Korean Journal of Plant Tissue Culture (식물조직배양학회지)

The Korean Society of Plant Biotechnology (한국식물생명공학회)
권호 표지

Visible and Fast Assay System for Tobacco Transformant Introduced with Adenosine Deaminase Marker Gene

Adenosine Deaminase 표지유전자로 형질전환된 연초의 신속한 Assay 방법

  • 양덕춘 (한국인삼연초연구원) ;
  • 김용환 (농업과학기술원 생화학과) ;
  • 임학태 (강원대학교 식품응용과학부) ;
  • 방극수 (익산대학교 생명공학과) ;
  • 배창휴 (순천대학교 농업과학연구소)
  • Published : 2001.05.01
Download PDF
⟨ Previous Next ⟩

Abstract

New visible and fast assay system have been developed for tobacco transformant introduced with adenosine deaminase (ADA) marker gene, which converts cytotoxic adenosine analogues to non-toxic inosine analogues and ammonia. Ammonia was changed to blue color in the solution of phenol-nitoprusside and alkaline-hypochlorite. It was possible to detect activity of ADA visibly on the holes of 96 well plate using tiny explant of transgenic tobacco leaves within 1 hour incubation time. As substrates of ADA enzyme from transgenic plant on the plate, a number of adenosine analogues such as 9-D-arabinofuranosyl adenine, cordycepin, 2'-deoxyadenosine, adenosine and xylofuranosyl adenine were possible for detection of ADA activity. Optimal condition of substrate for ADA enzyme was each 10 mM and pH 7.5 in adenosine solution. Especially, transgenic plant did not convert adenosine to inosine and ammonia in the presence of ADA inhibitor deoxycoformycin, which means that ammonia produced from transgenic plant is due to expression of ADA gene. Now, we show that this detection system can be easily, sensitively, fast and cheaply as well as visibly assayed in vitro as GUS gene system with very small size of transformant explant.

Adenosine deaminase 유전자를 연초의 형질전환용 표지유전자로 활용할 때 형질전환체 여부를 매우 빠르고 눈으로 직접 색깔을 확인할 수 있는 새로운 방법이 개발되었다. ADA 효소는 독성인 adenosine 유도체를 비독성인 inosine 유도체와 암모니아로 변환시키는데, 이때 형성된 암모니아를 phenol-nitoprusside와 alkaline-hypochlorite 용액을 이용하여 청색으로 변환시켜 96 well plate상에서 1시간 내에 형질전환체 여부를 쉽게 확인할 수 있게 되었다. ADA효소의 substrate로서 9-D-arabinofuranosyl adenine, cordycepin, 2'-deoxyadenosine, adenosine and xylofuranosyl adenine이 모두 가능하였으며, substrate 용액의 최적조건은 adenosine 10 mM과 pH 7.5이었다. 특히 형질전환체는 ADA효소의 inhibitor인 deoxycoformycin이 함유되어 있는 용액 속에서는 adenosine을 inosine과 암모니아로 변환시키지 못해 색깔의 변화가 없었는데, 이는 형질전환체에서 색깔의 변화는 ADA효소의 작용 때문에 일어나는 것을 의미한다. 따라서 본 연구결과는 ADA 표지유전자가 도입된 형질전환체의 확인에 있어서 GUS gene system과 같이 눈으로 직접 확인할 수 있을 뿐만 아니라 매우 작은 크기의 형질전환체 절편으로 쉽고, 빠르면, 값싸게 확인할 수 있게 되었다.

Keywords

References

  1. Methods in Enzymology v.153 Binary Ti vector for plant transformation and promoter analysis An G
  2. Nature v.304 A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation Bevan MW;Flavell RB;Chilton MD
  3. Korean J Plant Res v.11 A comparison of microtuberization efficiency between normal and adenosine deaminase transgenic potato plantlets cultured in vitro Choi KH;Yang DC;Jeon JH;Kim HS;Jung YH;Jung H
  4. Gene v.211 Modified binary plant transformation vectors with the wild type gene encoding NPT 2 Datla RSS;Hammerlindl JK;Panchuk B;Pelcher LE;Keller W
  5. EMBO J v.3 Engineering herbicide resistance in plants by expression of a detoxifying enzyme DeBlock M;Herrera-Estrella L;Montagu MV;Schell J;Zambryski P
  6. Nucleic Acids Res v.19 A simple and rapid method for the preparation of plant genomic DNA for PCR analysis Edwards K;Johnston C;Thompson C
  7. J Lab Clin Med v.76 A NADH linked kinetic assay for adenosine deaminase EC 3.5.4.4 activity Ellis G;Goldberg DM
  8. Ann Rev Biochem v.47 The role of adenosine and 2' - deoxyadenosine in mammalian cells Fox IH;Reckel S;Pilz R;Kalden JR
  9. Enzyme v.25 A new spectrophoto-metric assay for enzymes of purine metabolism: IV Determination of adenosine deaminase Heinz F;Reckel S;Pilz R;Kalden JR
  10. EMBO J v.2 Chimeric genes as dominant selectable markers in plant cells Herrera-Estrella L;De Block M;Montagu MV;Schell J
  11. Nature v.303 Expression of chimetric genes transferred into plant cells using a Ti-Plasmid derived vetor Herrera-Estrella L;De Block M;Montagu MV;Schell J
  12. Anal Chim Acta v.88 Automated adenosine deaminase enzyme determination with an ammoniasensing membrane electrode Hjemdahl-Monsen OE;Papastathopoulos DS;Rechnitz GA
  13. Nature v.303 A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaaciens Ti-plasmid Hoekema A;Hirsch PR;Hooykaas PJJ;Schilperoort RA
  14. Plant Molecular Biology v.5 Non-oncogenic plant vectors for use in the Agrobacterium binary vector system Hoekema A;Van Haaren MJJ;Fellinger AJ;Hooykaas PJJ;Schilperoort RA
  15. Ann Hum Genet v.32 Further data on the adenosine deaminase polymorphism and a report of a new phenotype Hopkinson DAH;Cook PJL; Harris H
  16. Plant Mol Biol Rep v.5 Assaying chimeric genes in plants: The GUS gene fusion system Jefferson RA
  17. Trend Genet v.1 Adenosine deaminase deficiency and severe combined immunodeficiencies Kellems RE;Yeung CY;Ingolia DE
  18. Biochem J v.50 The enzymic oxidation and assay of adenine Klenow J
  19. Clin Chim Acta v.63 Quantative measurement of adenosine deaminase from human erythrocytes Koerber W;Meisterernst EB;Herrmann G
  20. Science v.247 A regulatory gene as a novel visible marker for maize transformation Ludwig SR;Bower B;Beach L;Wessler SR
  21. Clin Chem v.9 Micromethod for the estimation of serum adenosine deaminase Martinek RG
  22. Anal Biochem v.105 A fast and simple radiometric assay for adenosine deaminase using reversed-phase thin-layer chromatography Meier W;Conscience JF
  23. Plant Molecular Biology v.14 The in vitro pattern of firefly luciferase expression in transgenic plants Schneider M;Ow DW;Howell SH
  24. Adv Exp Med Biol v.76 Adenosine deaminase: characterization of the molecular heterogeneity of the enzyme in human tissue Weyden MBV;Kelley WM
  25. Korean J Plant Tissue Culture v.22 Expression of mouse adenosine deaminase gene in transgenic tobacco (Nicotiana adenosine deaminase gene in transgenic tobacco (Nicotiana tabacum L.). Yang DC;Park JC;Choi KT;Lee JM
  26. Korean J Plant Tissue Culture v.22 Adenosine deaminase gene: possible selectable marker for tobacco transformation Yang DC;Han SS;Yoon ES
  27. Korean J Plant Tissue Culture v.23 Extraction and activity assay of mouse adenosine deaminase from transgenic tobacco (Nicotiana tabacum L.). Yang DC;Bang KS;Choi KH
  28. Korean Society of Plant tissue Culture v.23 Expression of adenosine deaminase gene in ginseng hairy root by genetic transformation Yang DC;Bang KS;Park JC;Choi KH;Choi KT
  29. Korean Sciety of Plant tissue Culture v.24 Plant Regeneration and expression of mouse adenosine deaminase gene in transgenic hot pepper (Casicum annuum L.) plant Yang DC;Lee KY;Yoo YS;Choi KH;Lim HT
  30. J Biol Chem v.260 Identification of fuction murine deaminase cDNA clones by complementation in E. Coli Yeung CY;Ingolia DE;Roth DB;Shoemaker C;Al-Ubaidi MR;Yen JY;Ching C;Bobomis C;Kaufman RJ;Kellems RE