AATAGTTTAA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 500 510 520 530 540 550 560 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TGATACTTTT AAATCTTTTA AATTTTATGT TTCTTTTAGA AAATAAAAAT TCAAAAAA-T TAAATATATT AF515784.1 .......... .......... .......... .......... .......... ........-. .......... DQ317599.1 .......... .......... .......... .......... .......... ........A. .......... 570 580 590 600 610 620 630 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TACAAAAACT ACAATCAAAC ACAACTTCAT ATATTAAAAG CAAAATATAT TTTGAAAATT TCAAGTGTCC AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 640 650 660 670 680 690 700 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TAACAAATAA GACAAGAGGA AAATGTACGA TGAGAGACAT AAAGAGAACT AATAATTGAG GAGTCCTATA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 710 720 730 740 750 760 770 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ATATATAATA AAGTTTATTA GTAAACTTAA TTATTAAGGA CTCCTAAAAT ATATGATAGG AGAAAATGAA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 780 790 800 810 820 830 840 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TGGTGAGAGA TATTGGAAAA CTTAATAATT AAGGATTTTA AAATATATGG TAAAAGATAG GCAAAGTATC AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... ......N... .......... .......... .......... 850 860 870 880 890 900 910 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter CATTATCCCC TTTTAACTTG AAGTCTAC-T AGGCGCATGT GAAAG-TTGA TTTTTTGTCA CGTCATATAG AF515784.1 .......... .......... ........-. .......... .....-.... .......... .......... DQ317599.1 .......... .......... ........C. .......... .....G.... .......... .......... 920 930 940 950 960 970 980 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter CTATAACGTA AAAAAAGAAA GTAAAATTTT TAATTTTTTT TAATATATGA CATATTTTAA ACGAAATATA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 990 1000 1010 1020 1030 1040 1050 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter GGACAAAATG TAAATGAATA GTAAAGGAAA CAAAGATTAA TACTTACTTT GTAAGAATTT AAGATAAATT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1060 1070 1080 1090 1100 1110 1120 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TAAAATTTAA TAGATCAACT TTACGTCTAG AAAGACCCTA TCTTAGAAGG AATTTCA-GA AATCGGCCCT AF515784.1 .......... .......... .......... ........AT ATC....... .......C.. .......... DQ317599.1 .......... .......... .......... ........AT ATC....... .......C.. .......... 1130 1140 1150 1160 1170 1180 1190 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTTTCAAAAA TAACTTTTAA ATAATGAATT TTAAATTTTA AGAAATAATT TCCAATGAAT AAATGACATG AF515784.1 .A...G.... .......... .......... .......... .........A .......... .......... DQ317599.1 .A........ .......... .......... .......... .........A .......... .......... 1200 1210 1220 1230 1240 1250 1260 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TAGCATTTTA CCTAAATATT TCAACTATTT TAATCCAATA TTAATTTGTT T-ATTCCCAA CAATAGAAAG AF515784.1 .......... .......... .......... .......... .......... .T........ .......... DQ317599.1 .......... .......... .......... .......... .......... .T........ .......... 1270 1280 1290 1300 1310 1320 1330 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TCTTGTGCAG ACATTTAATC TGACTTTTCC AGTACTAAAT ATTAATTTTC TGAAGATTTT CGGGTTTAGT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1340 1350 1360 1370 1380 1390 1400 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter CCACAAGTTT TAGTGAGAAG TTTTGCTCAA AATTTTAGGT GAGAAGGTTT GATATTTATC TTTTGTTAAA AF515784.1 .......... .......... .......... .-........ .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1410 1420 1430 1440 1450 1460 1470 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTAATTTATC TAGGTGACTA TTATTTATTT AAGTAGAAAT TCATATCATT ACTTTTGCCA ACTTGTAGTC AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1480 1490 1500 1510 1520 1530 1540 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ATAATAGGAG TAGGTGTATA TGATGAAGGA ATAAACAAGT TCAGTGAAGT GATTAAAATA AAATATAATT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1550 1560 1570 1580 1590 1600 1610 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TAGGTGTACA TCAAATAAAA ACCTTAAAGT TTAGAAAGGC ACCGAATAAT TTTGCATAGA AGATATTAGT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1620 1630 1640 1650 1660 1670 1680 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter AAATTTATAA AAATAAAAGA AATGTAGTTG TCAAGTTGTC TTCTTTTTTT TGGATAAAAA TAGCAGTTGG AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1690 1700 1710 1720 1730 1740 1750 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter CTTATGTCAT TCTTTTACAA CCTCCATGCC ACTTGTCCAA TTGTTGACAC TTAACTAATT AGTTTGATTC Hộp CAAT AF515784.1 .......... .......... .......... .......... ..A....... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1760 1770 1780 1790 1800 1810 1820 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter ATGTATGAAT ACTAAATAAT TTTTTAGGAC TGACTCAAAT ATTTTTATAT TATCATAGTA ATATTTATCT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1830 1840 1850 1860 1870 1880 1890 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter AATTTTTAGG ACCACTTATT ACTAAATAAT AAATTAACTA CTACTATATT ATTGTTGTGA AACAACAACG AF515784.1 .......... .......... ...T...... .......... .A........ .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1900 1910 1920 1930 1940 1950 1960 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TTTTGGTTGT TATGATGAAA CGTACACTAT ATCAGTATGA AAAATTCAAA ACGATTAGTA TAAATTATAT Hộp GATA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 1970 1980 1990 2000 2010 2020 2030 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TGAAAATTTG ATATTTTTCT ATTCTTAATC AGACGTATTG GGTTTCATAT TTTAAAAAGG GACTAAACTT AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 2040 2050 2060 2070 2080 2090 2100 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter AGAAGAGAAG TTTGTTTGAA ACTACTTTTG TCTCTTTCTT GTTCCCATTT CTCTCTTAGA TTTCAAAAAG Hộp TATA AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 2110 2120 2130 2140 2150 2160 2170 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| E8 promoter TGAACTACTT TATCTCTTTC TTTGTTCACA TTTTATTTTA TTCTATTATA AATATGGCAT CCTCATATTG AF515784.1 .......... .......... .......... .......... .......... .......... .......... DQ317599.1 .......... .......... .......... .......... .......... .......... .......... 2180 2190 2200 2210 ....|....| ....|....| ....|....| ....|....| ....|.... E8 promoter AGATTTTTAG AAATTATTCT AATCATTCAC AGTGCAAAAG AAGGGATCC AF515784.1 .......... .......... .......... .......... .-------- DQ317599.1 ........-- ---------- ---------- ---------- --------- Trình tự nền màu xanh lần lƣợt là: trình tự biểu hiện đặc hiệu ở quả (phụ thuộc ethylen): vị trí 1343-1489 và trình tự điều khiển quá trình chín của quả độc lập với ethylen: vị trí: 1943-2168. 2. So sánh trình tự nucleotide của promoter HSP 18.2 thu đƣợc với trình tự X17295.1 10 20 30 40 50 60 70 80 90 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro AAGCTTATGG TCATTTCTTC TGGTTCAAGC ATGACATGAA CAGGCAATAA ATAAGTTGAG ATTTTGATCA CAGTAACTGA TACTTGAATC Hộp CAAT Hộp GAGA X17295.1 ------.... .......... .......... .......... .......... .......... .......... .......... .......... 100 110 120 130 140 150 160 170 180 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro GAATCATTTA GATTTTTTTT TTTTTTAGTT TACTTGTTTA GTAAATATGT TGTCTATGTT TGTCACAAAA ACGTGGCTCA GTTCTTGTAT X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 190 200 210 220 230 240 250 260 270 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro ATATGGAGAC AAAAAAATCC ATTAAAAGAT TGTTGACATT CTCGGAAATT TAGTGCCAAC TGTTATTGCG AGAACTTACT ATAGTTTTCC X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 280 290 300 310 320 330 340 350 360 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro TTTGGCGAAA AGCTAATAAT CTTAAATCTT GATTTTGTCC TCTTTTCTCT GAGTTAGATT TTCTTAAATT CCACTTCCGA CCTATTAAGA X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 370 380 390 400 410 420 430 440 450 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro AATGGGCTTT TGCAAAGAAG ATCCGCTTCA CTGAGCCCGT ATCTCGAAGA GGATAATACA ACAACAAAGC AAAACGGCAC GTAGTTTTAA Hộp GAGA X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 460 470 480 490 500 510 520 530 540 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro TTGTAACCAA GGATTGCATT TCGGTCTTGT TTCAACAAAC GAAACTTCCT GAAATGCCAA GAAAAATCTG GTCATTTCAC CACAGTGATC AAC GAAACTTCCT G (HSE) CCT GAAATGCCAA G (HSE) CAA GAAAAATCTG G (HSE) X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 550 560 570 580 590 600 610 620 630 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro ATTGTGTATG TGTTCTAAAG ACTCCAAGCG AAGGTTTTAG AAAAAGGAGC ATTTTCTATT CTATTCAAGA AACTCGAAGA ACATTCTCTC CTAAAG ACTCCAAG (HSE) CAAGA AACTCGAAG (HSE) CAAGCG AAGGTTTT (HSE) X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 640 650 660 670 680 690 700 710 720 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 pro TTCATCCTCT AACTTCCCTA TAAATATGTC CTTTGCTAAT CAGATCAAAT CAGCAGGAAA ATCAAGAACC AAAAGTCTCC CGAAAAGCAA Hộp TATA X17295.1 .......... .......... .......... .......... .......... .......... .......... .......... .......... 730 ....|....| .. HSP 18.2 pro CGAACAGGAT CC X17295.1 ......------ HSE: heat shock elements 3. So sánh trình tự nucleotide của terminatorHSP 18.2 thu đƣợc với trình tự đã công bố 10 20 30 40 50 60 70 80 90 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 ter GAGCTCATAT GAAGATGAAG ATGAAATATT TGGTGTGTCA AATAAAAAGC TTGTGTGCTT AAGTTTGTGT TTTTTTCTTG GCTTGTTGTG HSP ter Nagaya ------.... .......... .......... .......... .......... .......... .......... .......... .......... 100 110 120 130 140 150 160 170 180 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| HSP 18.2 ter TTATGAATTT GTGGCTTTTT CTAATATTAA ATGAATGTAA GATCTCATTA TAATGAATAAACAAATGT↑TT CTATAATCCA TTGTGAATGT HSP ter Nagaya .......... .......... .......... .......... .......... .......... .......... .......... .......... 190 200 210 220 230 240 250 260 ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| .. HSP 18.2 ter TTTGTTGGAT CTCTTCTGCA GCATATAACT ACTGTATGTG CTATGGTATG GACTATGGAA TATGATTAAA GATAAGGAAT TC HSP ter Nagaya .......... .......... .......... .......... .......... .......... .......... ......------ ↑poly A site 4. Thành phần các môi trƣờng nuôi cấy in vitro cơ bản Loại môi trƣờng Thành phần MS lỏng MS (I-V) + Sucrose 30 mg/l; pH 5,8 MS đặc MS lỏng + 7,5 g Agar CCM MS (I - V) bổ sung BAP 2 mg/l, NAA 2 mg/l, sucrose 30 g/l và 7,5g agar, pH 5,8 STM MS (I - V) bổ sung zeatin 2 mg/l, sucrose 30 mg/l và 7,5g agar, pH 5,8 RM MS (I - V) bổ sung IBA 0,3 mg/l, sucrose 15 g/l và 7,5g agar, pH = 5,8 5. Thành phần các môi trƣờng nuôi khuẩn Môi trƣờng Thành phần LB đặc 5 g/l yeast extract + 5 g/l NaCl + 10 g/l tryptone + 15 g/l bacto agar, pH = 7,0 LB lỏng 5 g/l yeast extract + 5 g/l NaCl + 10 g/l tryptone, pH = 7,0 6. Trình tự cặp mồi M13 F/R (5’→3’) M13_F: GTAAAACGACGGCCAG; M13_R: CAGGAAACAGCTATGAC 7. Sơ đồ cấu trúc vector pBI121 8. Sơ đồ cấu trúc vector pBluescript II SK 9. Phƣơng trình đƣờng chuẩn 9.1. Phương trình đường chuẩn protein (theo Braford, 1976, sử dụng BSA làm chất chuẩn) 9.2. Phương trình đường chuẩn protein scFv gắn đuôi c-myc Nồng độ chất chuẩn: 0; 1,25; 2,5; 5; 10; 20 ng/μl Nồng độ chất chuẩn: 0; 10; 25; 50; 100; 150; 200 ng/μl y = 3503.6x - 18.094 R² = 0.9924 0 250 500 750 1000 1250 1500 1750 2000 2250 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 C (µ g /µ l) OD595nm y = 26.318x - 1.6454 R² = 0.9862 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 0 0.2 0.4 0.6 0.8 1 C ( n g /μ l) OD630nm y = 281.98x - 36 R² = 0.903 0 50 100 150 200 250 0 0.2 0.4 0.6 0.8 C ( n g /μ l) OD630nm 10. Thành phần đệm chiết DNA (theo Xin và Chen, 2012) STT Nồng độ gốc Nồng độ sử dụng 1 1 M Tris-HCl (pH8,0) 50 mM 2 5 M NaCl 300 mM 3 0,5 M EDTA (pH 8,0) 20 mM 4 CTAB 4% 5 Nƣớc khử ion, khử trùng 11. Cà chua chuyển gen: giai đoạn quả xanh và quả chín đỏ

Các file đính kèm theo tài liệu này:

  • pdfluan_an_nghien_cuu_bieu_hien_protein_tai_to_hop_miraculin_tr.pdf
Luận văn liên quan