Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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The Optimization for Functional Expression of Arabidopsis Thaliana AtPIP2-1 in Xenopus laevis Oocyte

Xenopus oocyte에서 애기장대 AtPIP2-1 활성측정을 위한 발현 최적화 조건 규명

  • Kim, Hyun-Mi (Bio-crop Development Division, National Academy of Agricultural Science, Rural Development Administration) ;
  • Hwang, Hyun-Sik (Bio-crop Development Division, National Academy of Agricultural Science, Rural Development Administration) ;
  • Lee, Suk-Chan (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Jo, Su-Hyun (Department of Physiology, Kangwon National University College of Medicine) ;
  • Kim, Beom-Gi (Bio-crop Development Division, National Academy of Agricultural Science, Rural Development Administration)
  • 김현미 (농촌진흥청 국립농업과학원 농업생명자원부 신작물개발과) ;
  • 황현식 (농촌진흥청 국립농업과학원 농업생명자원부 신작물개발과) ;
  • 이석찬 (성균관대학교 유전공학과) ;
  • 조수현 (강원대학교 의학전문대학원 생리학교실) ;
  • 김범기 (농촌진흥청 국립농업과학원 농업생명자원부 신작물개발과)
  • Received : 2010.09.09
  • Accepted : 2010.10.15
  • Published : 2010.12.31
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Abstract

We confirmed the hypo-osmotic shock strengths and duration, different type of vectors, and subcelluar localization to identify the optimum analysis condition of plant aquaporin activity in Xenopus ooctye using Arabidopsis thaliana AtPIP2-1 gene. Six minutes and 1/5ND buffer hypoosmotic shock treatment was the best condition to show the maximum swelling of Xenopus oocytes where AtPIP2-1 was expressed using pcDNA3.1 vector. AtPIP2-1 protein was expressed more efficiently in pGEMHE vector which has 5' and 3' UTR (untranslation region) of Xenopus ${\beta}$-GLOBIN gene in multiple cloning site than in pcDNA3.1 vector. Also green fluorescence of GFP fused to AtPIP2-1 was detected onto oocyte plasmamembrane where is the proper subcellular localization target of AtPIP2-1.

Xenopus oocyte을 이용하여 식물 aquaporin 단백질의 물 흡수 활성을 측정하기 위한 최적의 조건을 확립하기 위하여 애기장대의 AtPIP2-1유전자를 클로닝하여 cRNA 제작용 vector, buffer osmolarity, hypoosmotic shock 처리시간, 발현 단백질의 localization등을 검토한 결과, Xenopus ${\beta}$-globin 유전자의 5'과 3' UTR(untranlation region)'염기서열을 갖고 있는 pGEMHE vector가 단백질 생산에 더욱 효과적이며, 이 vector를 시용하였을 경우 hypoosomotic stress는 1/2ND buffer에서 6분간 처리시 가장 큰 차이를 볼 수 있었으며, 애기장대 AtPIP2-1단백질과 GFP를 결합시켜 발현시킬 경우 GFP가 plasmamembrane에 위치하는 것을 보아 올바른 subcelluar localization이 이루어지는 것을 확인할 수 있었다.

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References

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