Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Expression of an artificial gene encoding a repeated tripeptide lysyl-g1utamyl-tryptophan in Tobacco Plant

담배식물체에서 필수아미노산인 lysyl-glutamyl-tryptophan을 암호화하는 인공유전자의 발현

  • 이수영 (이화여자대학교 분자생명과학부) ;
  • 나경수 (대구공업대학 식품영양과) ;
  • 백형석 (부산대학교 미생물학과) ;
  • 박희성 (대구가톨릭대학교 생명자원학부) ;
  • 조훈식 (대구대학교 자연자원대학 생명자원학부) ;
  • 이용세 (대구대학교 자연자원대학 생명자원학부) ;
  • 최장원 (대구대학교 자연자원대학 생명자원학부)
  • Published : 2002.02.01
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Abstract

To investigate expression of the artificial gene encoding a repeated tripeptide lysyl-glutamyl-tryptophan in tobacco plant, the plant binary vector, pART404 has been constructed, which contains the duplicated CaMV 35S promoter, an artificial gene coding for repetitive polymer (Lys-Glu-Trp)$_{64}$, and nopaline synthase (nos) terminator. The recombinant expression vector was introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated trans-formation. The transgenic calli selected by kanamycin containing medium were then regenerated to whole plants. Southern blot analysis indicated that five transgenic plants (No. 1, 7, 9, 43, 45) showed the hybridizing signals at 1.1 kb of the expected size on EcoRI digestion and each of the transgenic plants contained 1 or 3 copies of the artificial gene inserted into its genome. By northern blot analysis, the size of the hybridized total RNA was estimated to be approximately 1.2 kb and the RNA appeared generally to have the integrity. Western blot indicated that the protein was detected at the position of 33 kDa and the expression level of the polypeptide in the transgenic plant (No. 45) was measured to approximately 0.1% of the total protein.

식물 단백질의 영양가 향상을 위한 일환으로 필수아미노산의 조성이 풍부한 인공단백질을 암호화하는 인공유전자를 담배 식물체에서 발현을 시도하기 위하여, 식물에서 외래유전자의 발현에 널리 사용되는 Cauliflower mosaic virus (CaMV)의 35S promoter를 이중으로 중첩되도록 하고, (Lys-Glu-Trp)이 64번 반복되는 인공유전자 및 nopaline synthase (nos) terminator를 갖고있는 binary vector pART4-4를 구성하였다. 이 재조합 플라스미드는 Agrobacterium tumefaciens를 이용한 형질전환에 의해 Nicotiann tabacum (Var. Xanthi)으로 도입되었다. Kanamycin이 포함된 신초 유도 배지 및 뿌리 유도배지를 이용하여 정상적으로 재생된 담배 식물체로부터 도입된 인공유전자의 발현을 분석하였다. 추출한 genomic DNA를 EcoRI으로 자른 다음 Southern blot 분석에 의하면, 효소 절단 시 예상되는 1.1 kb에서 band를 형성하였으며 각각의 형질전환 식물체에 인공유전자가 1 또는 3 개씩 도입되어 있음을 확인하였다. Northern blot 분석에 의하면 약 1.2 kb 전사체가 비교적 안정하게 발현되었으며, 잎, 줄기, 뿌리로부터 RNA를 분리하여 promoter의 조직 특이성 발현을 분석한 결과, 잎에서 생성되는 RNA가 줄기나 뿌리 조직보다 안정하게 발현되었다. 형질전환 식물체에서 Western blot에 의한 단백질 분석 결과, 잎에서 추출한 단백질로부터 원하는 크기인 33 kDa의 인공단백질이 생성됨을 확인하였으며 발현 수준은 전체 세포 단백질의 0.1%로서 낮은 수준이었다.

Keywords

References

  1. Plant Mol. Biol. v.18 Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine Altenbach, S. B.;C. Kuo;L. C. Staraci;K. W. Pearson;C. Wainwright;A. Georgescu;J. Townsend https://doi.org/10.1007/BF00034952
  2. Protein quality in humans Conference overview. Altschul, A. M.;Bodwell, C. E.(ed.);J. S. Adkins(ed.);D. T. Hopkins(ed.)
  3. Binary vectors, In Plant Gene Res/Manual An, G.;A. M. Ebert;S. Ha;Gelvin, S. B.(ed.);R. Schilperoot(ed.);D. Verma(ed.)
  4. Plant Physiol. v.85 Bacillus thruingiensis δ-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidopteran insects Barton, K. A.;H. R. Whiteley;N. S. Yang https://doi.org/10.1104/pp.85.4.1103
  5. Protein Eng. v.1 Expression of synthetic genes coding for completely new nutritionally rich, artificial protein Biernat, J;K. Koster https://doi.org/10.1093/protein/1.4.353
  6. Anal. Biochem. v.72 A rapid and sensitive method for the quantities of protein utilizing the principle of protein dye binding Braford, M. https://doi.org/10.1016/0003-2697(76)90527-3
  7. Proc. Natl. Acad. Sci. v.83 Functional analysis of regulatory elements in a plant embryo-specific gene Chen, Z. L.;M. A. Schuler;R. N. Beachy https://doi.org/10.1073/pnas.83.22.8560
  8. Nucleic Acids Res. v.8 The expression in E. coli of synthetic repeating polymeric genes coding for poly(L-aspartyl-L-phenylalanine) Doel, M. T.;M. Eaton;E. A. Cook;H. Lewis;T. Patel;N. H. Carey https://doi.org/10.1093/nar/8.20.4575
  9. Plant genetic transformation and gene expression Draper, J.;R. Scott;P.Amitage;R. Walden
  10. Mol. Gen. Genet. v.221 Expression of the 2S albumin from Bertholletia excelsa in Brassica napus Guerche, P.;E. R. P. de Almeida;M. A. Schwarztein;E. Gander;E. Krebbers;G. Pelletier https://doi.org/10.1007/BF00259393
  11. Nature v.303 A binary plant vector sta\rategy based on separation of vir- and T-region of Agrobacterium tumefaciens Hoekema, A.;P. Hirsch;P. Hooykass;R. A. Schilperoot https://doi.org/10.1038/303179a0
  12. Mol. Gen. Genet. v.163 Transfection and transformation of Agrobacterium tamefaciens Holster, M.;D. de Waele;A. Depicker;E. Messens;M. van Montagu;J. Schell https://doi.org/10.1007/BF00267408
  13. Science v.227 A simple method for transferring genes into plants Horsch, R. B.;J. E. Fry;N. L. Hoffman;D. Eichholtz;S. D. Rogers;R. T. Fraley https://doi.org/10.1126/science.227.4691.1229
  14. Appl. Microbiol. Biotechnol. v.21 Construction and expression of synthetic DNA fragments coding for polypeptides with elevated levels of essential amino acids Jaynes, J. M.;P. Langridage;K. Anderson;C. Bond;D. Sands;C. W. Newman;R. Newman https://doi.org/10.1007/BF00295122
  15. EMBO J. v.4 High level expression of introduced chimeric genes in regenerated transformed plants Jones, J. D. G.;P. Dunsmuir;J. Bedrook
  16. Science v.236 Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes Kay, R.;A. Chan;M. Daly;J. McPherson https://doi.org/10.1126/science.236.4806.1299
  17. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Laemmli, U. K. https://doi.org/10.1038/227680a0
  18. Korean Biochem. J. v.27 no.1 Construction and expression of artificial genes encoding polypeptides containing essential amino acids in E. coli Lee, S. Y.;M. A. Lee;S. Y. Lee
  19. Physiol. Plant v.15 A revised medium for rapid growth and bioassays with tobacco tissue cultures Murashige, T.;F. Skoog. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  20. Plant Mol. Biol. v.16 Normal and lysine containing zeins are unstable in transgenic tobacco seeds Ohtani, T.;G. Galili;J. C. Wallace;G. A. Tompson;B. A. Larkins https://doi.org/10.1007/BF00017922
  21. Trends in Biotechnol v.4 Improving grain protein quality by genetic engineering;Some biochemical consideration Rao, A. S. https://doi.org/10.1016/0167-7799(86)90135-6
  22. In Molecular Cloning, A Laboratory Manual Sambrook, J.;E. F. Fritsch;T. Maniatis
  23. Proc. Natl. Acad. Sci. v.74 DNA sequencing with chain terminating inhibitors Sanger, F.;S. Niklen;A. R. Coulson https://doi.org/10.1073/pnas.74.12.5463
  24. Plant Physiol. v.100 Threonine overproduction in transgenic tobacco plants expressing a mutant desensitized aspartate kinase of E. coli Shaul, O;G. Galili https://doi.org/10.1104/pp.100.3.1157
  25. Nucleic Acids Res. v.17 A small scale procedure for the rapid isolation of plant RNAs Verwoerd, T. C.;B. M. M. Dekker;A. Hoekema https://doi.org/10.1093/nar/17.6.2362
  26. Science v.240 Aggregation of lysine containing zeins into protein bodies in Xenopus Oocytes Wallace, J. C.;G. Galili;E. E. Kawata;R. E. Cuellar;M. A. Shotwell;B. A. Larkins https://doi.org/10.1126/science.2834822
  27. Plant Science v.64 Expression of a synthetic gene for improved protein quality in transformed potato plants Yang, M. S.;N. O. Espinoza;P. G. Nagpala;J. H. Dodds;F. F. White;K. L. Schnorr;J. M. Jaynes https://doi.org/10.1016/0168-9452(89)90156-8