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벼 성숙종자로부터 배상체 캘러스 형성 및 식물체 재분화에 DNA methylation 억제제인 5-azacytidine의 영향
이연희,이정숙,김수윤,손성한,김둘이,윤인선,권순종,서석철 한국식물생명공학회 2008 식물생명공학회지 Vol.35 No.2
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 GeneFishigTM 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.
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연구보문 : 형질전환체에서 도입유전자 구조와 메틸화가 유전자 발현에 미치는 영향
이연희 ( Yeon Hee Lee ),손성한 ( Seong Han Sohn ),서석철 ( Seok Chul Suh ),윤인선 ( In Sun Yoon ),김둘이 ( Dool Yi Kim ) 한국국제농업개발학회 2011 韓國國際農業開發學會誌 Vol.23 No.2
35S::hpt-35S::gus-mUbi1::bar 유전자로 형질전환된 벼에서 세대진전을 통한 유전자 구조 및 발현양상을 분석하였다. 1. 하이그로마이신 저항성을 나타낸 형질전환 벼(T0)에서 제초제 감수성을 나타낸 개체는 bar 유전자가 존재하지 않았거나 mUbi1 프로모터의 불완전한 삽입으로 인한 것을 알 수 있었다. GUS 발현은 도입 유전자의 copy 수에 따라 상당한 변이가 있었으며 3 copy 이상 도입된 형질전환체에서는 발현이 약하거나 silencing 현상이 일어났다. 2. 도입 유전자의 구조 및 상태를 알아보기 위하여 Hpa II와 Msp I 으로 genomic DNA를 절단 한 후 Southern 분석을 한 결과 여러 copy가 도입된 형질전환체에서 gus 및 35S 프로모터의 재배열 현상이 뚜렷하였다. 또한 일부 계통에서는 methylation 된 유전자를 확인하였고 demethylation 물질인 5-azaC 처리 후에 GUS 발현이 회복되었다. 3. T0에서 여러 copy의 유전자가 도입되어 발현이 없었던 형질전환체의 경우 후대 개체들에서 1 copy를 포함하고 유전자 발현이 정상적인 개체를 선발할 수 있었다. Transgene expression was analyzed in transgenic rice plants carrying multiple copies of TDNA(RB-35S::hpt-35S::gus-mUbi1::bar-LB) insertions. Only transgenic plants survived on the medium containing hygromycin were used for gus and bar gene expression. Herbicide susceptibility of transgenic lines was caused by incomplete integration of mUbi1::bar gene. Transgenic lines with more than three copies of T-DNA showed partial and complete silencing of gus transgene. In order to investigate the organization and methylation status of T-DNA, Southern blot analysis was carried out using two methylation sensitive enzymes, Hpa II and Msp I. Transgenic lines with multiple copies of T-DNA appeared to have some rearranged and methylated fragments of gus and 35S promoter, but not in transgenic lines with single copy. These results suggested that the methylation derived from these complicated structures of T-DNA induced high variable transgene expression among T2 and T3 progeny sublines. Therefore, in order to produce the GM crops with stable expression of target genes, the molecular characterization of T-DNA insertions should be carefully analyzed across the generation.
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