생명과학회지 (Journal of Life Science)

  • 제28권7호
  • /
  • Pages.835-840
  • /
  • 2018
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

AtCYP78A7 과발현 환경스트레스 내성 형질전환 벼의 단백질 진단 키트 개발

Development of a Kit for Diagnosing AtCYP78A7 Protein in Abiotic-tolerant Transgenic Rice Overexpressing AtCYP78A7

  • 남경희 (국립생태원 생태보전연구실) ;
  • 박정호 (한국생명공학연구원 바이오평가센터) ;
  • 백인순 (한국생명공학연구원 바이오평가센터) ;
  • 김호방 ((주)바이오메딕 생명과학연구소) ;
  • 김창기 (한국생명공학연구원 바이오평가센터)
  • Nam, Kyong-Hee (Division of Ecological Conservation, National Institute of Ecology) ;
  • Park, Jung-Ho (Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Pack, In-Soon (Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Ho Bang (Life Sciences Research Institute, Biomedic Co., Ltd.) ;
  • Kim, Chang-Gi (Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology)
  • 투고 : 2018.02.26
  • 심사 : 2018.07.05
  • 발행 : 2018.07.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

본 연구는 시토크롬 P450 단백질을 암호화하는 애기장대 유래의 AtCYP78A7을 과발현하는 형질전환 식물체로부터 AtCYP78A7 단백질을 특이적으로 인식하는 단일큰론 항체의 제조와 그 항체를 AtCYP78A7 단백질과 접촉시켜 항원-항체 복합체 형성을 검출함으로써 AtCYP78A7 단백질을 효소면역학적(ELISA) 방법으로 검출하는 진단 키트를 개발하기 위하여 수행하였다. 재조합한 GST-AtCYP78A7 단백질을 항원으로 사용하여 단일클론 항체를 분비하는 융합세포주를 제조한 후 비오틴화 및 페어링 테스트를 통해 포획항체와 검출항체를 선정하였으며, GST-AtCYP78A7 정제 단백질을 기준으로 일품벼, 화영벼, AtCYP78A7 과발현 벼(10B-5, 18A-4)의 용해물을 검출항원으로 사용하여 product test를 진행하였다. 그 결과 AtCYP78A7 단백질에 특이적으로 결합하는 4개의 단클론 항체(mAb 6A7, mAb 4C2, mAb 11H6, mAb 7E8)를 생산하였고, 포획항체 mAb 4C2와 검출항체 mAb 7E8-biotin의 조합으로 ELISA 키트를 개발하였다. 개발된 ELISA 키트를 이용한 벼 시료의 분석 결과 AtCYP78A7 과발현 벼는 전체 단백질 대비 AtCYP78A7 단백질의 비율이 0.1% 이상인 양성으로, 일품벼와 화영벼는 0.1% 미만인 음성으로 나타나 키트를 이용한 AtCYP78A7 단백질의 검출이 가능하였으며, 따라서 본 키트는 향후 AtCYP78A7를 과발현하는 형질전환 작물을 대상으로 하는 환경 모니터링 또는 인체 위해성 평가에 유용하게 활용될 수 있을 것으로 사료된다.

Quantitative determination of the protein expression levels is one of the most important parts in assessment of the safety of foods derived from genetically modified (GM) crops. Overexpression of AtCYP78A7, a gene encoding cytochrome P450 protein, has been reported to improve tolerance to abiotic stress, such as drought and salt stress, in transgenic rice (Oryza sativa L.). In the present study, an enzyme-linked immunosorbent assay (ELISA) kit for diagnosing AtCYP78A7 protein including AtCYP78A7-specific monoclonal antibody was developed. GST-AtCYP78A7 recombinant protein was induced and purified by affinity column. Four monoclonal antibodies (mAb 6A7, mAb 4C2, mAb 11H6, and mAb 7E8) against recombinant protein were also produced and biotinylated with avidin-HRP. After pairing test using GST-AtCYP78A7 protein and lysate of rice samples, mAb 4C2 and mAb 7E8 were selected as a capture antibody and a detecting antibody, respectively, for ELISA kit. Product test using rice samples indicated that percentages of detected protein in total protein were greater than 0.1% in AtCYP78A7-overexpressing transgenic rice (Line 10B-5 and 18A-4), whereas those in negative control non-transgenic rice (Ilpum and Hwayoung) were less than 0.1%. The ELISA kit developed in this study can be useful for the rapid detection and safety assessment of transgenic rice overexpressing AtCYP78A7.

키워드

참고문헌

  1. Ahmed, F. E. 2002. Detection of genetically modified organisms in foods. Trends Biotech. 20, 215-223. https://doi.org/10.1016/S0167-7799(01)01920-5
  2. Anklam, E., Gadani, F., Heinze, P., Pijnenburg, H. and Eede, G. V. D. 2002. Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. Eur. Food Res. Technol. 214, 3-26. https://doi.org/10.1007/s002170100415
  3. Chhapekar, S., Raghavendrarao, S., Pavan, G., Ramakrishna, C., Singh, V. K., Phanindra, M. L. V., Dhandapani, G., Sreevathsa, R. and Kumar, P. A. 2015. Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosate. Plant Cell Rep. 34, 721-731. https://doi.org/10.1007/s00299-014-1732-2
  4. Head, G., Brown, C. R., Groth, M. E. and Duan, J. J. 2001. Cry1Ab protein levels in phytophagous insects feeding on transgenic corn: implications for secondary exposure risk assessment. Entomol. Exp. Appl. 99, 37-45. https://doi.org/10.1046/j.1570-7458.2001.00799.x
  5. Ito, T. and Meyerowitz, E. M. 2000. Overexpression of a gene encoding a cytochrome P450, CYP78A9, induced large and seedless fruit in Arabidopsis. Plant Cell 12, 1541-1550. https://doi.org/10.1105/tpc.12.9.1541
  6. Kim, H. B. and Choi, S. B. 2012. Cytochrome P450 gene for increasing seed size or water stress resistance of plant. US Patent 8153862 B2.
  7. Kim, H. J., Lee, S. M., Kim, J. K., Ryu, T. H., Suh, S. C., and Cho, H. S. 2010. Expresseion of PAT and NPTII proteins during the developmental stages of a genetically modified pepper developed in Korea. J. Agric. Food Chem. 58, 10906-10910. https://doi.org/10.1021/jf1026907
  8. Korea Biosafety Clearing House. 2017. Biosafety White Paper 2017. Korea Biosafety Clearing House, Daejeon.
  9. Marx, U., Embleton, M. J., Fischer, R., Gruber, F. P., Hansson, U., Heuer, J., de Leeuw, W. A., Logtenberg, T., Merz, W., Portetelle, D., Romette, J. and Straughan, D. W. 1997. Monoclonal antibody production. The report and recommendations of ECVAM workshop 23. ATLA 25, 121-137.
  10. Nam, K. H., Kim, D. Y., Shin, H. J., Nam, K. J., An, J. H., Pack, I. S., Park, J. H., Jeong, S. C., Kim, H. B. and Kim, C. G. 2014. Drought stress-induced compositional changes in tolerant transgenic rice and its wild type. Food Chem. 153, 145-150. https://doi.org/10.1016/j.foodchem.2013.12.051
  11. Nam, K. H., Kim, D. Y., Shin, H. J., Pack, I. S., Park, J. H., Yoon, W. K., Kim, H. B. and Kim, C. G. 2018. Safety assessment of AtCYP78A7 protein expressed in genetically modified rice tolerant to abiotic stress. Kor. J. Agric. Sci. 45, 248-257.
  12. Nam, K. H., Shin, H. J., Pack, I. S., Park, J. H., Kim, H. B. and Kim, C. G. 2015. Growth stage-based metabolite profiling of drought-tolerant transgenic rice under well-watered and deficit conditions. Plant Omics J. 8, 587-594.
  13. Nam, K. H., Shin, H. J., Pack, I. S., Park, J. H., Kim, H. B. and Kim, C. G. 2016. Metabolomic changes in grains of well-watered and drought-stressed transgenic rice. J. Sci. Food Agri. 96, 807-814. https://doi.org/10.1002/jsfa.7152
  14. Rahnama, H., Nikmard, M., Abolhasani, M., Osfoori, R., Sanjarian, F. and Habashi, A. A. 2017. Immune analysis of cry1Ab-genetically modified potato by in-silico analysis and animal model. Food Sci. Biotechnol. 26, 1437-1445. https://doi.org/10.1007/s10068-017-0181-4
  15. Rui, Y. K., Yi, G. X., Zhao, J., Wang, B. M., Li, Z. H., Zhai, Z. X., He, Z. P. and Li, Q. X. 2005. Changes of Bt toxin in the rhizosphere of transgenic Bt cotton and its influence on soil functional bacteria. World J. Microb. Biot. 21, 1279-1284. https://doi.org/10.1007/s11274-005-2303-z
  16. Schuler, M. A. 1996. Plant cytochrome P450 monooxygenases. Crit. Rev. Plant Sci. 15, 235-284. https://doi.org/10.1080/07352689609701942
  17. Su, V. and Hsu, B. D. 2010. Transient expression of the Cytochrome p450 CYP78A2 enhances anthocyanin production in flowers. Plant Mol. Biol. Rep. 28, 302-308. https://doi.org/10.1007/s11105-009-0153-9
  18. Wan, P., Zhang, Y., Wu, K. and Huang, M. 2005. Seasonal expression profiles of insecticidal protein and control efficacy against Helicoverpa armigera for Bt cotton in the Yangtze River Valley of China. J. Econ. Entomol. 98, 195-201. https://doi.org/10.1093/jee/98.1.195
  19. Wang, Y., Ke, K., Li, Y., Han, L., Liu, Y., Hua, H. and Peng, Y. 2016. Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis (Lepidoptera: Crambidae). Insect Sci. 23, 78-87. https://doi.org/10.1111/1744-7917.12178
  20. Xu, J., Wang, X. Y. and Guo, W. Z. 2015. The cytochrome P450 superfamily: Key players in plant development and defense. J. Integr. Agr. 14, 1673-1686. https://doi.org/10.1016/S2095-3119(14)60980-1