Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Screening of salt-tolerance plants using transgenic Arabidopsis that express a salt cress cDNA library

Salt cress 유전자의 형질전환을 통한 내염성 식물체 선별

  • Baek, Dongwon (Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University) ;
  • Choi, Wonkyun (Bureau of Ecological Conservation Research, National Institute of Ecology) ;
  • Kang, Songhwa (Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University) ;
  • Shin, Gilok (Division of Applied Life Science (BK21plus program), Gyeongsang National University) ;
  • Park, Su Jung (Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University) ;
  • Kim, Chanmin (Division of Applied Life Science (BK21plus program), Gyeongsang National University) ;
  • Park, Hyeong Cheol (Bureau of Ecological Conservation Research, National Institute of Ecology) ;
  • Yun, Dae-Jin (Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University)
  • 백동원 (경상대학교 식물생명공학연구소) ;
  • 최원균 (국립생태원 생태보전연구본부) ;
  • 강송화 (경상대학교 식물생명공학연구소) ;
  • 신길옥 (경상대학교 응용생명과학부 (BK21plus program)) ;
  • 박수정 (경상대학교 식물생명공학연구소) ;
  • 김찬민 (경상대학교 응용생명과학부 (BK21plus program)) ;
  • 박형철 (국립생태원 생태보전연구본부) ;
  • 윤대진 (경상대학교 식물생명공학연구소)
  • Received : 2014.04.29
  • Accepted : 2014.05.26
  • Published : 2014.06.30
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Abstract

Salt cress (Thellungiella halophila or Thellungiella parvula), species closely related to Arabidopsis thaliana, represents an extremophile adapted to harsh saline environments. To isolate salt-tolerance genes from this species, we constructed a cDNA library from roots and leaves of salt cress plants treated with 200 mM NaCl. This cDNA library was subsequently shuttled into the destination binary vector [driven by the cauliflower mosaic virus (CaMV) 35S promoter] designed for plant transformation and expression via recombination- assisted cloning. In total, 305,400 pools of transgenic BASTA-resistant lines were generated in Arabidopsis using either T. halophila or T. parvula cDNA libraries. These were used for functional screening of genes involved in salt tolerance. Among these pools, 168,500 pools were used for primary screening to date from which 7,157 lines showed apparent salt tolerant-phenotypes in the initial screen. A secondary screen has now identified 165 salt tolerant transgenic lines using 1,551 (10.6%) lines that emerged in the first screen. The prevalent phenotype in these lines includes accelerated seed germination often accompanied by faster root growth compared to WT Arabidopsis under salt stress condition. In addition, other lines showed non-typical development of stems and flowers compared to WT Arabidopsis. Based on the close relationship of the tolerant species to the target species we suggest this approach as an appropriate method for the large-scale identification of salt tolerance genes from salt cress.

식물생명공학 연구에 있어서 모델 식물인 Arabidopsis thaliana (애기장대)와 아주 유사한 염생 식물인 salt cress (Thellungiella halophila 또는 Thellungiella parvula)는 고염 스트레스에 대하여 강한 내성을 가지고 있다. 본 연구에서는 고염에 저항성을 가지는 유용유전자를 선별하기 위하여, 200 mM NaCl을 처리한 T. halophila 또는 T. parvula 식물로부터 mRNA를 분리하여 cDNA library를 작성하였다. Full length cDNA library를 Agrobacteria-methods 형질전환 방법에 필요한 binary vector pool을 작성하고 Arabidopsis에 형질전환 시켰다. 형질전환되어진 Arabidopsis를 항생제 Basta 선별을 통하여 형질전환체 pool을 구축하였다(305,400 종자). 이와 같은 방법을 통해 구축 되어진 pool중에서 168,500 종자를 이용하여 고염 스트레스 조건하에서 종자 발아율과 뿌리 생장 촉진되는 현상이 나타나는 내염성 형질전환체를 1차 선별하였다(7,157 개체; 4.24%). 1차 선별된 형질전환체 중에서 1,551개체를 이용하여 2차 선별을 수행하여 내염성 형질전환체 165 개체를 확보하였다(10.6%). 선별된 형질전환체 중 대부분 개체는 고염 스트레스에 대응하여 종자 발아 및 뿌리 생장 모두 야생형보다 촉진된 표현형을 보였다. 그 중 몇 몇의 형질전환체는 야생형에 비하여 특이적인 기관 발달 현상이 나타났다. 예를 들면, 꽃과 줄기의 발달이 야생형과는 다른 표현형을 보이는 형질전환체가 선별되었다. 이렇게 스트레스에 내성을 가지는 형질전환체로부터 유전자 분리 방법을 통하여 해당 유용유전자를 확보할 수가 있다. 본 연구에서는 향후 halopyte 식물체를 이용하여 고염 뿐만 아니라 다양한 환경스트레스(건조, 냉해, 고열, 호르몬 등) 신호전달에 관여하는 유용유전자를 보다 쉽게 확보하는 방법을 제시함으로서, 환경재해극복에 관여하는 신호전달 기작을 보다 쉽게 이해할 수 있을 것으로 사료된다.

Keywords

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