생명과학회지 (Journal of Life Science)

  • 제22권12호
  • /
  • Pages.1614-1620
  • /
  • 2012
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

Snowdrop lectin (Galanthus nivalis agglutinin: GNA) 유전자 도입에 의한 벼멸구 저항성 형질전환 벼 개발

Transgenic Rice Expressing Snowdrop Lectin (Galanthus nivalis agglutinin; GNA) Shows Resistance to Rice Brown Planthopper (Nilaparvata lugens Stål)

  • 이수인 (국립농업과학원 농업생명자원부) ;
  • 윤인선 (국립농업과학원 농업생명자원부) ;
  • 김진아 (국립농업과학원 농업생명자원부) ;
  • 홍준기 (국립농업과학원 농업생명자원부) ;
  • 박범석 (국립농업과학원 농업생명자원부) ;
  • 이연희 (국립농업과학원 농업생명자원부)
  • Lee, Soo In (Department of Agricultural Biotechnology, National Academy of Agricultural Science (NAAS), RDA) ;
  • Yoon, In Sun (Department of Agricultural Biotechnology, National Academy of Agricultural Science (NAAS), RDA) ;
  • Kim, Jin A (Department of Agricultural Biotechnology, National Academy of Agricultural Science (NAAS), RDA) ;
  • Hong, Joon Ki (Department of Agricultural Biotechnology, National Academy of Agricultural Science (NAAS), RDA) ;
  • Park, Beom-Seok (Department of Agricultural Biotechnology, National Academy of Agricultural Science (NAAS), RDA) ;
  • Lee, Yeon-Hee (Department of Agricultural Biotechnology, National Academy of Agricultural Science (NAAS), RDA)
  • 투고 : 2012.11.12
  • 심사 : 2012.11.16
  • 발행 : 2012.12.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

유전자총(Particle bombardment) 방법에 의해 snowdrop lectin (Galanthus nivalis agglutinin; GNA) 유전자가 도입된 해충저항성 형질전환 벼를 개발하였다. GNA 유전자가 도입된 많은 형질전환 식물체가 재분화되었으며, 형질전환 벼의 GNA 유전자의 integration, expression 및 inheritance는 Southern 및 western analysis분석 방법에 의해 증명되었다. 벼 genome 내 도입 유전자 수는 one 혹은 five copies이었다. 형질전환 $R_1$과 $R_2$ 식물체의 GNA 단백질 발현 분석 결과, 함량은 총 단백질 중 0.01%부터 2.0%까지 포함하고 있었다. 형질전환 식물체 중 GNA 과발현 형질전환 벼를 이용한 생물 검정 결과, 대조구와 비교하여 벼의 주요 해충인 벼멸구(Nilaparvata lugens St${\aa}$l)에 저항성을 나타내었다. 위의 실험 결과를 통해, 해충저항성 유전자인 GNA가 도입된 형질전환 벼는 해충의 증식을 억제하는데 이용될 수 있을 것이다.

Transgenic rice plants with increased resistance to rice brown planthopper (Nilaparvata lugens St${\aa}$l) were generated by particle bombardment-mediated transformation of plants with a gene encoding snowdrop lectin (Galanthus nivalis agglutinin; GNA) under control of the rice Rubisco small subunit (rbcS) promoter.. A large number of transgenic rice plants containing the GNA gene were generated. The integration, expression, and inheritance of this gene in the $R_1$ and $R_2$ generations were demonstrated by Southern and western blot analyses. The plants contained one to five copies of the transgene. The GNA protein comprised approximately 0.01-2.0% of total soluble protein in the $R_1$ and $R_2$ transgenic plants. Insect bioassays and feeding studies showed that the GNA protein expressed in the $R_2$ transgenic rice plants reduced the survival of brown planthoppers. The introduction of GNA into rice plants therefore can help to control insect pests.

키워드

참고문헌

  1. Bell, H. A., Fitches, E. C., Bell, J., Edwards, J. P., Gatehouse, J. A. and Gatehouse, A. M. R. 2001. Transgenic GNA expressing potato plants augment the beneficial biocontrol of Lacanobia oleracea (Lepidoptera; Noctuidae) by the parasitoid Eulophus pennicornis (Hymenoptera; Eulophidae). Transgenic Res. 10, 35-42. https://doi.org/10.1023/A:1008923103515
  2. Bharathi, Y., Vijaya Komar, S., Pasalu, I. C., Balachandran, S. M., Reddy, V. D. and Rao, K. V. 2011. Pyramided rice lines harbouring Allium sativum (asal) and Galanthus nivalis (gna) lectin genes impart enhanced resistance against major sap-sucking pests. J. Biotechnol. 152, 63-71. https://doi.org/10.1016/j.jbiotec.2011.01.021
  3. Dellaporta, S. L., Wood, J. and Hicks, J. B. 1983. A plant DNA minipreparation version II. Plant Mol. Biol. Rep. 1, 19-21. https://doi.org/10.1007/BF02712670
  4. Down, R. E., Ford, L., Woodhouse, S. D., Majerus, M. E. N., Gatehouse, J. A. and Gatehouse, A. M. R. 2003. Tritrophic interactions between transgenic potato expressing snowdrop lectin. Transgenic Res. 12, 229-241. https://doi.org/10.1023/A:1022904805028
  5. Foissac, X., Thi Loc, N., Christou, P., Gatehouse, A. M. R. and Gatehouse, J. A. 2000. Resistance to green leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA). J. Insect Physiol. 46, 573-583. https://doi.org/10.1016/S0022-1910(99)00143-2
  6. Gatehouse, A. M. R., Davison, G. M., Newell, C. A., Merryweather, A., Hamiton, W. D. O. Burgess, E. P. J., Gilbert, R. J. C. and Gatehouse, J. A. 1997. Transgenic potato plants with enhanced resistance to the tomato moth Lacanobia oleracea: growth room trials. Mol. Breed 3, 49-63. https://doi.org/10.1023/A:1009600321838
  7. Gatehouse, A. M. R. and Gatehouse, J. A. 1998. Identifying proteins with insecticidal activity: use of encoding genes to produce insect resistant transgenic crops. Pest. Sci. 52, 165-175. https://doi.org/10.1002/(SICI)1096-9063(199802)52:2<165::AID-PS679>3.0.CO;2-7
  8. Haq, S. K., Muhammad, S. and Khan, R. H. 2004. Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection. Arch. Biochem. Biophy. 431, 145-159. https://doi.org/10.1016/j.abb.2004.07.022
  9. Heinrichs, E. A., Medrano, F. G. and Rapuas, H. R. 1985. Genetic Evaluation for Insect Resistance in Rice, pp. 71-170, International Rice Research Institute, Lasbanos, Philippines.
  10. Herdt, T. W. 1991. Research priorities for rice biotechnology. In Khush, G. S. and Toenniessen, G. H. (eds.), Rice Biotechnology, pp. 19-54, CAB International, Wallingford, UK.
  11. Hilder, V. A., Powell, K. S., Gatehouse, A. M. R., Gatehouse, J. A., Gatehouse, L. N., Shi, Y., Hamilton, W. D. O., Merryweather, A. Newell, C. A., Timans, J. C., Peumans, W. J., van Damme, E. and Boulter, D. 1995. Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids. Transgenic Res. 4, 18-25. https://doi.org/10.1007/BF01976497
  12. Ishii, T., Brar, D. S., Multani, D. S. and Khush, G. S. 1994. Molecular tagging of genes for brown planthopper resistance and earliness introgressed from Oryza australiensis into cultivated rice, O. sativa. Genome 37, 217-221. https://doi.org/10.1139/g94-030
  13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  14. Lee, S. I. and Lee, S.-H. 2004. Expression of phosphinothricin acetyltransferase gene in transgenic rice plants. J. Life Sci. 14, 368-373. https://doi.org/10.5352/JLS.2004.14.2.368
  15. Lee, S. I., Lee, S.-H., Koo, J. C., Chun, H. J., Lim, C. O., Mun, J. H., Song, Y. H. and Cho, M. J. 1999. Soybean Kunitz trypsin inhibitor (SKTI) confers resistance to the brown planthopper (Nilaparvata lugens Stål) in transgenic rice. Mol. Breed. 5, 1-9. https://doi.org/10.1023/A:1009660712382
  16. Lee, S. I., Shon, Y. G., Kim, C. Y., Lim, C. O., Choi, Y. J., Kim, H-I., Lee, S. Y. and Lee, S.-H. 2003. A routine system for generation of fertile transgenic rice plants using biolistic method. J. Plant Biotechnol. 5, 163-168.
  17. Ling, K. C. 1972. Rice Virus Disease, pp. 11-33, International Rice Research Institute, Lasbanos, Philippines.
  18. Miao, J., Wu, Y., Xu, W., Hu, L., Tu, Z. and Xu, Q. 2011. The impact of transgenic wheat expressing GNA (snowdrop lectin) on the aphids Sitobion avenae, Schizaphis granium, and Rhopalosiphum padi. Environ. Entomol. 40, 743-748. https://doi.org/10.1603/EN10261
  19. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  20. Nagadhara, D., Ramesh, S., Pasalu, I. C., Kondala Rao, Y., Sarma, N. P., Reddy, V. D. and Rao, K. V. 2004. Transgenic rice plants expressing the snowdrop lectin gene (gna) exhibit high-level resistance to the whitebacked planthpper (Sogatella furcifera). Theor. Appl. Genet. 109, 1399-1405. https://doi.org/10.1007/s00122-004-1750-5
  21. Powell, K. S., Gatehouse, A. M. R., Hilder, V. A. and Gatehouse, J. A. 1995. Antifeedant effects of plant lectins and an enzyme on the adult stage of the rice brown planthopper, Nilaparvata lugens. Entomol. Exp. Appl. 75, 51-59. https://doi.org/10.1111/j.1570-7458.1995.tb01909.x
  22. Pusztai, A. J. 1991. Plant Lectins. Chemistry and Pharmacology of Natural Products Series, pp. 263. Cambridge University Press, Cambridge.
  23. Rao, K. V., Rathore, K. S., Horges, T. K., Fu, X., Stoger, E., Sudhaker, D., Williams, S., Christou, P., Bharathi, M., Brown, D., Powell, K. S., Spence, J., Gatehouse, A. M. R. and Gatehouse, J. A. 1998. Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper. Plant J. 15, 469-477. https://doi.org/10.1046/j.1365-313X.1998.00226.x
  24. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  25. Schledzawaki, K. and Mendel, R. R. 1994. Quantitative transient gene expression: comparison of the promoters for maize polyubiquitin 1, rice actin 1, maize-derived EMU and CaMV 35S in cells of barley, maize and tabacco. Transgenic Res. 3, 249-255. https://doi.org/10.1007/BF02336778
  26. Schuler, T. H., Poppy, G. M., Kerry, B. P. and Denholm, I. 1998. Insect-resistant transgenic plants. Trends Biotechnol. 16, 168-175. https://doi.org/10.1016/S0167-7799(97)01171-2
  27. Sudhakar, D., Fu, X., Stoger, E., Williams, S., Spence, J., Brown, D. P., Bharathi, M., Gatehouse, J. A. and Christou, P. 1998. Expression and immunolocalization of the snowdrop lectin insecticidal protein GNA, in transgenic rice plants. Transgenic Res. 7, 371-378. https://doi.org/10.1023/A:1008856703464
  28. Stoger, E., Williams, S., Christou, P., Down, R. E. and Gatehouse, J. A. 1999. Expression of the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: effects on predation by the grain aphid Sitobion avenae. Mol. Breed. 5, 65-73. https://doi.org/10.1023/A:1009616413886
  29. Sun, X., Wu, A. and Tang, K. 2002. Transgenic rice lines with enhanced resistance to the small brown planthopper. Crop Protection 21, 511-514. https://doi.org/10.1016/S0261-2194(01)00127-2
  30. Tang, K. X., Tinjuangjun, P., Xu, Y. N., Sun, F. X., Gatehouse, J. A., Ronald, P. C. Qi, H. X., Lu, X. G., Christou, P. and Kohli, A. 1999. Partcle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests. Planta 208, 552-563. https://doi.org/10.1007/s004250050593
  31. van Damme, E. J. M., DeClerg, H., Claessens, F., Hemschoote, K., Peeters, B. and Peumans, W. 1991. Molecular cloning and characterization of multiple isoforms of the snowdrop (Galanthus nivalis L.) lectin. Planta 186, 35-43. https://doi.org/10.1007/BF00201495
  32. van Damme, E. J. M., Allen, A. K. and Peumans, W. J. 1987. Isolation and characterization of a lectin with exclusive specificity towards mannose from snowdrop lectin (Galanthus nivalis) bulbs. FEBS Lett. 215, 140-144. https://doi.org/10.1016/0014-5793(87)80129-1
  33. Wakefield, M. E., Bell, H. A., Fitches, E. C., Edwards, J. P. and Gatehouse, A. M. R. 2006. Effects of Galanthus nivalis agglutinin (GNA) expressed in tomato leaves on larvae of the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae) and the effect of GNA on the development of the endoparasitoid Meteorus gyrator (Hymenoptera: Braconidae). Bull. Entomol. Res. 96, 43-52. https://doi.org/10.1079/BER2005396
  34. Wang, Z., Zhang, K., Sun, X., Tang, K. and Zhang, J. 2005. Enhancement of resistance to aphids by introducing the snowdrop lectin gene gna into maize plants. J. Biosci. 30, 627-638. https://doi.org/10.1007/BF02703563