Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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The development of transgenic maize expressing Actinobacillus pleuropneumoniae ApxIIA gene using Agrobacterium

아그로박테리움을 이용한 Actinobacillus pleuropneumoniae ApxIIA (ApxII toxin) 유전자 발현 옥수수 형질전환체 개발

  • Kim, Hyun-A (Department of Medicinal Plant Resources, Nambu University) ;
  • Yoo, Han-Sang (Department of Infectious Diseases, College of Veterinary, Seoul National University) ;
  • Yang, Moon-Sik (Division of Biological Science, Chonbuk National University) ;
  • Kwon, Suk-Yoon (Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Jin-Seog (Korea Research Institute of Chemical Technology) ;
  • Choi, Pil-Son (Department of Medicinal Plant Resources, Nambu University)
  • Received : 2010.08.18
  • Accepted : 2010.08.27
  • Published : 2010.09.30
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Abstract

To develop edible vaccines for swine, the embryogenic calli (type II) derived from HiII genotype were inoculated with A. tumefaciens strain C58C1 containing the binary vector pMYV611, 613, 616, and V621, 622 and 623 respectively. Six of those vectors carry nptII gene which confers resistance to paromomycin and apxIIA gene producing ApxII toxin which is generated in various serum types of A. pleuropneumoniae as a target gene. The 4,120 callus clones for pMYV611, 5,959 callus clones for pMYV613, 7,581 callus clones for pMYV616, 52,329 callus clones for V621, 48,948 callus clones for V622, and 56,188 callus clones for V623 were inoculated. The frequency of positive response clone was confirmed into range of 2.3% - 4.4% for each vectors by NPTII ELISA kit assay, and the selected callus clones of them were finally 3 callus clones from pMYV611 (0.07%), 4 callus clones from pMYV613 (0.07%), 2 callus clones from pMYV616 (0.03%), 51 callus clones from V621 (0.1%), 72 callus clones from V622 (0.15%), and 102 callus clones from V623 (0.18%) respectively. From the selected callus clones of each binary vector, the integration of the apxIIA gene into maize genome was detected from 2 plants of pMYV613 and 2 plants of V623 by Southern blot analysis.

돼지 흉막폐렴백신을 개발하기 위해 옥수수 HiII genotype 으로부터 유도한 type II형의 배발생캘러스를 식물발현벡터 pMYV611, pMYV613, pMYV616, V621, V622 및 V623로 형질전환시킨 Agrobacterium (C58C1)과 공동배양 하였다. 이들 식물발현벡터는 paromomycin 항생제 저항 유전자인 NPTII 선발마커와 표적 유전자로서 흉막폐렴균의 여러 가지 혈청을 생산하는 apxIIA유전자로 재조합하여 구축하였다. 식물발현벡터pMYV611, pMYV613, pMYV616, V621, V622 및V623의 경우 각각 4,120개, 5,959개, 7,581개, 52,329개, 48,948개 및 56,188개의 캘러스 클론을 Agrobacterium과 공동한 후 NPTII assay kit에 의해 nptII유전자의 발현빈도를 조사한 결과 각 벡터별로 2.3-4.4%의 캘러스 클론에서 항체결합 양성반응을 보였고, 이들 중 최종적으로 선발된 형질전환 캘러스 클론은 pMYV611에서 3개 (0.07%), pMYV613에서 4개 (0.07%), pMYV616에서 2개 (0.02%), V621에서 51개 (0.1%), V622에서 72개 (0.15%) 및 V623에서 102개 (0.18%)를 각각 얻었다. 형질전환된 캘러스 클론으로부터 재분화된 식물체에서 유전자 도입여부를 Southern 분석으로 통해 확인한 결과 pMYV613에서 2개 식물체 및 V623에서 얻은 2개 식물체에서 각각 확인되었다.

Keywords

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