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감미단백질 관련 브라제인, 타우마틴 및 미라쿨린 유전자를 이용한 형질전환 상추 육성 및 발현분석

Stable expression and characterization of brazzein, thaumatin and miraculin genes related to sweet protein in transgenic lettuce

  • 정여진 (국립한경대학교 원예생명과학과) ;
  • 강권규 (국립한경대학교 원예생명과학과)
  • Jung, Yeo Jin (Department of Horticultural Life Science, Hankyong National University) ;
  • Kang, Kwon Kyoo (Department of Horticultural Life Science, Hankyong National University)
  • 투고 : 2018.08.17
  • 심사 : 2018.09.11
  • 발행 : 2018.09.30

초록

감미료(sweetener)는 단맛을 느끼게 하는 첨가물 중 하나로 인공감미료와 설탕이 대표적이며, 단맛의 특성을 지닌 감미 단백질도 잘 알려져 있다. 본 연구는 천연 감미 단백질, brazzein, thaumatin 및 miraculin의 안정적인 생산을 위해 Agrobacterium방법으로 상추 세포를 형질 전환시켰다. 감미 단백질을 코딩하는 합성 유전자들은 상시적으로 발현하는 프로모터의 제어 하에 상추에 형질전환 하였다. 형질전환한 잎을 이용하여 RT-PCR 및 Western blot 분석을 실시한 결과 감미 단백질이 안정적으로 발현하는 것을 확인하였다. 형질전환 상추에서 발현한 단백질은 단맛을 유발하는 단백질 활성을 가지고 있었다. 이러한 결과는 유전자 재조합 감미 단백질들은 형질전환한 상추에 잘 발현된 것을 보여 주며, 이들 생산 시스템은 식물로부터 감미단백질 생산을 위한 좋은 대안이 될 수 있을 것으로 생각된다.

Sweetener is one of the additives that makes you feel sweet. Artificial sweeteners and sugar are typical examples, and sweetness proteins with sweetness characteristics have been widely studied. These studies elucidated the transformation lettuce cells with Agrobacterium method for stable production of natural sweet proteins, brazzein, thaumatin, and miraculin. In this paper, we report use of a plant expression system for production of sweet proteins. A synthetic gene encoding sweet proteins was placed under the control of constitutive promoters and transferred to lettuce. High and genetically stable expression of sweetener was confirmed in leaves by RT-PCR and Western blot analysis. Sweet proteins expressed in transgenic lettuce had sweetness-inducing activity. Results demonstrate recombinant sweet proteins correctly processed in transgenic lettuce plants, and that this production system could be a viable alternative to production from the native plant.

키워드

참고문헌

  1. Akter S, Huq MA, Jung YJ, Cho YG, Kang KK (2016) Application of sweet and taste modifying genes for development in plants:current status and prospects. J Plant Biotechnol 43:397-404 https://doi.org/10.5010/JPB.2016.43.4.397
  2. Assadi-Porter FM, Aceti DJ, Cheng H, Markley JL (2000) Efficient production of recombinant brazzein, a small, heat-stable, sweettasting protein of plant origin. Arch Biochem Biophys 376:252-258 https://doi.org/10.1006/abbi.2000.1725
  3. Assadi-Porter FM, Aceti DJ, Markley JL (2000) Sweetness Determinant Sites of Brazzein, a Small, Heat-Stable, Sweet-Tasting Protein. Arch Biochem Biophys 376:259-265. https://doi.org/10.1006/abbi.2000.1726
  4. Assadi-Porter FM, Patry S, Markley JL (2008) Efficient and rapid protein expression and purification of small high disulfide containing sweet protein brazzein in E. coli. Protein Expr Purif 58:263-268 https://doi.org/10.1016/j.pep.2007.11.009
  5. Berlec A, Jevnikar Z, Majhenic AC, Rogelj I, Strukelj B (2006) Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria. Appl microbiol biot 73:158-165 https://doi.org/10.1007/s00253-006-0438-y
  6. Berlec A, Strukelj B (2009) Large increase in brazzein expression achieved by changing the plasmid/strain combination of the NICE system in Lactococcus lactis. Lett Appl Microbiol 48:750-755
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  8. Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6:219-226 https://doi.org/10.1016/S1360-1385(01)01922-7
  9. Faus I (2000) Recent developments in the characterization and biotechnological production of sweet-tasting proteins. Appl Microbiol Biot 53:145-151 https://doi.org/10.1007/s002530050001
  10. Farbman AI, Ogden-Ogle CK, Hellekant G, Simmons SR, Albrecht RM, Van Der Wel H: Labeling of sweet taste binding sites using a colloidal gold-labeled sweet protein, thaumatin. Scanning Microsc. 1987, 1 (1): 351-7
  11. Howard JA, Hood E (2005) Bioindustrial and biopharmaceutical products produced in plants. Adv Agron 85:91-124
  12. Hwang-Bo J, Kyung Ok Jang, Hayoung Chung, Jong-Hwa Park, Tae Hoon Lee, Jiyoung Kim and In Sik Chung (2016) Antiinflammatory Effect of Lactuca sativa L. Extract in Human Umbilical Vein Endothelial Cells and Improvement of Lipid Levels in Mice Fed a High-fat Diet. Korean J. Food Nutr. 29(6): 998-1007 https://doi.org/10.9799/ksfan.2016.29.6.998
  13. Hood EE, Jilka JM (1999) Plant-based production of xenogenic proteins. Curr Opin Biotech 10:382-386 https://doi.org/10.1016/S0958-1669(99)80069-X
  14. Hood EE, Woodard SL, Horn ME (2002) Monoclonal antibody manufacturing on transgenic plants-myths and realities. Curr Opin Biotech 13:630-635 https://doi.org/10.1016/S0958-1669(02)00351-8
  15. Horn ME, Woodard SL, Howard JA (2004) Plant molecular farming:systems and products. Plant Cell Rep 22:711-720 https://doi.org/10.1007/s00299-004-0767-1
  16. Jo HJ, Noh JS, Kong KH (2013) Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis. Protein Expres Purif 90:84-9 https://doi.org/10.1016/j.pep.2013.05.001
  17. Jung YJ, Bae SS, Lee GJ, Seo PJ, Cho Y G, Kang KK (2017) A novel method for high-frequency genome editing in rice, using the CRISPR/Cas9 system. J Plant Biotechnol 44:89-96 https://doi.org/10.5010/JPB.2017.44.1.089
  18. Jung YJ, Nogoy FM, Cho Y G, Kang KK (2015) Development of high tryptophan GM rice and its transcriptome analysis. J Plant Biotechnol 42:186-195 https://doi.org/10.5010/JPB.2015.42.3.186
  19. Jung YJ, Nou IS, Kang KK (2014) Overexpression of Oshsp16.9 gene encoding small heat shock protein enhances tolerance to abiotic stresses in rice. Plant Breed Biotech 2(4):370-379 https://doi.org/10.9787/PBB.2014.2.4.370
  20. Kant R (2005) Sweet proteins, potential replacement for artificial low calorie sweeteners. Nutrition 4:5 https://doi.org/10.1186/1475-2891-4-5
  21. Kurihara Y, Beidler LM (1968) Taste-modifying protein from miracle fruit. Science 161:1241-1243 https://doi.org/10.1126/science.161.3847.1241
  22. Lamphear BJ, Barker DK, Brooks CA, Delaney DE, Lane JR, Beifuss K, Love R, Thompson K, Mayor J, Clough R, Harkey R, Poage M, Drees C, Horn ME, Streatfield SJ, Nikolov Z, Woodard SL, Hood EE, Jilka JM, Howard JA (2005) Expression of the sweet protein brazzein in maize for production of a new commercial sweetener. Plant Biotechnology Journal 3:103-114
  23. Lee JJ, Kong JN, Do HD, Jo DH, Kong KH (2010) Design and efficient soluble expression of a sweet protein, brazzein and minor-form mutant. B Kor Chem Soc 31:3830-3833. https://doi.org/10.5012/bkcs.2010.31.12.3830
  24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 https://doi.org/10.1038/227680a0
  25. Lee YR, Akter S, Lee IH, Jung YJ, Park SY, Cho Y-G, Kang KK, Jung YJ (2018) Stable expression of brazzein protein, a new type of alternative sweetener in transgenic rice. J Plant Biotechnol. 45:63-70 https://doi.org/10.5010/JPB.2018.45.1.063
  26. Liu X, Hu Z, Maeda S, Aiuchi T, Nakaya K, Kurihara Y (1993) Purification, complete amino acid sequence and structure characterization of the heat stable sweet protein, mabinlin II. Eur J Biochem 211:281-287 https://doi.org/10.1111/j.1432-1033.1993.tb19896.x
  27. Price JM, Biava CG, Oser BL, Vogin EE, Steinfield J, Ley HL (1970) 'Bladder tumors in rats fed cyclohexylamine or high doses of a mixture of cyclamate and saccharin'. Science 167:1131-1132 https://doi.org/10.1126/science.167.3921.1131
  28. Masuda T, Kitabatake N (2006) Developments in biotechnological production of sweet proteins. J Biosci Bioeng 102:375-389 https://doi.org/10.1263/jbb.102.375
  29. Masuda T, Ueno Y, Kitabatake N (2001) Sweetness and enzymatic activity of lysozyme. J Agr Food Chem 49:4937-4941 https://doi.org/10.1021/jf010404q
  30. Ming D, Hellekant G (1994) Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra byamarazzeana B. FEBS Lett 355:106-108 https://doi.org/10.1016/0014-5793(94)01184-2
  31. Moris JA, Cagan RH (1972) Purification of monellin, the sweet principal for Dioscoreophyllum cumminsii. Biochim Biophys Acta 261:114-122 https://doi.org/10.1016/0304-4165(72)90320-0
  32. Rachid A, Belloir C, Chevalier J, Desmetz C, Miller ML, et al. (2009) Optimization of the Production of Recombinant Brazzein Secreted by the Yeast Pichia pastoris. Chem senses 34:A80-A80
  33. Ren YL, Zhou YW, Ye YH. 2004. Chemical components of Lactuca and their bioactivites. Acta Pharmaceutica Sinica 39:954-960
  34. Shirasuka Y, Nakajima K, Asakura T, Yamashita H, Yamamoto A, Hata S, Nagata S, Abo M, Sorimachi H, Abe K (2004) Neoculin as a new taste-modifying protein occurring in the fruit of Curculigo latifolia. Biosci Biotech Bioch 68:1403-1407 https://doi.org/10.1271/bbb.68.1403
  35. Sun HJ, Cui ML, Ma B, Ezura H (2006) Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce. FEBS Lett 580:620-626 https://doi.org/10.1016/j.febslet.2005.12.080
  36. Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5:2-15 https://doi.org/10.1111/j.1467-7652.2006.00216.x
  37. Sun HJ, Kataoka H, Yano M, Ezura H (2007) Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants. Plant Biotechnol J 5:768-777 https://doi.org/10.1111/j.1467-7652.2007.00283.x
  38. Streatfield SJ, Howard JA (2003) Plant-based vaccines. Int J Parasitol 33:479-493 https://doi.org/10.1016/S0020-7519(03)00052-3
  39. Thole V, Alves SC, Worland B, Bevan MW, Vain P (2009) A protocol for efficiently retrieving and characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA insertional mutants. Nature 4:650-661
  40. Van der Wel H (1972) Isolation and characterization of the sweet principal for Dioscoreophyllum cumminsii (Stapf) Diels. FEBS Lett 21:88-90 https://doi.org/10.1016/0014-5793(72)80170-4
  41. Van der Wel H and Loeve K (1972) Isolation and characterization of thaumatin I and II, the sweet-tasting proteins from Thaumatococcus daniellii Benth. Eur J Biochem 31:221-225 https://doi.org/10.1111/j.1432-1033.1972.tb02522.x
  42. Van der Wel H, Larson G, Hladik A, Hladik CM, Hellekant G, Glaser D (1989) Isolation and characterization of pentadin, the sweet principle of Pentadiplandra brazzeana Baillon. Chem Senses 264:6655-6659
  43. Yan S, Song H, Pang D, Zou Q, Li L, Yan Q, Fan N, Zhao X, Yu H, Li Z, Wang H, Gao F, Ouyang H, Lai L (2013) Expression of Plant Sweet Protein Brazzein in the Milk of Transgenic Mice. PloS one 8:e76769 https://doi.org/10.1371/journal.pone.0076769
  44. Yamashita H, Theeraship A, Nakaya T, Nakamura Y, Kurihara Y (1990) Purification and complete amino acid sequence of a new type of sweet protein with taste-modifying activity, curculin. J Biol Chem 265:15770-15775
  45. Yoshida K, Shinmyo A (2000) Transgenic expression systems in plants, a natural bioreactor. J Biosci Bioeng 90:353-362 https://doi.org/10.1016/S1389-1723(01)80001-3
  46. Zemanek EC, Wasserman BP: Issues and advances in the use of transgenic organisms for the production of thaumatin, the intensely sweet protein from Thaumatococcus danielli. Crit Rev Food Sci Nutr. 1995, 35(5):455-66 https://doi.org/10.1080/10408399509527709