Horticultural Science & Technology (원예과학기술지)

Korean Society of Horticultural Science (한국원예학회)
권호 표지

Detection Method for Genetically Modified Carnation Plants Using Multiplex Polymerase Chain Reaction

Multiplex PCR을 이용한 유전자변형 카네이션의 검출법

Kim, Jae-Hwan;Lee, Jea-Eun;Yoo, Sang-Jin;Park, Young-Doo;Kim, Hae-Yeong
김재환;이재은;유상진;박영두;김해영

  • Published : 20100400
⟨ Previous Next ⟩

Abstract

A multiplex PCR method was developed to simultaneously detect two transformation vectors (pCGP1470 and pCGP1991) used in the development of GM carnation plants (Moonvista, Moonaqua, Moonshadow, Moonlite, Moonshade, and Moodust). To screen for GM carnation plants, one specific primer was designed from acetolactate synthase (SuRB/ALS) terminator sequence. The anthocyanidin synthase (ANS) was also used as an endogenous reference gene of carnation in PCR detection. The primer pair ANS-KF/KR producing 100 bp amplicon was used to amplify the ANS gene and no amplified product was observed in any of the 10 different plants used as a template. The primer pair C1991-KF/KR was designed to amplify the junction sequence between dihydroflavonol reductase (DFR) gene and DFR terminator in pCGP1991. To confirm pCGP1470, the primer pair C1470-KF/KR was designed to cover the junction sequence between Mac promoter and DFR gene. The detection limit of the multiplex PCR method is approximately 1%. This result indicates that this multiplex PCR method could be a useful tool for monitoring unauthorized GM carnation in Korea.

유전자변형 카네이션(Moonvista, Moonaqua, Moonshadow, Moonlite, Moonshade, 및 Moodust) 개발에 이용된 형질전환 벡터(pCGP1470, pCGP1991)를 동시에 검출할 수 있는 multiplex PCR이 개발되었다. 유전자변형 카네이션을 스크리닝하기 위해 acetolactate synthase(SuRB/ALS) terminator의 염기 서열로부터 한 쌍의 primer를 제작하였다. Anthocyanidin synthase(ANS)를 카네이션의 내재유전자로 PCR 검사법에 이용하였다. 100bp의 PCR 산물을 얻을 수 있는 ANS-KF/KR primer가 ANS 유전자를 증폭시키는데 이용되었고, 이것을 이용하여 11개의 다른 식물에 대해서 PCR한 결과 카네이션에서만 특이적인 증폭산물이 확인되었다. C1991-KF/KR primer가 형질전환 벡터 pCGP1991에 삽입된 Dihydroflavonol reductase(DFR) 유전자와 DFR terminator 사이의 염기서열을 확인할 수 있도록 제작되었고, 형질전환 벡터 pCGP1470를 확인하기 위해 C1470-KF/KR primer가 Mac promoter와 DFR 유전자 사이의 염기서열을 확인할 수 있도록 제작되었다. 본 연구에서 개발된 multiplex PCR의 검정한계치는 약 1%이며, 이러한 multiplex PCR이 국내 미승인 유전자변형 카네이션을 모니터링 하는데 유용하게 사용될 수 있을 것이다.

Keywords

References

  1. European Commission (EC). 2003. 'Commission Regulation (EC) No. 1830/2003 of September 22, 2003, Concerning the traceability and labeling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive 2001/18/EC' Off. J. Eur. Commun. No. L 268:24-28
  2. Hern$\'{a}}$ndez, M., D. Rodr$\'{\i}$guez-L$\'{a}}$zaro, D. Zhang, T. Esteve, M. Pla, and S. Prat. 2005. Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21. J. Agric. Food Chem. 53:3333-3337 https://doi.org/10.1021/jf049192y
  3. James, C. 2009. Global status of commercialized transgenic crops. ISAAA Briefs No. 39
  4. Kim, H.Y. and J.H. Kim. 2007. Allergenicity of genetically modified crops. Pediatr. Allergy Respir. Dis. 17:166-172
  5. Kim, J.H., S.A. Kim, Y.J. Seo, W.Y. Lee, S.H. Park, and H.Y. Kim. 2008. Multiplex PCR detection of the MON1445, MON15985, MON88913, and LLcotton25 varieties of GM cotton. Food Sci. Biotechnol. 17:829-832
  6. Kim, J.H., S.H. Park, and H.Y. Kim. 2009. Multiplex PCR detection of four events of GM maize (Event 3272, LY038, MIR162, and MON88017). J. Kor. Soc. Appl. Biol. Chem. 52:105-107 https://doi.org/10.3839/jksabc.2009.020
  7. Matsuoka, T. 2001. GMO labeling and detection methods in Japan. In: APEC- JIRCAS Joint Symposium and Workshop on Agricultural Biotechnology. September 6, Amari Watergate Hotel, Bangkok, Thailand. JIRCAS, Japan
  8. Ministry of Agriculture & Forestry (MAF). 2000. Guidelines for labeling of genetically modified agricultural products. Notification No. 31
  9. Ministry of Agriculture & Forestry (MAF). 2008. Flower production in Korea. p. 1-190
  10. Mol, J., E. Cornish, J. Mason, and R. Koes. 1999. Novel coloured flowers. Curr. Opin. Biotechnol. 10:198-201 https://doi.org/10.1016/S0958-1669(99)80035-4
  11. Tanaka, Y. 2006. Flower colour and cytochromes P450. Phytochem. Rev. 5:283-291 https://doi.org/10.1007/s11101-006-9003-7
  12. Tanaka, Y., Y. Katsumoto, F. Brugliera, and J. Mason. 2005. Genetic engineering in floriculture. Plant Cell Tissue Organ Cult. 80:1-24 https://doi.org/10.1007/s11240-004-0739-8
  13. Yu, S.N., M.H. Lee, J.H. Jung, and M.U. Chang. 2006. The viruses in carnation in Korea. Kor. J. Hort. Sci. Technol. 24:507-514