Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Analysis of junction between T-DNA and plant genome in insect resistance GM Chinese cabbage

해충저항성 GM 배추에서 T-DNA와 식물체 게놈의 인접 부위 분석

  • Lim, Sun-Hyung (Biosafety Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Park, Seung-Hye (Biosafety Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Kim, Jung-Hwan (Biosafety Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Kim, Na-Young (Biosafety Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Won, So-Youn (Biosafety Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Lee, Si-Myung (Biosafety Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Shin, Kong-Sik (Biosafety Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Woo, Hee-Jong (Biosafety Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Kim, Dong-Hern (Biosafety Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Cho, Hyun-Suk (Biosafety Division, National Institute of Agricultural Biotechnology, RDA)
  • 임선형 (농업생명공학연구원 생물안전성과) ;
  • 박승혜 (농업생명공학연구원 생물안전성과) ;
  • 김정환 (농업생명공학연구원 생물안전성과) ;
  • 김나영 (농업생명공학연구원 생물안전성과) ;
  • 원소윤 (농업생명공학연구원 생물안전성과) ;
  • 이시명 (농업생명공학연구원 생물안전성과) ;
  • 신공식 (농업생명공학연구원 생물안전성과) ;
  • 우희종 (농업생명공학연구원 생물안전성과) ;
  • 김동헌 (농업생명공학연구원 생물안전성과) ;
  • 조현석 (농업생명공학연구원 생물안전성과)
  • Published : 2008.06.30
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Abstract

The Agrobacterium-mediated transformation has been successfully used method to introduce foreign genes into some monocotyledonous as well as a large number of dicotyledonous plants genome, We developed transgenic Chinese cabbage plants with insect-resistance gene, modified CryIAc, by Agrobacterium-transformation and confirmed transgene copy number by Southern blot analysis. We confirmed that twenty-nine out of 46 transgenic Chinese cabbage plants have single copy of CryIAc. To obtain the sequences information on the transferred DNA (T-DNA) integration into plant genome, we analyzed left border (LB) flanking sequences by genome walking (GW) PCR method. Out of 46 transgenic Chinese cabbage plants examined, 37 carried the vector backbone sequences. This result indicates that the transfer of the vector backbone from the binary vectors resulted mainly from inefficient termination of LB site. Analysis of T-DNA LB flanking region of 9 transgenic Chinese cabbage plants without vector backbone revealed that all LB ends were not conserved and nucleotides up to 36bp from the LB cleavage site were deleted.

아그로박테리움을 이용한 형질전환은 대다수의 쌍자엽과 몇몇 단자엽 식물의 게놈내로 외래유전자를 도입하는 성공적인 방법이다. 해충저항성 CryIAc 유전자가 도입된 배추형 질전환체를 아그로박테리움 형질전환법을 통해 얻은 후, 도입유전자의 수를 Southern 분석을 통하여 확인하였다. 배추형질전환체 46개 중에서 29개는 1 copy의 CryIAc 유전자가 도입된 것으로 확인되었다. 식물체의 게놈내로 T-DNA 결합에 관한 정보를 얻기 위해서 LB 인접서열을 genome walking PCR 방법을 통하여 분석하였다. 46개의 배추형질전환체중에서 37개는 운반체의 backbone 염기서열을 지니는 것으로 확인되었다. 이러한 결과는 도입 운반체의 LB 지점에서 제대로 종결이 이루어지지 않아서 운반체의 backbone 염기서열이 운반된 것으로 보여진다. 운반체의 backbone 염기서열이 도입되지 않은 9개체의 배추형질전환체를 LB 인접서열을 분석한 결과, 모든 LB 부위는 절단부위가 보존되지 않았고, 절단부위에서 36bp까지도 결실이 확인되었다.

Keywords

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