• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.134-134
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• 2017

Recently, host-induced gene silencing (HIGS) system has been successfully applied into development of resistant crops against insects, fungal and viral pathogens. To test HIGS-mediated resistance in rice against rice blast fungus, Magnaporthe oryzae, we first tested possibility of movement of small non-coding RNA from rice cells to rice blast fungus. The rice blast fungus expressing GFP transgene were inoculated to transgenic rice plants ectopically expressing dsRNAi construct targeting fungal GFP gene. Expression of dsRNAi construct for GFP gene in transgenic plants significantly suppressed GFP expression in infected fungal cells indicating that small RNAs generated in plant cells can move into infected fungal cells and efficiently suppress the expression of fungal GFP gene. Consistent with these results, expression of dsRNAi constructs against 3 fungal pathogenic genes of M. oryzae in transgenic rice specifically and efficiently suppressed not only the expression of fungal pathogenic genes, but also fungal infection. The conidia of M. oryzae applied on leaf sheath of transgenic rice expressing dsRNAs against 3 fungal pathogenic genes showed abnormal development of primary hyphae and malfunction of appressorium, which is consistent with the phenotypes of corresponding fungal knock-out mutants. Taken these results together, here, we suggest a novel strategy for development of antifungal crops by means of HIGS system.

• Biomolecules & Therapeutics
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• 제22권4호
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• pp.267-274
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• 2014

Drug development and preclinical trials are challenging processes and more than 80% to 90% of drug candidates fail to gain approval from the United States Food and Drug Administration. Predictive and efficient tools are required to discover high quality targets and increase the probability of success in the process of new drug development. One such solution to the challenges faced in the development of new drugs and combination therapies is the use of low-cost and experimentally manageable in vivo animal models. Since the 1980's, scientists have been able to genetically modify the mouse genome by removing or replacing a specific gene, which has improved the identification and validation of target genes of interest. Now genetically engineered mouse models (GEMMs) are widely used and have proved to be a powerful tool in drug discovery processes. This review particularly covers recent fascinating technologies for drug discovery and preclinical trials, targeted transgenesis and RNAi mouse, including application and combination of inducible system. Improvements in technologies and the development of new GEMMs are expected to guide future applications of these models to drug discovery and preclinical trials.

• Molecules and Cells
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• 제26권4호
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• pp.344-349
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• 2008

5-Fluorouracil (5-FU), a pyrimidine antagonist, has a long history in cancer treatment. The targeted pyrimidine biosynthesis pathway includes dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU. Using Caenorhabditis elegans as a model system to study the functional and resistance mechanisms of anti-cancer drugs, we examined these two genes in order to determine the extent of molecular conservation between C. elegans and humans. Overexpression of the worm DPD and TS homologs (DPYD-1 and Y110A7A.4, respectively) suppressed germ cell death following 5-FU exposure. In addition, DPYD-1 depletion by RNAi resulted in 5-FU sensitivity, while treatment with Y110A7A.4 RNAi and 5-FU resulted in similar patterns of embryonic death. Thus, the pathway of 5-FU function appears to be highly conserved between C. elegans and humans at the molecular level.

• 한국환경농학회지
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• 제36권4호
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• pp.241-248
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• 2017

본 연구는 최근 RNAi 기반 LMO의 연구 개발이 활발히 진행됨에 따라 향후 이러한 기술을 이용한 LMO의 유해성 및 자연생태계 위해성평가가 필요할 때 실험실 수준에서 dsRNA를 대량으로 발현시키는 시스템을 확립하고, 수분(화분)매개 곤충인 꿀벌을 대상으로 유해성평가 시험을 수행하는 방법을 제시하고자 하였다. L4440 vector에 Snf7과 GFP 유전자를 클로닝한 plasmid를 HT115 (DE3) 대장균에 형질 전환한 후 온도, 배양시간, IPTG 농도를 각기 다르게 하여 최적의 발현조건을 탐색한 결과 $37^{\circ}C$, 0.4 mM IPTG, 4시간의 배양시간에서 가장 많은 양의 dsRNA가 발현됨을 확인하였다. 국내 외 제시된 꿀벌 위해성평가 가이드라인을 바탕으로 대장균에서 분리한 dsRNA를 꿀벌 성충에 급성섭식으로 처리한 결과 생사율과 일반중독증상에서 차이를 보이지 않는 것으로 보아 대장균으로부터 분리한 Snf7 dsRNA와 GFP dsRNA는 꿀벌 성충에 유해하지 않음을 알 수 있었다. 본 연구를 통해 dsRNA 물질의 유해성평가 및 자연생태계 위해성 평가를 위한 대량 추출 방법과 위해성평가 대상종의 사육 및 물질 처리 방법을 확립하여 향후 이뤄질 dsRNA의 꿀벌 위해성평가에 활용될 것으로 사료된다.

• BMB Reports
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• 제37권3호
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• pp.383-385
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• 2004

RNA interference (RNAi) is a process of sequence-specific gene silencing, which is initiated by double-stranded RNA (dsRNA). RNAi may also serve as an antiviral system in vertebrates. This study describes the inhibition of herpesvirus-6B (HHV-6B) replication by short interference RNAs (siRNAs) that are targeted to the U38 sequence that encodes DNA polymerase. When virus-infected SupT1 cells were treated by siRNA, these cells blocked the cytopathic effect (CPE) and detected the HHV-6B antibody-negative in indirect immunofluorescence assays (IFA). Our result suggests that RNAi can efficiently block Herpesvirus-6B replication.

• BMB Reports
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• 제37권1호
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• pp.107-113
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• 2004

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.

• 한국동물학회:학술대회논문집
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• 한국동물학회 2003년도 한국생물과학협회 학술발표대회
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• pp.168.3-168
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• 2003

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• 한국작물학회지
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• 제54권3호
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• pp.299-306
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• 2009

콩과식물의 뿌리혹 형성을 조절하는 신호물질의 확인을 위해 신팔달콩2호의 줄기 수액 단백질 중에서 B. japonicum USDA110의 접종 후 2.5일(DAI)에 20 kDa의 SKTI 단백질이 증가하였다가 7 DAI에는 감소되면서 6 kDa의 작은 크기의 단백질이 증가되었다. 이러한 단백질의 차등발현은 조사한 3종의 콩에서 모두 유사하게 나타났으며 특히 대원콩에서 가장 두드러졌다. Western 분석으로 7 DAI에서 증가하는 6 kDa 단백질이 SKTI 항체와 특이적 반응을 하는 것으로 확인하여 SKTI가 절단되어 생긴 펩타이드로 추정되었다. 이러한 결과를 통해 20 KDa의 SKTI단백질이 콩의 뿌리혹 착생 초기단계인 2.5 DAI에 영향을 주고, 7 DAI로 진행되면서 6 kDa의 작은 크기의 단백질로 분해되어 그 양이 감소하는 것으로 생각된다. RNAi를 이용하여 유전자 기능이 억제된 형질전환된 모상근의 뿌리혹을 실제 형질전환이 확인된 모상근에 착생된 뿌리혹의 수를 비교한 결과 비재조합 A. rhizogenes을 접종시킨 대조구에 비해 SKTI RNAi 유전자를 형질전환한 모상근에서 모상근 당 착생된 뿌리혹 수가 감소되었다. 실시간 PCR 방법으로 형질전환된 모상근의 SKTI 전사체 수준에서도 상응하는 차이를 확인하였다. 이에 정확한 기작을 알 수 없지만 SKTI유전자가 뿌리혹 형성 초기에 뿌리혹 형성과정에 직간접적으로 관련하고 있음을 확인하였다. Sesbania rostrata의 뿌리혹 발생과정의 Protease 저해제와 같이 뿌리 혹 내의 감염세포 대 비감염세포의 비율을 조절하는 SKTI 발현 억제는 이러한 균형을 교란하여 뿌리혹의 생성을 억제하는 것으로 추정된다.

• Entomological Research
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• 제48권6호
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• pp.533-539
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• 2018

The RNA interference (RNAi) has been considered as an important genetic tool and applied to develop a new living modified (LM) crop trait which is an improvement of nutrient quality or pest management. The RNAi of DvSnf7 has been used for resistance to LM maize and the Western Corn Rootworm which is a major agricultural pest for the US Corn Belt. Most of the environmental risk assessments (ERA) of double strand RNA (dsRNA) have been performed using in vitro transcript products, and not in vivo expressed product. A large amount of dsRNA was required for the acute toxicity assay of water fleas. Therefore development of massive dsRNA purification techniques is critical. Daphnia, a freshwater microcrustacean, is a model organism for studying cellular and molecular mechanism involved in life history traits and ecotoxicology. In this study, we established the massive dsRNA purification method using Escherichia coli and implemented acute toxicity assays to Daphnia magna. As a result, the present RNase A and DNase I, dsRNA was efficiently purified without any special techniques or equipment. Even though purified dsRNA existed during the acute toxicity test, lethality or abnormal behavior were not observed in D. magna. These results indicated that GFP and DvSnf7 dsRNA were not significantly affected to D. magna due to their lack of sequence matching in its genome. The purification method of dsRNA and the acute toxicity assay of water fleas using purified dsRNA would be suitable for the toxicological studies of LMOs to aquatic non-target organisms.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
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• pp.64-66
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• 2005

Photosynthetic microbes possess a wealth of photoactive proteins including chlorophyll-based pigments, phototropin-related blue light receptors, phytochromes, and cryptochromes. Surprisingly, recent genome sequencing projects discovered additional photoactive proteins, retinal-based rhodopsins, in cyanobacterial and algal genera. Most of these newly found rhodopsin genes and retinal synthase have not been expressed and their functions are unknown. Analysis of the Anabaena and Chlamyrhodopsin with retinal synthase revealed that they have sensory functions, which, based on our work with haloarchaeal rhodopsins, may use a variety of signaling mechanisms. Anabaena rhodopsin is believed to be sensory, shown to interact with a soluble transducer and the putative function is either chromatic adaptation or circadian rhythm. Chlamydomonas rhodopsins are involved in phototaxis and photophobic responses based on electrical measurements by RNAi experiment. In order to analyze the protein, we developed a sensory rhodopsin expression system in E. coli. The opsin in E. coil bound endogenous all-trans retinal to form a pigment and can be observed on the plate. Using this system we could identify retinal synthase in Anabaena PCC 7120. We conclude that Anabaena D475 dioxygenase functions as a retinal synthase to the Anabaena rhodopsin in the cell.