Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Agrobacterium-Mediated Genetic Transformation of Pepper for the Development of Blight Resistant Cultivar

고추의 역병 저항성 품종 개발을 위하여 Agrobacterium tumefaciens를 이용한 elicitin 유전자 도입

  • Kwon, Tae-Ryong (Gyeoungbuk Agricutural Technology Administration) ;
  • Lee, Moon-Jung (Gyeoungbuk Agricutural Technology Administration) ;
  • Han, Jung-Sul (National Horticultural Research Institute, Rural Development Administration) ;
  • Shin, Dong-Hyun (Department of Agronomy, College of Agriculture and Life Sciences, Kyungpook National University) ;
  • Oh, Jung-Youl (Department of Horticulture, Sangju National University) ;
  • Kim, Kyung-Min (Department of Horticulture, Sangju National University) ;
  • Kim, Chang-Kil (Department of Horticulture, Sangju National University)
  • Published : 2007.03.31
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Abstract

The study was carried out to develop transformants resisting to Phyophthora blight disease in the domestic pepper cultivar Subicho. In transforming of syn600 promoter with elicitin gene using Agrobacterium (LBA4404/pBI101 syn600-syn${\alpha}$-elicitin) to cotyledons of pepper, rate of shoot formation in 'Subicho' was 11.1% in medium containing 3 mg/L zeatin and 0.05 mg/L NAA, and also 12.8% in medium containing combination of 4 mg/L zeatin and 0.05 mg/L MAA. For PCR reaction using elicitin gene primer of transformants regenerated from cotyledons, we detected a specific band of 536 bp, and also showed strong signal at position of 536 bp in accordance with NPTII gene used as probe in Southern blot. Transformants pepper shown resistance to blight fungus was inoculated to seedlings of the $T_{1}\;and\;T_{2}$ transformants by concentration (density: zoo spore $10^{3}/mL$).

고추 형질전환은 Agrobacterium (LBA4404/pBI101 cyc600-syna-elicitin)을 이용한 cyc600 promoter에 구축된 elicitin 유전자의 형질전환시 shoot의 형성율은 수비초의 경우 3 mg/L zeatin과 0.05 mg/L NAA 함유 배지에서 11.1%, 4 mg/L zeatin과 0.05 mg/L NAA 함유 배지에서 12.8%였다. 전배양 3일, 공동배양 $3{\sim}4$일에서 재분화율이 높았다. 자엽으로부터 재분화된 형질전환체의 NPTII 유전자의 primer를 이용한 PCR 반응에서 형질전환된 재분화 식물체는 536 bp의 밴드를 확인하였다. Membrane에 blot하여 NPTII gene을 probe로 사용하여 Southern blot 분석에서 고추형질전환 식물체는 536 bp 부위에 강한 signal을 보였다. elicitin 유전자를 이용한 역병 저항성 형질전환체 수비초 $T_{0}$세대의 생육은 계통간에 다소 차이는 보였고, 계통 모두 생육은 저조한 반면 개화 및 수정 등의 임성은 정상적이었다. 자식을 통해 채종한 $T_{1}$ 식물체의 유전자 도입을 확인하기 위하여 PCR을 수행한 결과 $T_{0}$ 식물체 $S1{\sim}S5$ 계통에서 elicitin 밴드가 나타나 형질전환체임을 확인하였고, $T_{1}$식물체인 S1-1 등 7계통에서 elicitin 밴드가 나타났고, S1-2 등 4계통에서는 밴드가 나타나지 않아 형질전환체가 후대에서 분리가 일어남을 확인할 수 있었다. Syn ${\alpha}$ clone을 이용하여 Southern blot analysis를 한 결과 band가 나타난 300 bp 정도의 위치에서 blot이 나오는 것을 관찰할 수 있었다. 위의 결과에서 cyc600 promoter-syn ${\alpha}$의 construct가 수비초의 genomic DNA에 삽입되었는 것을 확인할 수 있었다. 고추형질전환체의 유묘에 역병균을 접종한 결과 유주포자 $10^{3}$개/mL에서 형질전환체의 저항성 계통선발이 가능하였다.

Keywords

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