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CpBV-ELP1 발현 재조합 벡큘로바이러스의 대량 증식과 파밤나방 방제 기술

Mass Production of a Recombinant Baculovirus Expressing CpBV-ELP1 and Control of the Beet Armyworm, Spodoptera exigua

  • 박아름 (안동대학교 자연과학대학 생명자원과학과) ;
  • 김용균 (안동대학교 자연과학대학 생명자원과학과)
  • Park, Arum (Department of Bioresource Sciences, College of Natural Sciences, Andong National University) ;
  • Kim, Yonggyun (Department of Bioresource Sciences, College of Natural Sciences, Andong National University)
  • 투고 : 2015.06.08
  • 심사 : 2015.08.20
  • 발행 : 2015.09.30

초록

프루텔고치벌 브라코바이러스(CpBV)는 배추좀나방(Plutella xylostella)의 어린 유충에 기생하는 프루텔고치벌(Cotesia plutellae) 공생 폴리드나바이러스이다. 일부 CpBV 유전자들은 피기생체 유충의 면역반응을 낮추는 데 중요한 역할을 담당한다. 본 연구는 기주의 면역작용을 억제하는 CpBV 유전자를 이용하여 살충력이 높은 재조합 벡큘로바이러스를 개발하는 데 응용할 목적으로 수행되었다. 기보고 자료에 기초하여 CpBV-ELP1를 발현하는 재조합 벡큘로바이러스(AcMNPV-ELP1)를 선발하여 파밤나방(Spodoptera exigua) 충체에서 증식시켰다. 충체에서 증식된 재조합바이러스들은 파밤나방 유충 한 마리 당 $5{\times}10^{10}$ polyhedral inclusion body (PIB)를 생산하였다. 배양된 AcMNPV-ELP1은 파밤나방 유충에 높은 병원성을 보였다. 특히 파밤나방 1령과 노숙령기에 높은 살충력을 나타냈다. 파밤나방 유충이 가해하는 배추에 재조합 벡큘로바이러스($5{\times}10^6PIB/mL$)를 살포한 결과 처리 7일 후에 화학 농약인 테부페노자이드 보다 높은 방제효과(약 88%)를 나타냈다. 이 결과는 충체에서 대량배양된 AcMNPV-ELP1이 높은 병원성을 나타내 새로운 미생물방제제로 응용될 수 있음을 제시한다.

Cotesia plutellae bracovirus (CpBV) is a polydnavirus symbiotic to C. plutellae parasitizing young larvae of the diamondback moth, Plutella xylostella. Several CpBV genes play important roles in suppressing immune responses of the parasitized larvae. This study tested a hypothesis that the CpBV genes inducing host immunosuppression could be applied to develop a potent recombinant baculovirus. Based on a previous study, a recombinant baculovirus expressing CpBV-ELP1 (AcMNPV-ELP1) was selected and multiplied using larvae of the beet armyworm, Spodoptera exigua. The recombinant viruses were produced in a yield of $5{\times}10^{10}$ polyhedral inclusion body (PIB)/larva. The cultured AcMNPV-ELP1 exhibited a much higher pathogenicity against S. exigua larvae. However, its insecticidal activity was varied among larval instars of S. exigua, in which first and late instars were high susceptible. Spray of the recombinant baculovirus ($5{\times}10^6PIB/mL$) exhibited higher control efficacy (${\approx}$ 88%) against S. exigua larvae infesting cabbage than a chemical insecticide, tebufenozide, at 7 days after treatment. These results indicate that AcMNPV-ELP1 mass-cultured using host insect system is highly pathogenic and can be applied to develop a novel microbial control agent.

키워드

참고문헌

  1. Bae, S. and Y. Kim (2004) Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella. Comp. Biochem. Physiol. A 138:39-44. https://doi.org/10.1016/j.cbpb.2004.02.018
  2. Barandoc, K. P. and Y. Kim (2009) A technique of segment expression and RNA interference (SERI) reveals a specific physiological function of a cysteine-rich protein gene encoded in Cotesia plutellae bracovirus. J. Microbiol. Biotechnol. 19:610-615.
  3. Bezier, A., M. Annaheim, J. Herbiniere, C. Wetterwald, G. Gyapay, S. Bernard-Samain, P. Wincker, I. Roditi, M. Heller, M. Belghazi, R. Pfister-Wilhem, G. Periquet, C. Dupuy, E. Huguet, A.N. Volkoff, B. Lanzrein and J. M. Drezen (2009) Polydnaviruses of braconid wasps derive from an ancestral nudivirus. Science 323:926-930. https://doi.org/10.1126/science.1166788
  4. Chen, Y., F. Gao, X. Ye, S. Wei, M. Shi, H. Zheng and X. Chen (2011) Deep sequencing of Cotesia vestalis bracovirus reveals the complexity of a polydnavirus genome. Virology 414:42-50. https://doi.org/10.1016/j.virol.2011.03.009
  5. Choi, J. Y., J. Y. Rho, J. N. Kang, H. J. Shim, S. D. Woo, B. R. Jin, M. S. Li and Y. H. Je (2005) Genomic segments cloning analysis of Cotesia plutellae polydnavirus using plasmid capture system. Biochem. Biophys. Res. Commun. 322: 487-493.
  6. Goh, H. K., S. G. Lee, B. P. Lee, K. M. Choi and J. H. Kim (1991) Simple mass-rearing of beet armyworm, Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae), on an artificial diet. Kor. J. Appl. Entomol. 29:180-183.
  7. Hepat, R. and Y. Kim (2011) Transient expression of a viral histone H4 inhibits expression of cellular and humoral immune-associated genes in Tribolium castaneum. Biochem. Biophys. Res. Commun. 415:279-283. https://doi.org/10.1016/j.bbrc.2011.10.040
  8. Hepat, R., J. J. Song, D. Lee and Y. Kim (2013) A viral histone H4 joins to eukaryotic nucleosomes and alters host gene expression. J. Virol. 87:11223-11230. https://doi.org/10.1128/JVI.01759-13
  9. Jia, B., Y. Liu, Y. C. Zhu, X. Liu, C. Gao and J. Shen (2008) Inheritance, fitness cost and mechanism of resistance to tebufenozide in Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae). Pest Manag. Sci. 65:996-1002.
  10. Kim, S., J. Park, D. Kim, H. Choi, S. Kim and I. Hwang (2004) Effects of different temperatures on pathogenicity of Spodoptera exigua nucleopolyhedrovirus (SeNPV). Kor. J. Appl. Entomol. 43:329-332.
  11. Kim, Y., J. Y. Choi and Y. H. Je (2007) Cotesia plutellae bracovirus genome and its function in altering insect physiology. J. Asia Pac. Entomol. 10:181-191. https://doi.org/10.1016/S1226-8615(08)60351-9
  12. Kim, Y., B. Kwon, S. Bae, J. Y. Choi and Y. H. Je (2008) Enhanced pathogenicity of baculovirus using immunosuppressive genes derived from Cotesia plutellae bracovirus. Kor. J. Pestic. Sci. 12:283-290.
  13. Kim, Y., J. Lee, S. Kang and S. Han (1997) Variation in insecticide susceptibilities of the beet armyworm, Spodoptera exigua (Hubner): esterase and acetylcholinesterase activities. Kor. J. Appl. Entomol. 36:172-178.
  14. Kim, Y., J. Lee, S. Kang and S. Han (1998) Age variation in insecticide susceptibility and biochemical changes of beet armyworm, Spodoptera exigua (Hubner). J. Asia Pac. Entomol. 1:109-113. https://doi.org/10.1016/S1226-8615(08)60012-6
  15. Kim, Y. and Y. Son (2006) Parasitism of Cotesia plutellae alters morphological and biochemical characters of diamondback moth, Plutella xylostella. J. Asia Pac. Entomol. 9:37-42. https://doi.org/10.1016/S1226-8615(08)60273-3
  16. Kwon, B. and Y. Kim (2008) Transient expression of an EP1-like gene encoded in Cotesia plutellae bracovirus suppresses the hemocyte population in the diamondback moth, Plutella xylostella. Dev. Comp. Immunol. 32:932-942. https://doi.org/10.1016/j.dci.2008.01.005
  17. Lee, S. and Y. Kim (2008) Two homologous parasitism-specific proteins encoded in Cotesia plutellae bracovirus and their expression profiles in parasitized Plutella xylostella. Arch. Insect Biochem. Physiol. 67:157-171. https://doi.org/10.1002/arch.20218
  18. Moar, W. J., M. Pusztai-Carey, H. Van Faassen, D. Bosch, R. Frutos, C. Rang, K. Luo and M. J. Adang (1995) Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae). Appl. Environ. Microbiol. 61:2086-2092.
  19. SAS Institute, Inc. (1989) SAS/STAT user's guide, Release 6.03, Ed. Cary, N.C.
  20. Strand, M. R. (2010) Polydnaviruses, In Insect Virology; Asgari, S. and K.N. Johnson (Eds.); Caister Academic Press, Norwich, pp. 171-197.
  21. Volkoff, A. N., V. Jouan, S. Urbach, S. Samain, M. Bergoin, P. Wincker, E. Demettre, F. Cousserans, B. Provost, F. Coulibaly, F. Legeai, C. Beliveau, M. Cusson, G. Gyapay and J. M. Drezen (2010) Analysis of virion structural components reveals vestiges of the ancestral ichnovirus genome. PLoS Pathog. 6:e1000923. https://doi.org/10.1371/journal.ppat.1000923
  22. Webb, B. A., N. E. Beckage, Y. Hayakawa, P. J. Krell, B. Lanzrein, D. B. Stoltz, M. R. Strand and M. D. Summers (2000) Polydnaviridae, In Virus Taxonomy; van Regenmortel, M. H. V., C. M. Faquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lennon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle and R. B. Wickner (Eds.); Academic Press, New York, pp. 253-260.
  23. Webb, B. A. and M. R. Strand (2005) The biology and genomics of polydnaviruses. In: Gilbert, L., Iatrou, K., Gill, S.S. (Eds.), Comprehensive Molecular Insect Science. Elsevier Inc., San Diego. pp. 323-360.
  24. Wing, K. D., R. A. Slawecki and G. R. Carlson (1988) RH-5849, a non-steroidal ecdysone agonist: effects on larval Lepidoptera. Science 241:470-472. https://doi.org/10.1126/science.241.4864.470
  25. Wyler, T. and B. Lanzrein (2003) Ovary development and polydnavirus morphogenesis in the parasitic wasp Chelonus inanitus. II. Ultrastructural analysis of calyx cell development, virion formation and release. J. Gen. Virol. 84, 1151-1163. https://doi.org/10.1099/vir.0.18830-0