Horticultural Science & Technology (원예과학기술지)

Korean Society of Horticultural Science (한국원예학회)
권호 표지

Cloning and Characterization of a 5-Enolpyruvyl Shikimate 3-Phosphate Synthase (EPSPS) Gene from Korean Lawn Grass (Zoysia japonica)

들잔디 5-Enolpyruvyl Shikimate 3-Phosphate Synthase(EPSPS) 유전자 클로닝 및 특성

  • Lee, Hye-Jung (Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute) ;
  • Lee, Geung-Joo (Department of Horticulture, Mokpo National University) ;
  • Kim, Dong-Sub (Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute) ;
  • Kim, Jin-Beak (Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute) ;
  • Ku, Ja-Hyeong (Department of Horticulture, Chungnam National University) ;
  • Kang, Si-Yong (Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute)
  • 이혜정 (한국원자력연구원 정읍 방사선과학연구소) ;
  • 이긍주 (목포대학교 원예과학과) ;
  • 김동섭 (한국원자력연구원 정읍 방사선과학연구소) ;
  • 김진백 (한국원자력연구원 정읍 방사선과학연구소) ;
  • 구자형 (충남대학교 원예학과) ;
  • 강시용 (한국원자력연구원 정읍 방사선과학연구소)
  • Received : 2010.02.12
  • Accepted : 2010.04.09
  • Published : 2010.08.31
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Abstract

This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$ $japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.

본 연구에서는 들잔디와 돌연변이체에서 Glyphosate 내성 관련 유전자인 EPSPS를 코딩하는 cDNA를 분리하여, 시퀸스 비교분석과 발현 양상 차이 등에 관하여 조사하였다. 5'/3' RACE를 통하여 밝혀진 EPSPS의 cDNA는 각각 1176bp의 open reading frame으로 이루어져 있으며 391개의 아미노산을 코딩하고 있었다. 이는 보고된 다른 EPSPS 유전자들과 높은 유사성을 가지고 있다. Genomic southern 결과 들잔디 내에 EPSPS 유전자는 단일copy로 존재하였다. Wild type과 제초제 내성 변이체의 EPSPS 유전자는 6개의 아미노산 시퀀스의 차이를 보였으며 기존에 보고된 EPSPS active site에서 시퀀스의 차이를 나타냈다. 한편 glyphosate의 독성기작의 하나인 EPSPS 효소활성 저해 작용을 알아본 결과, 내성 개체에서 높은(1.5배 이상) EPSPS 활성을 확인할 수 있었다. Northern 분석과 RT-PCR로 제초제 처리 시간에 따른 EPSPS의 발현량을 살펴본 결과, 제초제 처리 후 시간이 경과할수록 발현량이 증가하다가 7일 이후에는 감소하였고, wild type에 비해 glyphosate 내성 선발개체에서 전체적인 발현량이 높음을 알 수 있었다. 본 연구 결과 선발된 glyphosate 내성 돌연변이체는 높은 EPSPS 효소활성 증가 및 EPSPS active site의 아미노산 시퀀스 변화를 통해 원할하고 지속적인 방향족 아미노산의 합성이 가능한 것으로 보여졌다. 본 실험을 통해 얻어진 glyphosate 내성 잔디 돌연변이체와 방법은 앞으로 유용한 제초제 저항성 돌연변이 품종개발 연구에 활용될 수 있을 것으로 기대된다.

Keywords

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