Journal of Plant Biotechnology

  • 제40권1호
  • /
  • Pages.18-26
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  • 2013
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  • 1229-2818(pISSN)
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  • 2384-1397(eISSN)
한국식물생명공학회 (The Korean Society of Plant Biotechnology)
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해충저항성 유전자변형 벼 Agb0101에 대한 PCR 검정

Qualitative and quantitative PCR detection of insect-resistant genetically modified rice Agb0101 developed in korea

  • 신공식 (국립농업과학원 생물안전성과) ;
  • 이진형 (국립농업과학원 생물안전성과) ;
  • 임명호 (국립농업과학원 생물안전성과) ;
  • 우희종 (국립농업과학원 생물안전성과) ;
  • 친양 (국립농업과학원 생물안전성과) ;
  • 서석철 (국립농업과학원 생물안전성과) ;
  • 권순종 (국립농업과학원 생물안전성과) ;
  • 조현석 (국립농업과학원 생물안전성과)
  • Shin, Kong-Sik (National Academy of Agricultural Science, Rural Development Administration) ;
  • Lee, Jin-Hyoung (National Academy of Agricultural Science, Rural Development Administration) ;
  • Lim, Myung-Ho (National Academy of Agricultural Science, Rural Development Administration) ;
  • Woo, Hee-Jong (National Academy of Agricultural Science, Rural Development Administration) ;
  • Qin, Yang (National Academy of Agricultural Science, Rural Development Administration) ;
  • Suh, Seok-Cheol (National Academy of Agricultural Science, Rural Development Administration) ;
  • Kweon, Soon-Jong (National Academy of Agricultural Science, Rural Development Administration) ;
  • Cho, Hyun-Suk (National Academy of Agricultural Science, Rural Development Administration)
  • 투고 : 2012.12.11
  • 심사 : 2012.12.13
  • 발행 : 2013.03.31
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초록

살충성 유전자 mcry1Ac1을 포함하고 있는 해충저항성 유전자변형(GM) 벼 Agb0101이 국내에서 개발되었다. 향후 Agb0101 벼의 환경방출에 따른 모니터링과 이력추적을 위해서는 신뢰성 있는 검출방법의 개발이 필요하다. 따라서, 본 연구에서 해충저항성 GM벼의 사후 안전관리를 위한 정성적 및 정량적 PCR 검정 방법을 개발하였다. 벼 녹말분지효소 유전자 RBE4를 PCR 분석의 내재유전자로 사용하였고, 이의 primer쌍 RBEgh-1/-2는 101bp의 PCR 증폭산물을 형성하였다. 정성 PCR 분석을 위해서 삽입된 T-DNA를 바탕으로 특이 primer를 제작하였고, 이벤트 특이적 검출 primer의 경우 Agb0101의 도입유전자 및 벼 염색체 DNA 사이의 5' 또는 3' 인접염기부위를 정확하게 특이적으로 PCR 증폭하였다. 반면, 대조구인 각종 작물, 국내 벼 품종 및 Agb0101과 동일 형질전환 벡터를 갖는 해충저항성 벼에서는 어떠한 PCR 증폭산물도 형성하지 않았다. 표준물질로써 내재유전자 및 이벤트 특이적 단편으로 제조된 pRBECrR을 이용한 real-time PCR 분석에 의해서 정량한계(LOQ)가 10 copies 농도의 범위인 것으로 확인되었고, 이의 유효성을 검증하기 위하여 상이한 농도의 Agb0101시료(10, 5, 3 및 1%)를 real-time PCR 분석하여 정량검정에 대한 표준편차 및 상대표준편차가 각각 0.06 ~ 0.40 및 3.80 ~ 7.01%의 낮은 범위에 포함되는 것을 확인할 수 있었다. 이들 결과로 본 연구에서 개발된 정성 및 정량 PCR 검정 방법이 해충저항성 GM벼 Agb0101의 모니터링 및 이력추적에 효과적으로 이용될 수 있을 것으로 본다.

Genetically modified (GM) rice Agb0101, which expresses the insecticidal toxin modified cry1Ac (mcry1Ac1) gene, was developed by the Rural Development Administration in Korea. To monitor the probable release of Agb0101 in the future, it is necessary to develop a reliable detection method. Here, we developed the PCR detection method for monitoring and tracing of GM rice. The primer pair (RBEgh-1/-2) from a starch branching enzyme (RBE4) gene was designed as an endogenous reference, giving rise to an expected PCR amplicon of 101 bp. For the qualitative PCR detection, construct- and event-specific primers were designed on the basis of integration sequence of T-DNA. Event-specific PCRs amplified specifically 5'- or 3'-junction region spanning the native genome DNA and the integrated gene construct, while none of amplified product was shown on crops, rice varieties, and other insect-resistant transgenic rice lines. The event-specific real-time PCR method was performed using TaqMan probe and plasmid pRBECrR containing both rice endogenous gene RBE4 sequence and 5'-junction sequence as the reference molecule. The absolute limit of quantification (LOQ) of real-time PCR was established with around 10 copies for one plasmid molecule pRBECrR. Thereafter, the different amounts of transgenic rice (1, 3, 5, and 10%, respectively) were quantified by using the established real-time PCR method, with a range below 19.55% of the accuracy expressed as bias, 0.06-0.40 of standard deviation (SD) and 3.80-7.01% of relative standard deviations (RSD), respectively. These results indicate that the qualitative and quantitative PCR methods could be used effectively to detect the event Agb0101 in monitoring and traceability.

키워드

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