Journal of Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Qualitative PCR Detection of Stack Gene GM Rice (LS28 X Cry1Ac) Developed in Korea

국내개발 stack gene GM 벼(LS28 X Cry1Ac)에 대한 정성 PCR 분석

  • 신공식 (농촌진흥청 국립농업과학원) ;
  • 박종현 (농촌진흥청 국립농업과학원) ;
  • 이진형 (농촌진흥청 국립농업과학원) ;
  • 이시명 (농촌진흥청 국립농업과학원) ;
  • 우희종 (농촌진흥청 국립농업과학원) ;
  • 임선형 (농촌진흥청 국립농업과학원) ;
  • 김해영 (경희대학교 생명공학원) ;
  • 서석철 (농촌진흥청 국립농업과학원) ;
  • 권순종 (농촌진흥청 국립농업과학원)
  • Published : 2009.03.31
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Abstract

For the development of qualitative PCR detection method of genetically modified (CM) rice, rice species-specific gene, OsCc-1 (rice cytochrome c gene), was selected as suitable far use as an endogenous gene in rice. The primer pair OsCytC-5'/3'with 111 bp amplicon was used for PCR amplification of the rice endogenous gene, OsCc-1 and no amplified product was observed from 8 different crops as templates. Qualitative PCR method was carried out with stack traits of L528$\times$CryIAc1 GM rice developed in Korea. For the qualitative PCRs, some primer pairs were designed with a construct-specific and event-specific type based on T-DNA and junction sequences of T-DNA in GM rice. Actck-5'/3' amplifying between actin promoter and OsCK1 gene introduced in LS28 gave rise to an amplicon 306 bp; also, CrLB-5'/3' from CryIAcl and CKRB-5'/3'amplifying the junction region of T-DNA and genome sequence from LS28 as event-specific primers gave rise to an amplicon 142 bp and 91 bp, respectively. These primer pairs for the detection of event-specific targets not produced PCR amplicons on non-CM rice and various crops in contrast to event lines. Therefore, in this study we verified that event-specific primers were effective to specifically detect stack trait lines and demonstrated that this method presented could be provided with the detection-method data for risk assessment analysis of GM rice to be commercialized.

후대교배종 CM 벼의 정성 PCR 검정방법을 개발하기 위하여, 벼의 내재유전자로써 OsCs-J(rice cytochrome c gene)을 선발하여 OsCytC-5'/3'의 primer쌍을 제작하고, 벼를 포함한 서로 다른 8개 작물에 대하여 PCR을 수행한 결과 벼에 특이적으로 증폭되는 111 bp의 반응 산물을 확인하였다. 국내 개발된 LS28$\times$CryIAc1 GM 벼의 검정 분석으로 정성 PCR 반응을 수행하였다. 정성 PCR을 위하여 GM 벼에 도입된 T-DNA 및 게놈상의 도입유전자 삽입부위의 인접서열을 바탕으로 구조 및 계통 특이적인 검정 primer 쌍을 제작하였다. ActCk-5'/3' primer 쌍을 이용하여 LS28의 T-DNA 내의 actin 프로모터와 OsCK1 유전자 사이를 증폭시켜 306 bp의 PCR 반응 산물을 얻을 수 있었으며, 또한 계통특이 primer 쌍인 CryIAc1 GM 벼유래의 CrLB-5'/3' 및 LS28 GM 벼 유래의 CKRB-5'/3'를 이용한 PCR 반응으로 각각 142 bp와 91 bp의 도입유전자의 인접서열 부위의 특이적인 증폭 산물을 확인할 수 있었다. 계통 특이적 검정을 위한 이들 개발 primer 쌍들은 event 계통과 대조적으로 non-GM 벼와 다양한 작물에 대하여 어떠한 특이적인 PCR 증폭 산물을 형성하지 않았다. 따라서 본 연구에서 계통특이 primer를 이용하여 후대교배종 GM 벼 계통, L528$\times$CryIAc1을 특이적으로 검출할 수 있음을 확인하였고, 제시된 방법이 GM 벼의 실용화를 위한 위해성평가의 검정방법 자료로 제공될 수 있음을 확인하였다.

Keywords

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