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RISS 인기검색어
- 검색키워드
- 저자 : 임선형(Sun Hyung Lim)이종열(Jong Yeol Lee) (검색결과 3 건)
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김진선(Jin Sun Kim),임선형(Sun Hyung Lim),김영미(Young Mi Kim),이종열(Jong Yeol Lee) 한국육종학회 2018 한국육종학회지 Vol.50 No.3
Promoters are essential regulatory elements for efficiently expressing a gene of interest in a target tissue of a organism. Therefore, the identification of a suitable promoter is important in plant biotechnology. In this study, four promoters were selected and identified to be constitutively or tissue-specifically expressed in Brassica rapa bacterial artificial chromosome (BAC) clones using the results of transcriptomic analysis of Brassica rapa and open-source database information on Arabidopsis thaliana. The 2 kb region of the 5′ upstream was isolated from the Brassica rapa genomic DNA for each promoter. The four promoters were then fused to β-glucuronidase (gus) and green fluorescent protein (gfp) genes, and the recombinant transgenes were introduced into Arabidopsis. As a result of histochemical GUS staining, GFP fluorescence and RT-PCR, the gus gene was observed to be expressed constitutively in all tissues using all four promoters. A GUS activity assay using fluorescent 4-MUG revealed that the BR11 promoter showed similar activity to the CaMV35S promoter, while the BR4, BR15, and BR16 promoters showed 1.5, 4, and 18-fold higher activities than the CaMV35S promoter, respectively. These results indicate that these four promoters could be used to incorporate useful genes with enhanced function into crops of interest.
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김진선(Jin Sun Kim),임선형(Sun Hyung Lim),강상호(Sang Ho Kang),김영미(Young Mi Kim),이종열(Jong Yeol Lee) 한국육종학회 2018 한국육종학회지 Vol.50 No.1
목표유전자를 목적 조직에 효율적으로 발현 시키기 위해 조직 특이적 프로모터를 동정하고 적용하는 것은 생명공학을 활용한 작물형질개량 연구의 주요 핵심요소 중 하나이다. 애기장대의 전사체 분석과 공개된 생명정보데이터를 이용하여 뿌리 특이적으로 발현되는 프로모터 7종을 선발하였다. 애기장대 genomic DNA로부터 해당 유전자 CDS의 5‘ 상류 2kb 영역을 PCR 증폭하고 Gateway cloning system을 이용하여 프로모터 부위를 β-glucuronidase(gus)와 green fluorescent protein(gfp) 유전자가 연결된 식물형질전환용 프로모터::리포터 운반체인 pBGWFS7에 클로닝하였다. 제작된 운반체들을 각각 Agrobacterium tumefaciens를 이용하여 애기장대에 형질전환한 후 T₂ 종자까지 선발, 확보 한 후, T₂ 종자를 발아시킨 후 유식물체부터 성숙식물체까지 잎, 줄기, 뿌리, 꽃, 꼬투리 등 각 기관별로 GUS 염색, RT-PCR, GFP 형광을 뿌리 조직에서 분석하였다. 그 결과 5종의 프로모터가 CaMV35S 프로모터에 비해 뿌리조직에서 강한 발현 유도성을 확인하였다. 이들 뿌리 특이 프로모터가 선충 피해가 큰 작물인 토마토 선충 저항성 작물 육성에의 적용성을 검정하기 위해 토마토 형질전환을 수행한 결과, 2종의 프로모터가 토마토 뿌리에서 특이적으로 활성을 보여주었고, 이는 실제 타 작물에서 도 적용 가능함을 시사한다. To identify and apply tissue-specific promoters is one of the major challenges in plants genetic engineering for optimizing efficient expression of interest genes in appropriate tissues. In this research, open-source database information of Arabidopsis thaliana was adapted to determine root-specific expressed promoter region. A total seven sequences that might function as a root-specific promoter element were initially isolated from Arabidopsis genomic DNA. Then seven promoters were cloned into pBGWFS7 in which β-glucuronidase (gus) and green fluorescent protein (gfp) genes were linked. The GUS activities were measured in different tissues of transgenic Arabidopsis by both histochemical GUS staining and fluorescent 4 methylumbelliferyl β-D-glucuronide (MUG) assay. To confirm root-specific expression, GFP-confocal microscope analysis was conducted in Arabidopsis transgenic plant. As a result, the five promoters showed strong GUS activity in the root tissue as compared with the CaMV35S promoter. To test crop application availability as a root-specific promoter, seven promoters were introduced into tomato plants and confirmed transient expression using Agrobacterium rhizogenesis ARqua1 root-nodule inducible strain. Two promoters showed that gus genes were specifically expressed in roots of transgenic tomato plant. Taken together, the novel seven promoters showed specific activity in root suggesting that it is applicable in crop improvement.
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밀 반수체 계통에서 오메가-5 글리아딘 돌연변이의 선발 및 동정
장유란(You Ran Jang),강천식(Chon Sik Kang),임선형(Sun Hyung Lim)이종열(Jong Yeol Lee) 한국육종학회 2018 한국육종학회지 Vol.50 No.3
Gliadin proteins are a major component of gluten proteins and important determinants of bread-making quality by conferring the viscosity and extensibility of dough, but also present significant health problems for consumers with wheat-related diseases like celiac disease or wheat allergies. In order to solve this problem, we conducted RP-HPLC analysis to profile gliadin fractions for screening the mutants deficient in gliadins from 122 wheat doubled-haploid (DH) lines cultivated by the National Institute of Crop Science. Comparing the RP-HPLC chromatogram of 122 DH lines with those of the respective parents, we found that some peaks of omega-5 gliadin were not present in 28 DH lines. Further analysis using SDS-PAGE and A-PAGE showed that the omega-5 gliadin in the parental varieties had two to three bands, but only one band in the absent 28 DH lines. The relative expression levels of all gliadin groups in the parental and mutant lines were also examined by RP-HPLC. Our study contributes to establishing a method for the rapid screening and identification of mutants missing gliadins as major epitopes of wheat-related disease in many wheat genetic resources and breeding lines as valuable information to other researchers.
KCI
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