Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Introduction of VP6 Gene into Potato Plant by Agrobacterium-mediated Transformation and Analysis of VP6 Expression in Transgenic Potatoes

Rotavirus VP6 유전자의 감자식물체내로의 도입과 형질전환체의 발현분석

  • 염정원 (한국생명공학연구원 식물세포공학실) ;
  • 전재흥 (한국생명공학연구원 식물세포공학실) ;
  • 정재열 (한국생명공학연구원 식물세포공학실) ;
  • 이병찬 (한국생명공학연구원 식물세포공학실) ;
  • 강원진 (한국생명공학연구원 식물세포공학실) ;
  • 김미선 (한국생명공학연구원 식물세포공학실) ;
  • 김철중 (충남대학교 수의과대학) ;
  • 정혁 (한국생명공학연구원 식물세포공학실) ;
  • 김현순 (한국생명공학연구원 식물세포공학실)
  • Published : 2002.06.01
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Abstract

A VP6 fragments was subcloned with BamHI in the binary pMBP-1 vector under Califlower Mosaic Virus (CaMV) 355 promoter and neomycin phosphotransferase II (npt II) gene. The recombinant binary vector was mobilized into Agrobacterium-tumefaciens LBA4404 by the freeze-thaw method and potato (Solanum tubensum L. cv Desiree) was transformed by modified leaf-disc cocultivation. Shoots were induced on MS medium with 0.01 mg/L NAA, 0.1 mg/L GA$_3$, 2.0 mg/L Zeatin, 100.0 mg/L kanamycin, 500.0 mg/L carbenicillin. In order to identify the copy number of VP6 into potato plant, total genomic DNA was isolated from transgenic potato and analysed by Southern blotting. Genomic DNA and total mRNA analysis demonstrated the incorporation of the foreign gene into the potato genome, as well as their transcription.

바이러스 설사병의 원인인 VP6유전자를 감자에 형질전환 시키기 위하여 CaMV 35S promoter와 kanamycin 항생제 내성을 갖는 식물발현벡터 pMBP-1에 subcloning하고, 이 재조합 벡터를 A. tumefaciens LBA4404에 도입시킨 후, freeze haw방법을 이용하여 감자에 형질전환 시켰다. 공동배양된 감자의 잎절편은 2,4-D가 2.0 mg/L첨가된 배지에서 2일간 배양 후, 0.01 mg/L NAA, 0.1 mg/L GA$_3$, 2.0 mg/L Zeatin, 100.0 mg/L kanamycin, 500.0 mg/L carbenicillin이 첨가된 선발배지에서 재분화시켰다. 이 때 유도된 신초는 100.0 mg/L의 kanamycin이 포함된 배지에 옮겨준 후, 왕성한 생육을 위해 MS 기본배지에서 다시 배양하였다. 기내배양시 외부유전자의 도입에 의한 외형적인 변화는 찾을 수 없었으며, 형질전환체는 NPT primer를 사용한 PCR방법으로 1차선별 하였다. DIG 표지된 probe를 이용하여 total RNA를 분석한 결과 개체별로 발현양의 차이는 있었으나, 95% 이상의 안정성을 보였고, genomic DNA를 추출해 Southern blot hybridization했을 경우 1~3개의 copy수를 보임으로써 형질전환 식물체에 외래유전자인 VP6유전자가 안정적으로 도입되었음이 확인되었다.

Keywords

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