Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Qualitative PCR Detection of vitamin E-enriched GM Perilla

비타민 E 강화 유전자변형 들깨에 대한 정성 PCR 분석법

  • Kim, Jae-Hwan (Department of Food Science and Biotechnology, Kyung Hee University) ;
  • Ahn, Ji-Hye (Department of Food Science and Biotechnology, Kyung Hee University) ;
  • Song, Hee-Sung (Department of Food Science and Biotechnology, Kyung Hee University) ;
  • Kim, Kyung-Hwan (Division of Cell and Genetics, National Institute of Agricultural Biotechnology) ;
  • Kim, Dong-Hern (Division of Biosafety, National Institute of Agricultural Biotechnology) ;
  • Kim, Hae-Yeong (Department of Food Science and Biotechnology, Kyung Hee University)
  • 김재환 (경희대학교 생명공학원) ;
  • 안지혜 (경희대학교 생명공학원) ;
  • 송희성 (경희대학교 생명공학원) ;
  • 김경환 (농촌진흥청 농업생명공학연구원 세포유전과) ;
  • 김동헌 (농촌진흥청 농업생명공학연구원 생물안전성과) ;
  • 김해영 (경희대학교 생명공학원)
  • Published : 2006.09.30
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Abstract

For the development of a qualitative PCR detection method for genetically modified perilla (Perilla frutescens), perilla species-specific gene, KAS-I (Beta-ketoacyl-ACP synthase I), was selected and validated as suitable for the use as an endogenous reference gene in perilla. Primer specificity was first tested by the means of qualitative PCR analysis. The primer pair Pfru3-F/R amplifying the perilla endogenous gene, KAS-I, gave rise to an amplicon 95 bp. No amplified product was observed when DNA samples from 15 different plants were used as templates. Qualitative PCR detection method was assayed with vitamin E-enriched GM Perilla developed in Korea. For the qualitative PCR detection method, the construct-specific detection primer pairs were constructed. The primer pair TMTO-F/R amplifying the junction region of TMT (${\gamma}$-tocopherol methyltransferase) gene and OCS (Octopine synthase) terminator introduced in GM perilla gave rise to an amplicon 148 bp.

국내에서 개발된 비타민 E 강화 유전자변형 들깨의 정성 PCR 분석법의 개발을 위해 들깨의 내재 유전자로써 KAS-I (Beta-ketoacyl-ACP synthase I)를 선별하였고, 이러한 내재유전자를 특이적으로 증폭시킬 수 있는Primer(Pfru3-F/R)쌍을 이용한 PCR에서 95 bp의 PCR증폭 산물을 얻었으며, 들깨를 포함한 16개 작물에 대해 PCR을 수행한 결과에서 들깨만이 특이적으로 증폭되는 것을 확인하였다. 또한, 비타민 E 강화 유전자변형 들깨에 삽입된 TMT(${\gamma}$-tocopherol methyltransferase) 유전자와 OCS(Octopine synthase) terminator 연결 부위를 증폭시켜 148 bp의 PCR 산물을 얻을 수 있는 primer(TMTO-F/R)를 제작하였으며, 이러한 두 쌍의 primer를 이용하여 국내 개발된 비타민 E 강화 유전자변형 들깨의 PCR 정성 분석법을 확립하였다.

Keywords

References

  1. James, C. (2005) Executive Summary of Global Status of Commercialized Biotech/GM Crops: 2005. ISAAA Briefs No.34. ISAAA: Ithaca, NY
  2. Kim, K. H., Hwang, S. K., Kim, D. H., Lee, J. Y. and Hwang Y. S. (2002) Molecular cloning and characterization of $\gamma$-tocopherol methyltransferase cDNA from Perilla frutescens. J. Korean Soc. Agric. Chem. Biotechnol. 45(4), 203-206
  3. Shigeoka, S., Ishiko, H., Nakano, Y. and Mitsunaga, T. (1992) Isolation and properties of gamma-tocopherol methyltransferase in Euglena gracilis. Biochim Biophys. Acta 1128, 220-226 https://doi.org/10.1016/0005-2760(92)90311-I
  4. Kim, K. H., Lee, Y. H., Kim, D. H., Park, Y. H., Lee, J. Y.Hwang, Y. S. and Kim, Y. H. (2004) Agrobacterium-mediated genetic transformation of Perilla frutescens. Plant Cell Rep. 23, 386-390 https://doi.org/10.1007/s00299-004-0825-8
  5. Erin, A. N., Skrypin, V. V. and Kagan, V. E. (1985) Formation of alpha-tocopherol complexes with fatty acids Nature of complexes. Biochim. Biophys. Acta 815, 209-214 https://doi.org/10.1016/0005-2736(85)90291-3
  6. Hwang, S. K. and Kim, Y. M. (2000) A simple and reliable method for preparation of cross-contamination free plant genomic DNA for PCR-based detection of transgenes. J. Biochem. Mol. Biol. 33(6), 537-546
  7. Hwang, S. K. and Hwang, Y. S. (2000) Molecular cloning and functional expression of Perilla frutescens 3-ketoacyl-[acyl carrier protein] synthase III. Mol. Cells 10, 375-381
  8. Kim, S. K., Kim, K. H. and Hwang, Y. S. (2000) Molecular cloning and expression analysis of 3-Ketoacyl-ACP synthases in the immature seeds of Perilla frutescens. Mol. Cells 10(5), 533-539 https://doi.org/10.1007/s10059-000-0533-3
  9. Kim, J. H., Song, H. S., Jee, S. M., Ryu, T. H., Kim, D. H. and Kim, H. Y. (2005) Qualitative PCR detection of GM rices (Milyang 204 and Iksan 483) developed in Korea. J. Korean Soc. Appl. Biol. Chem. 48(4), 335-338