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서양뒤영벌 야외개체군에서 Real-Time PCR을 이용한 Nosema ceranae의 검출
이대원,Lee, Dae-Weon 한국미생물학회 2013 미생물학회지 Vol.49 No.3
The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA (SSU rRNA) gene of N. ceranae was successfully amplified among examined genes and sequenced, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentration as low as $0.85ng/{\mu}l$ genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris. 서양뒤영벌(Bombus terrestris)은 꿀벌의 봉군붕괴증후군(colony collapse disorder)에 대한 대체 화분매개곤충으로서 농업분야에서 중요한 역할을 하고 있다. 최근 서양뒤영벌에서 바이러스, 세균, 응애 등의 여러 병원체와 기생체가 발견되었고, 이들은 서양뒤영벌의 수명과 생식력 등에 영향을 주는 것이 알려져 있다. 서양뒤영벌 야외개체군에서 Nosema spp.를 탐지하기 위해, 서양뒤영벌 성충으로부터 genomic DNA를 추출하여 Nosema spp. 유전자들에 대해 polymerase chain reaction (PCR)을 수행하였다. 이들 유전자 중에서 small subunit ribosomal RNA (SSU rRNA) 유전자만이 증폭되었고, 염기서열분석을 통해 N. ceranae로 확인된 것은 조사된 야외개체군에서 N. ceranae가 서양뒤영벌의 주된 감염체임을 보여준다. Quantitative real-time PCR (qRT-PCR)을 이용하여 SSU rRNA 유전자를 탐지하기 위해, 먼저 PCR을 통해 SSU rRNA 유전자의 2개 영역에 대한 유전자 특이적 증폭을 확인하였다. qRT-PCR을 이용하여 각 개체에서 얻은 genomic DNA의 순차적인 농도희석를 통해 $0.85ng/{\mu}l$ 이하의 genomic DNA 농도에서도 SSU rRNA 유전자가 성공적으로 증폭되는 것이 확인되었다. 이러한 실험 결과, qRT-PCR를 이용한 N. ceranae 특이 유전자 증폭은 서양뒤영벌의 병원체 감염 진단 뿐만 아니라 생태계 위해성 평가에도 활용될 수 있을 것으로 사료된다.
이대원,박현우,진병래,정영호,박영목,강석권,Lee, Dae-Weon,Park, Hyun-Woo,Jin, Byung-Rae,Chung, Young-Ho,Park, Young-Mok,Kang, Seok-Kwon 한국미생물 · 생명공학회 1996 한국미생물·생명공학회지 Vol.24 No.2
Bacillus thuringiensis subsp. morrisoni PG-14 is a gram-positive soil bacterium producing mosquitocidal parasporal inclusions composed of several crystal proteins. Among these crystal protein genes, cryIVD gene is one of major component which has 72 kDa in size. However, these parasporal inclusions sink quickly from the surface of water where mosquito larval feeding occurred. To develope mosquitocidal cyanobacterium, therefore, we constructed the expression vector, pCYASK 5-1 harboring cryIVD gene. The expression vector, pCYASK5-1 was transformed into the cyanobacterium Syne- chocystis PCC6803 reported as a natural mosquito larval food source and the transformants were selected with kanamycin. Expression of IVD gene in transformant was characterized by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis. The mosquitocidal activity of a transformant was determined with Culex tritaeniorhynchus. The results showed that, the transformed cyanobacterium is toxic to mosquito larvae and will be expected as a potential agent that is used for mosquito control.
대체제설제인 EFD-1과 PC-10의 밀, 보리, 시금치 생육에 미치는 영향
이대원 ( Dae Weon Lee ) 한국환경농학회 2015 한국환경농학회지 Vol.34 No.1
BACKGROUND: During the winter, the use of deicers was rapidly increased for reduction of traffic accidents as well as injuries in Korea, whose components are largely comprised of calcium chloride and sodium chloride. Recently, to reduce the adverse effects of chloride-deciers such as pollution of water and soil, and decrease of agricultural productivity, the attention of eco-friendly deciers have been increased. This study aimed to investigate biological effects of magnesium chloride deicer (PC-10) and organic acid deicer (EFD-1) against wheat, barley, and spinach. METHODS AND RESULTS: We examined the effect of two deciers, PC-10 and EFD-1 on the seed germination and growth of wheat, barley and spinach. EFD-1 showed higher suppression of the germination than PC-10 among tested crop seeds. In demage index of the seedlings of the crops, there was no symptoms in spinach such as spotting and color change of leaves. EFD-1 showed much stronger inhibitory effect on the germination of tested crop seeds than PC-10 when crops were exposed continuously to tested deciers in soils. The growth and shoot and root in examined crops was relatively higher in PC-10 treatment than in EFD-1 treatment when compared to the control. The biomass decrease was found in all examined conditions of deciers. PC-10 showed 23-35% reduction of biomass whereas EFD-1 exhibited 39-84% loss in all examined crops at over 2% concentration. CONCLUSION: These results suggest that the effects of deicers used in this study by inputting into soil against growing tested crops cause the reduction of germination of seeds, growth, and biomass compared to the control.