Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Effects of 5-azacytidine, a DNA methylation inhibitor, on embryogenic callus formation and shoot regeneration from rice mature seeds

벼 성숙종자로부터 배상체 캘러스 형성 및 식물체 재분화에 DNA methylation 억제제인 5-azacytidine의 영향

  • Lee, Yeon-Hee (National Institute of Agricultural Biotechnology, RDA) ;
  • Lee, Jung-Sook (National Institute of Agricultural Biotechnology, RDA) ;
  • Kim, Soo-Yun (National Institute of Agricultural Biotechnology, RDA) ;
  • Sohn, Seong-Han (National Institute of Agricultural Biotechnology, RDA) ;
  • Kim, Dool-Yi (National Institute of Agricultural Biotechnology, RDA) ;
  • Yoon, In-Sun (National Institute of Agricultural Biotechnology, RDA) ;
  • Kweon, Soon-Jong (National Institute of Agricultural Biotechnology, RDA) ;
  • Suh, Seok-Chul (National Institute of Agricultural Biotechnology, RDA)
  • 이연희 (농촌진흥청 농업생명공학연구원) ;
  • 이정숙 (농촌진흥청 농업생명공학연구원) ;
  • 김수윤 (농촌진흥청 농업생명공학연구원) ;
  • 손성한 (농촌진흥청 농업생명공학연구원) ;
  • 김둘이 (농촌진흥청 농업생명공학연구원) ;
  • 윤인선 (농촌진흥청 농업생명공학연구원) ;
  • 권순종 (농촌진흥청 농업생명공학연구원) ;
  • 서석철 (농촌진흥청 농업생명공학연구원)
  • Published : 2008.06.30
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Abstract

The modification of DNA and histone plays an important role for gene expression in plant development. The objective of this research is to observe the effects of methylation on the gene expression during dedifferentiation from rice mature seeds to callus and differentiation from callus to shoots. The embryogenic callus with ability to shoot regeneration was not induced on the N6A medium supplemented with 5-azacytidine and abnormal callus with brown color was formed. When the normal rice callus was placed on the regeneration MSRA medium supplemented with 5-azacytidine, the shoot regeneration was inhibited. The results showed that 5-azacytidine, DNA demethylating agent, had negative effects on normal embryogenic callus formation and shoot regeneration. This suggested that DNA methylation of some genes was required for normal cell dedifferentiation and differentiation in tissue culture. The microarray and $GeneFishig^{TM}$ DEG screening were used to observe the gene transcript profile in callus induction and regeneration on N6A (N6 medium + 5-azaC) and MSRA (MS regeneration medium + 5-azaC). Subsets of genes were up-regulated or down-regulated in response to 5-azaC treatments. The genes related with epigenetic regulation, electron transport, nucleic acid metabolism and response to stress were up and down regulated. The different expression of some genes (germin like protein etc.) during callus induction and shoot regeneration was confirmed using RT-PCR and northern blot analysis.

DNA와 histone 단백질의 변형은 식물 발달에 상당히 중요한 역할을 하는 것으로 알려져 있다. 식물 조직 배양 및 식물 발달 단계에서 methylation의 영향을 알아보고자 벼 종자로부터 캘러스 형성 및 식물체 재분화 단계에서 demethylation 물질인 5-azacytidine을 처리하여 유전자 발현 양상을 분석하였다. 식물체로의 재분화 능력이 있는 벼 배상체 캘러스는 5-azaC가 첨가된 H6A 배지에서는 형성되지 않았으며 갈색을 띠는 캘러스가 형성되었다. 또한 정상적인 캘러스를 5-azaC가 첨가된 MSRA 재분화 배지에서 배양했을 때도 대조구와는 달리 식물체 재분화는 이루어지지 않았다. 이러한 결과는 5-azaC가 정상적인 배상체 캘러스 및 shoot 분화에 부정적인 영향을 미친다는 것을 나타냈으며 따라서 DNA methylation이 식물 조직배양에서의 정상적인 세포 dedifferentiation과 differentiation에 필수 요인이라는 것을 알 수 있었다. 벼 캘러스 형성 및 재분화 과정 동안의 methylation 영향을 알아보고자 각 단계별로 5-azaC를 처리 후 $GeneFishig^{TM}$ DEG와 DNA chip을 사용하여 유전자 발현 양상을 분석하였다. Epigenetic regulation, 전자전달, 핵산대사, 스트레스 반응에 관여하는 일부 유전자들의 발현이 증가하거나 감소하는 것을 알 수 있었다. 발현 차이가 있는 일부 유전자를 클로닝하여 확인하였고 RT-PCR 및 northern 분석으로 각 단계에서의 발현 차이를 할인하였다.

Keywords

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