Journal of the Korean Society of International Agriculture (한국국제농업개발학회지)

The Korean Society of International Agriculture (한국국제농업개발학회)
권호 표지

Detection of High ${\alpha}$-tocopherol GM Perilla Developed in Korea

알파토코페놀 고 함유 유전자변형 들깨에 대한 검정법 개발

  • 신공식 (농촌진흥청 국립농업과학원) ;
  • 우희종 (농촌진흥청 국립농업과학원) ;
  • 이기종 (농촌진흥청 국립농업과학원) ;
  • 김경환 (농촌진흥청 감사담당관실) ;
  • 권순종 (농촌진흥청 국립농업과학원) ;
  • 서석철 (농촌진흥청 국립농업과학원)
  • Received : 2010.10.22
  • Accepted : 2010.12.24
  • Published : 2010.12.30
⟨ Previous Next ⟩

Abstract

Qualitative and quantitative PCR methods were performed to examine the detection of ${\gamma}$-TMT (${\gamma}$-tocopherol methyltransferase) inserted into genetically modified (GM) perilla (Perilla frutescens) developed in Korea. Several primer pairs were prepared on introduced genes and an endogenous reference gene in perilla. Specificity of primers first was tested by the means of qualitative PCR analysis. Primer pair KASI02-1/2 was used to amplify the endogenous gene, KAS-I, and gave rise to an amplicon 195 bp. PCR amplification using the construct-specific primer pairs, TMTocs-1/2 and VicTM-1/2 also was performed for GM perilla. TMTocs-1/2 and VicTM-1/2 primers gave rise to an amplicon 191 bp and 109 bp, respectively. In contrast, no amplified product was observed when DNA samples from 6 different plants and 3 GM crops were used as templates. For quantitative detection, test samples containing 0.3, 1, and 1.5% genetically modified perilla were measured by real-time PCR using the plasmid DNA, pKAViTM as standard material. This result showed real-time PCR method was applicable to detect GM perilla quantitatively.

알파토코페놀을 증가시키기 위해서 ${\gamma}$-TMT(${\gamma}$-tocopherol methyltransferase) 유전자로 형질전환 된 유전자변형 들깨가 개발되었고, 도입유전자의 검출법 개발을 위하여 정성 및 정량 PCR 분석을 수행하였다. 도입유전자 및 들깨 내재유전자를 바탕으로 하여 몇 개의 검출 primer쌍을 제조하였으며, 정성 PCR 방법으로 primer의 특이성을 조사하였다. 들깨 내재 유전자, KAS-I의 증폭을 위해서 KASI02-1/2 primer를 사용하여 195 bp 크기의 PCR 증폭산물을 얻었으며, 도입유전자에 대하여 구조 특이적 primer, TMTocs-1/2 및 VicTM-1/2를 이용하여 유전자변형들깨를 포함한 6개 작물과 국내 개발된 3개 유전자변형 작물에 대해 PCR을 수행한 결과에서 각각 191 bp 및 109 bp의 PCR 산물이 유전자변형 들깨에서만 특이적으로 증폭되는 것을 확인하였다. 정량 검출을 위해서 plasmid, pKAViTM을 제조하였고, 이를 표준시료로 하여, 0.3, 1 및 1.5%로 조제된 유전자변형 들깨를 real-time PCR로 분석함으로써 유의성 있는 결과를 얻을 수 있었으며, 이의 방법이 유전자변형 들깨를 정량적으로 검정하는데 적용될 수 있음을 확인하였다.

Keywords

Acknowledgement

Supported by : 농촌진흥청

References

  1. 이기종, 권순종, 김동헌, 김명식, 이경렬, 박용환, 김정봉, 김경환, 이부영. $\alpha$-토코페롤 고 함유 형질전환 콩 및 그의 제조방법. 대한민국 특허, 1020080110858 (2008).
  2. 이병규, 김경환, 이장용, 유승희 김선림, 김율호, 안병옥, 허한순. $\alpha$-토코페롤 고 함유 들깨를 생산하는 방법 및 이 방법에 의해 생산된 들깨. 대한민국 특허, 1020030077776 (2003).
  3. Huang, H.Y. and Pan, T.M. 2004. Detection of genetically modified maize MON810 and NK603 by multiplex and real-time polymerase chain reaction methods. J. Agric. Food Chem. 52 : 3264-3268. https://doi.org/10.1021/jf049944o
  4. James, D. 2009. Global status of commercialized biotech/GM crops: 2009. ISAAA Briefs No. 39-2009.
  5. Kim, J.H., Ahn, j.H., Song, H.S., Kim, K.H., Kim, D.H. and Kim, H.Y. 2006. Qualitative PCR detection of vitamin E-enriched GM perilla. J. Korean Soc. Appl. Biol. Chem. 49 : 192-195.
  6. Kim, K.H., Hwang, S.K., Kim, D.H., Lee, J.Y., Kim, Y.H. and Hwang, Y.S. 2002. Molecular cloning and characterization of - tocopherol methyltransferase cDNA from Perilla frutescens. J Korean Soc Agric Chem Btiotechnol 45 : 203-206.
  7. Kuribara, H., shindo, Y., Matsuoka, T., Takubo, K., Futo, S., Aoki, N., Hirao, T., Akiyama, H., Goda, Y., Toyoda, M. and Hino, A. 2002. Novel reference molecules for quantitation of genetically modified maize and soybean. J. AOAC Int. 85 : 1077-1089.
  8. Lee, S.h., Kong, S.H., Park, Y.H., Min, D.M. and Kim, Y.M. 2006. Quantitative analysis of two genetically modified maize lines by real-time PCR. J. Microbiol. Biotechnol. 16 : 205-211.
  9. Min, D.M., Kim, M.Y., Jung, S.I., Heo, M.S., Kim, J.K. and Kim, H.Y. 2004. Quantitative analysis of genetically modified soybean in processed foods using real-time PCR. Korean J. Food Sci. Technol. 36:723-727.
  10. Pan, A., Yang, L., Xu, S., Yin, C., Zhang, K., Wang, Z. and Zhang, D. 2006. Event-specific qualitative and quantitative PCR detection of MON863 maize based upon the 3'-transgene integration sequence. J Cereal Sci 43 : 250-257. https://doi.org/10.1016/j.jcs.2005.10.003
  11. Rho, J.K., Lee, T., Jung, S.I., Kim, T.S., Park, Y.H. and Kim, Y.M. 2004. Qualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes. J Agric Food Chem 52 : 3269-3274. https://doi.org/10.1021/jf0499020
  12. Shindo, H., Goda, Y., Toyoda, M. and Hino, A. 2002. Validation of real-time PCR analysis for line-specific quantitation of genetically modified maize and soybean using new reference molecules. J. AOAC Int. 85 : 1119-1126.
  13. Shin, K.S., Lee, S.M., Lim, S.H., Woo, H.J., Cho, H.S., Lee, K.R., Lee, M.C., Kweon, S.J. and Suh, S.C. 2009. Molecular biological characteristics and analysis using the specific markers of leaf folder-resistant GM rice. J. Plant Biotechnol. 36 : 115-123. https://doi.org/10.5010/JPB.2009.36.2.115
  14. Taverniers, I., Windels, P., Vaitilingom, M., Milcamps, A., Van Brckstaele, E., Van Den Eede, G. and Ee Loose, M. 2005. Event-specific plasmid standard and real-time PCR methods for transgenic Bt11, Bt176, and GA21 maize and transgenic GT73 canola. J Agric Food Chem 53 : 3041-3052. https://doi.org/10.1021/jf0483467
  15. Tavva, V.S., Kim, Y.H., Kagan I.A., Dinkins R.d. Kim, K.H. and Collins, G.B. 2007. Increased alpha-tocopherol content in soybean seed overexpressing the the Perilla frutescens gamma-tocopherol methyltransferase gene. Plant Cell Rep. 26 : 61-70.
  16. Toyota, A., Akiyama, H., Sugimura, M., Watanabe, T., Kikuchi, H., Kanamori, H., Hino, A., Esaka, M. and Maitani, T. 2006. Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard. Biosci. Biotecnol. Biochem. 70 : 821-827. https://doi.org/10.1271/bbb.70.821
  17. Woo, H.J., Lim, S.H., Lee, K.J., Won, S.Y., Kim, T.S., Cho, H.S. and Jin, Y.M. 2006. Current development status on the genetically modified crops in korea. Korean J Intl Agri 18 : 221-229.
  18. Woo, H.J., Sung, J.K., Kim, J.B., Kim, N.Y., Lee, S.M., Shin, K.S., Lim, S.H., Suh, S.C., Kim, K.H. and Cho, Y.G. 2008. Transgenic tobacco with -TMT of perilla showed increased salt resistance and altered pigment synthesis. J Plant biotechnol 35 : 329-335. https://doi.org/10.5010/JPB.2008.35.4.329