Korean Journal of Environmental Agriculture (한국환경농학회지)

The Korean Society of Environmental Agriculture (한국환경농학회)
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Acute Oral Toxicity of dsRNA to Honey Bee, Apis mellifera

꿀벌에 대한 dsRNA의 급성섭식독성 평가

  • Lim, Hye Song (Division of Ecological Conservatioin, Bureau of Ecological Research, National Institute of Ecology) ;
  • Jung, Young Jun (Division of Ecological Conservatioin, Bureau of Ecological Research, National Institute of Ecology) ;
  • Kim, Il Ryong (Division of Ecological Conservatioin, Bureau of Ecological Research, National Institute of Ecology) ;
  • Kim, Jin (Central Research Institute) ;
  • Ryu, Sungmin (Central Research Institute) ;
  • Kim, Banni (Graduate School of Future Convergence Technology, Hankyong National University) ;
  • Lee, Jung Ro (Division of Ecological Conservatioin, Bureau of Ecological Research, National Institute of Ecology) ;
  • Choi, Wonkyun (Division of Ecological Conservatioin, Bureau of Ecological Research, National Institute of Ecology)
  • 임혜송 (국립생태원 생태연구본부 생태보전연구실) ;
  • 정영준 (국립생태원 생태연구본부 생태보전연구실) ;
  • 김일룡 (국립생태원 생태연구본부 생태보전연구실) ;
  • 김진 (경농 중앙연구소) ;
  • 유성민 (경농 중앙연구소) ;
  • 김반니 (한경대학교 미래융합기술대학원) ;
  • 이중로 (국립생태원 생태연구본부 생태보전연구실) ;
  • 최원균 (국립생태원 생태연구본부 생태보전연구실)
  • Received : 2017.10.18
  • Accepted : 2017.11.22
  • Published : 2017.12.31
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Abstract

BACKGROUND: RNA interference (RNAi) eliminates or decreases gene expression by disrupting the target mRNA or by interfering with translation. Recently, RNAi technique was applied to generate new crop traits which provide protection against pests. To establish the environmental risk assessment protocol of RNAi LMO in lab scale, we developed dsRNA expression system using E. coli and tested acute oral toxicity assay to honey. METHOD AND RESULTS: The dsRNA expression vector, L4440, was chosen and cloned 240 bp of Snf7 and GFP gene fragment. To develop the maximum dsRNA induction condition in E. coli, we tested induction time, temperature and IPTG concentration in media. To estimate the risk assessment of dsRNA to honey bee, it has been selected and cultured with dsRNA supplement for 48 hours according to OECD guideline. As a result, the optimum condition of dsRNA induction was $37^{\circ}C$, 4 hours and 0.4 mM IPTG concentration and the difference between Snf7 and GFP dsRNA molecules from E. coli was not significant in survival and behavior to honey bee. Furthermore, blast search results indicated that effective match of predicted dsRNA fragments were not existed in honey bee genome. CONCLUSION: In this study, we developed and tested the acute oral toxicity of dsRNA using E. coli expression system to honey bee.

본 연구는 최근 RNAi 기반 LMO의 연구 개발이 활발히 진행됨에 따라 향후 이러한 기술을 이용한 LMO의 유해성 및 자연생태계 위해성평가가 필요할 때 실험실 수준에서 dsRNA를 대량으로 발현시키는 시스템을 확립하고, 수분(화분)매개 곤충인 꿀벌을 대상으로 유해성평가 시험을 수행하는 방법을 제시하고자 하였다. L4440 vector에 Snf7과 GFP 유전자를 클로닝한 plasmid를 HT115 (DE3) 대장균에 형질 전환한 후 온도, 배양시간, IPTG 농도를 각기 다르게 하여 최적의 발현조건을 탐색한 결과 $37^{\circ}C$, 0.4 mM IPTG, 4시간의 배양시간에서 가장 많은 양의 dsRNA가 발현됨을 확인하였다. 국내 외 제시된 꿀벌 위해성평가 가이드라인을 바탕으로 대장균에서 분리한 dsRNA를 꿀벌 성충에 급성섭식으로 처리한 결과 생사율과 일반중독증상에서 차이를 보이지 않는 것으로 보아 대장균으로부터 분리한 Snf7 dsRNA와 GFP dsRNA는 꿀벌 성충에 유해하지 않음을 알 수 있었다. 본 연구를 통해 dsRNA 물질의 유해성평가 및 자연생태계 위해성 평가를 위한 대량 추출 방법과 위해성평가 대상종의 사육 및 물질 처리 방법을 확립하여 향후 이뤄질 dsRNA의 꿀벌 위해성평가에 활용될 것으로 사료된다.

Keywords

References

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