Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Optimal RNA Extraction Methods and Development of Synthetic Clones for Seven Strawberry Viruses

딸기바이러스 진단을 위한 최적의 RNA 추출 방법 및 주요 7종 딸기바이 러스의 진단법 개발

  • Kwon, Sun-Jung (Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Yoon, Ju-Yeon (Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Cho, In-Sook (Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Chung, Bong-Nam (Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration)
  • 권선정 (국립원예특작과학원 원예특작환경과) ;
  • 윤주연 (국립원예특작과학원 원예특작환경과) ;
  • 조인숙 (국립원예특작과학원 원예특작환경과) ;
  • 정봉남 (국립원예특작과학원 원예특작환경과)
  • Received : 2020.08.13
  • Accepted : 2020.09.12
  • Published : 2020.09.30
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Abstract

Most strawberry viruses exist relatively low titers in tissues, and strawberry tissues include high levels of contamination by polysaccharides and phenolic compounds. These traits make the efficiency of strawberry diagnosis difficult. In this study, we tested different commercially available kits and reagents to secure optimal RNA extraction methods to determine virus detection from strawberry leaves. Total RNA was isolated from leaves of strawberry mottle virus (SMoV)-infected strawberry cultivar 'Mihong'. The efficiency of total RNA for virus diagnosis was confirmed through SMoV detection by one-step or two-step reverse transcription and polymerase chain reaction (RT-PCR). Among those, the RNeasy plant RNA kit was best to isolate RNA and the isolated RNA was good enough for further applications. To ensure a reliable detection for strawberry viruses, synthetic diagnosis clones for major seven strawberry viruses such as strawberry mild yellow edge virus, SMoV, strawberry latent ring spot virus, strawberry crinkle virus, strawberry pallidosis associated virus, strawberry vein banding virus and strawberry necrotic spot virus have been constructed. Based on the synthetic genes in each clone, primer sets for seven strawberry viruses were designed and tested an RT-PCR condition through a simultaneous application of the same annealing temperature that allowed to achieve an efficient and convenient diagnosis.

바이러스의 정확한 진단법 확립은 바이러스의 피해 및 확산을 예방하는데 매우 중요하게 작용한다. 대부분의 딸기 바이러스는 조직내에 낮은 역가로 분포하여 진단이 어렵고, 특히 딸기 조직은 다당류 및 페놀화합물의 함유가 많아 RNA 추출이 어려운 것으로 알려져 있다. 딸기 우량묘 생산에 필요한 바이러스 검정기술을 확립하기 위해 본 연구에서는 딸기 잎에서 바이러스 진단을 위해 가장 최적의 RNA 추출방법 정립을 위해 다양한 상용 키트와 시약을 이용하여 RNA 추출효율 비교하였다. 바이러스 진단을 통한 RNA 추출효율을 분석하기 위해 SMoV 감염주인 미홍 딸기 품종을 이용하여 다양한 단계에서 잎조직으로부터 RNA를 추출하고 바이러스 진단을 수행하였다. 식물 RNA 추출 방법 가운데 상업용으로 판매되는 RNeasy plant mini kit (Qiagen)를 이용하는 경우 본 연구에서 살펴본 one-step 또는 two-step RT-PCR 방법과 무관하게 SMoV의 검출이 잘 되었다. 또한, 딸기 우량묘의 바이러스 검정에 대한 신뢰있는 진단방법을 구축하기 위해 주요 딸기 바이러스인 strawberry mild yellow edge virus (SMYEV), strawberry mottle virus (SMoV), strawberry latent ringspot virus (SLRSV), strawberry crinkle virus (SCV), strawberry pallidosis associated virus (SPaV), strawberry vein banding virus (SVBV) 및 strawberry necrotic shock virus (SNSV) 7종에 대한 유전자 합성을 통해 진단클론을 제작하였다. 각 클론의 합성유전자를 기반으로 7종의 딸기바이러스 프라이머 세트를 설계하고 편리한 진단법 수행을 위해 동일한 PCR 조건을 설정하였다.

Keywords

References

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