Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Development of transgenic cucumbers expressing Arabidopsis Nit gene

애기장대 Nit유전자 발현 오이 형질전환체 개발

  • Jang, Hyun A (Plant Systems Engineering Research Center, KRIBB) ;
  • Lim, Ka Min (Department of Oriental Pharmaceutical Development, Nambu University) ;
  • Kim, Hyun A (Plant Systems Engineering Research Center, KRIBB) ;
  • Park, Yeon-Il (Department of Biology, Chungnam National Univ.) ;
  • Kwon, Suk Yoon (Plant Systems Engineering Research Center, KRIBB) ;
  • Choi, Pil Son (Department of Oriental Pharmaceutical Development, Nambu University)
  • 장현아 (한국생명공학연구원 식물시스템공학연구센터) ;
  • 임가민 (남부대학교 한방제약개발학과 약용식물형질전환연구소) ;
  • 김현아 (한국생명공학연구원 식물시스템공학연구센터) ;
  • 박연일 (충남대학교 생물학과) ;
  • 권석윤 (한국생명공학연구원 식물시스템공학연구센터) ;
  • 최필선 (남부대학교 한방제약개발학과 약용식물형질전환연구소)
  • Received : 2013.10.24
  • Accepted : 2013.11.10
  • Published : 2013.12.31
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Abstract

To produce transgenic cucumber expressing Nit gene coffering abiotic resistance, the cotyledonary-node explants of cucumber (cv. Eunsung) were inoculated with A. tumefaciens transformed with pPZP211 or pCAMBIA2300 carrying Nit gene, that has cis-acting element involved in resistance to various abiotic environmental stresses. After co-cultivation, the procedures of selection, shoot initiation, shoot elongation, and plant regeneration were followed by cotyledonary-node transformation method (CTM, Jang et al. 2011). The putative transgenic plants were selected when shoots were grown to a length greater than 3 cm from the cotyledonary-node explants on selection medium supplemented with 100 mg/L paromomycin as a selectable agent. The confirmation of transgenic cucumber was based on the genomic PCR, Southern blot analysis, RT-PCR, and Northern blot analysis. A 105 shoots (4.12%) selected from the selection mediums were obtained from 2,547 explants inoculated. Of them, putative transgenic plants were only confirmed with 45 plants (1.77%) by genomic PCR analysis. Transgenic plants showed that the Nit genes integrated into each genome of 39 plants (1.53%) by Southern blot analysis, and the expression of gene integrated into cucumber genome was only confirmed at 6 plants (0.24%) by RT-PCR and Northern blot analysis. These results lead us to speculate that the genes were successfully integrated and expressed in each genome of transgenic cucumber.

환경스트레스 저항성 오이 형질전환체 생산을 위해서 오이 "Eunsung" 품종의 자엽절 절편을 Nit유전자를 포함하는 pPZP211와 pCAMBIA2300 발현벡터로 각각 형질전환된 Agrobacterium과 공동 배양하였다. 공동배양 후 형질전환체 선발, 형질전환체 유도, 신장, 유식물체 생산 등은 자엽절 절편을 이용하는 CTM방법(Jang et al. 2011)에 따라 수행하였다. 발현벡터에 따라 선발배지에 100 mg/L paromomycin을 첨가하여 선발과정을 거쳤으며, 선발배지에서 3 cm크기의 shoot를 유도한 후 PCR, Southern, RT-PCR 및 Northern분석을 통해 형질전환 여부를 확인하였다. 공동배양 한 2,547개의 자엽절 절편으로부터 105개체(4.12%)가 선발배지로부터 얻어졌으며, 그들 중 45개체(1.77%)만이 Nit유전자의 PCR product를 얻을 수 있었다. 오이 genome에 Nit유전자의 도입여부를 확인하기 위하여 45개체에 대한 Southern분석을 수행한 결과 각각 39개체(1.53%)서 확인할 수 있었으며, 이중 오직 6개체(0.24%)에서만 Nit유전자가 안정적으로 발현되고 있음을 RT-PCR과 Northern분석을 통해 확인하였다. 이러한 결과는 Nit유전자가 오이 genome에 안정적으로 도입 및 발현되고 있음을 보여 주고 있음을 알 수 있었다.

Keywords

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