• International Journal of Industrial Entomology and Biomaterials
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• v.12 no.1
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• pp.15-19
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• 2006

RNA interference has been used as a powerful tool in preventing virus proliferation in many species. In this study, we injected the dsRNA in vitro transcripts into Bombyx mori to investigate the resistance to B. mori nuclear polyhedrosis virus (BmNPV). Through vivisectional observation and real-time quantities PCR analysis, we found that these dsRNA can prevent the BmNPV to a certain extent, and delay the viruses' proliferation.

• The Plant Pathology Journal
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• v.15 no.2
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• pp.119-123
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• 1999

Chrysanthemums showing leaf mottling were collected from three southern locations in Korea in 1998. Two kinds of viruses were isolated from the leaves and were identified as tomato aspermy virus ch-TAV) and chrysanthemum virus B (ch-CVB), according to their host range, morphology, intracellular location, agar gel double diffusion test, and double-stranded RNA (dsRNA) analysis. The purified ch-TAV was spherical particles of approximately 29 nm in diameter and ch-CVB was filamentous particles of 685 nm long. Inclusion bodies were not observed in ch-TAV and/or ch-CVB infected chrysanthemum. ch-TAV showed positive serological reaction with TAV antiserum (ATCC-127) but not with CMV-pepper antiserum. In dsRNA analysis, four kinds of viral dsRNA were observed on ch-TAV and one viral dsRNA was shown on ch-CVB. Rate of co-infection with TAV and CVB in commercial chrysanthemums was 20.9%. On the other hand, infection with CVB alone was 97.2%. However, chrysanthemums naturally infected with TAV alone were not found.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.4
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• pp.456-464
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• 2010

RNA interference (RNAi) is an acknowledged useful and effective tool to study gene function in various cells. Here, we suppressed the Connexin 43 (Cx 43) gene expression during in vitro development of ovine pre-implantation embryos using the RNAi method. The 353 bp Cx 43 double-stranded RNA was microinjected into in vitro fertilized ovine zygotes, and the levels of target mRNA and protein were investigated. Control groups included uninjected zygotes or those injected with RNase-free water. The dsRNA injection resulted in the specific reduction of Cx 43 transcripts as analyzed by quantitative real-time RT-PCR and decreased protein levels as shown by Western blot analysis at the blastocyst stage. Microinjection of Cx 43 dsRNA led to 20.3%, 21.7% and 34.5% blastocyst rates and 19.2%, 37.5% and 41.3% hatched blastocyst rates in Cx 43 dsRNA-injected, water-injected and uninjected groups, respectively. Then the RNAi could not significantly affect cell number and cell death rates of blastocysts. Therefore, suppression of Cx 43 dsRNA and proteins did not apparently affect the development potential of ovine pre-implantation embryos but may play a role in embryo quality. RNAi technology is a promising approach to study gene function in early ovine embryogenesis.

• Molecules and Cells
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• v.23 no.3
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• pp.304-315
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• 2007

Fusarium graminearum causes a serious scab disease of small grains in Korea. The nucleotide sequence of the genomic RNA of a double-stranded RNA (dsRNA) virus, Fusarium graminearum virus-DK21 (FgV-DK21), from F. graminearum strain DK21, which is associated with hypovirulence in F. graminearum, was determined and compared to the genome sequences of other mycoviruses, including Cryponectria hypoviruses. The FgV-DK21 dsRNA consists of 6,624 nucleotides, excluding the 3'-terminal poly(A) tail. The viral genome has 53- and 46-nucleotide 5' and 3' untranslated regions (UTRs), respectively, and five putative open reading frames. A phylogenetic analysis of the deduced amino acid sequence of ORF1, which encodes a putative RNA-dependent RNA polymerase, and those of other mycoviruses revealed that this organism forms a distinct virus clade with other hypoviruses, and is more distantly related to other mycoviruses (3.8 to 24.0% identity). However, pairwise sequence comparisons of the nucleotide and deduced amino acid sequences of ORFs 2 through 5 revealed no close relationships to other protein sequences currently available in GenBank. Analyses of RNA accumulation by Northern blot and primer extension indicated that these putative gene products are expressed from at least two different subgenomic RNAs (sgRNAs), in contrast to the cases in other hypoviruses. This study suggests the existence of a new, as yet unassigned, genus of mycoviruses that exhibits a potex-like genome organization and sgRNA accumulation.

• Plant Biotechnology Reports
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• v.3 no.2
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• pp.139-145
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• 2009

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we isolated NtMET1 from Nicotiana tabacum cv. Havana (SR1) and obtain transgenic plants that reduced MET1 expression level with the double-strand RNA (dsRNA) MET1 gene. Transgenic tobacco plants showed dwarf and abnormal flower development when compared with the wild type. Using methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in transformed plants and the wild type were compared. MseI/HpaII selection primers showed an interesting polymorphism, and 153 DNA bands of interest were detected. Among these, 30 selective fragments were sequenced and analyzed with a BLAST search by successful MSAP modifications. The homology search showed that the transposons and tandem repeated sequences were related to the phenotypes. These results suggested that the decreased degree of methylation by dsRNA strategy caused abnormal growth and development in N. tabacum.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.139.1-139
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• 2003

Tissues from woody plant contain higher amount of phenolic compounds and polysaccharides, which give inhibitory effects on reverse transcriptase and/or Taq ploymerase. The common multiple-step protocols using several additives to inhibit polyphenoic compounds during nucleic acid extraction are time consuming and laborious. Sodium sulfite (Na$_2$SO$_3$) was used as inhibitor of polyphenolic oxidases in extraction buffer and compare it's effect between commercial RNA extraction kit and small-scale double-stranded RNA (dsRNA) extraction by RT-PCR. During nucleic acid extraction procedure, addition of 0.5％-1.5％ (w/v) sodium sulfite to Iysis buffer or STE buffer resulted in lighter color change than extracts without sodium sulfite and improve the RT-PCR detection. When commercial RNA extraction kit used, optimal concentration of sodium sulfite were variable according to the host plant. However, using dsRNA as RT-PCR template, 1.5％ sodium sulfite in STE buffer improves the detection of both viruses and unspecific amplifications were reduced significantly, Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

• Molecules and Cells
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• v.27 no.6
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• pp.689-695
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• 2009

Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of non-specific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without non-specific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.

• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.37-37
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• 2015

dsRNA was found in malformed cultures of Lentinula edodes strain FMRI0339, one of the three most popular sawdust cultivated commercial strains of shiitake, and was also found in healthy-looking fruiting bodies and actively growing mycelia. Cloning of the partial genome of the dsRNA revealed the presence of the RdRp sequence of a novel L. edodes mycovirus (LeV), and sequence comparison of the cloned amplicon showed an identical sequence to known RdRp genes of LeV found in strain HKA. The meiotic stability of dsRNA was examined by measuring the ratio of the presence of dsRNA among sexual monokaryotic progeny. More than 40% of the monokaryotic progeny still contained the dsRNA, indicating the persistence of dsRNA during sexual reproduction. Comparing the mycelia growth of monokaryotic progeny suggested that, although variations in the growth rate existed among progeny and virus infection was observed in highly actively growing progeny, there appeared to be a tendency toward a lower frequency of virus incidence in actively growing progeny. This study attempted to cure the edible mushroom L. edodes strain FMRI0339 of the L. edodes mycovirus (LeV) in order to obtain an isogenic virus-free fungal strain as well as a virus-infected strain for comparison. Mycelial fragmentation, followed by being spread on a plate with serial dilutions resulted in a virus-free colony. Viral absence was confirmed with gel electrophoresis after dsRNA-specific virus purification, Northern blot analysis, and PCR using reverse transcriptase (RT-PCR). Once cured, all of fungal cultures remained virus-free over the next two years. Interestingly, the viral titer of LeV varied depending on the culture condition. The titer from the plate culture showed at least a 20-fold higher concentration than that grown in the liquid culture. However, the reduced virus titer in the liquid culture was recovered by transferring the mycelia to a plate containing the same medium. In addition, oxygen-depleted culture conditions resulted in a significant decrease of viral concentration, but not to the extent seen in the submerged liquid culture. Although no $discernable phenotypic changes in colony morphology were observed, virus-cured strains showed significantly higher growth rates and mycelial mass than virus-infected strains. We were also explored effects of LeV on fruiting body formation and mushroom yield. The fruiting body formation yield of virus-free L. edodes was larger than virus-infected L. edodes. These results indicate that LeV infection has a deleterious effect on mycelial growth and fruiting body formation. In addition, we have been investigated host-parasite interaction between L. edodes and its mycovirus interaction to study viral mechanism by establishment of proteomics.

• The Plant Pathology Journal
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• v.22 no.1
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• pp.68-74
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• 2006

A new virus with a dsRNA genome was isolated and characterized from the Suhan-:neutari strain (ASI 2504) of Pleurotus ostreatus, which was characterized as long and slightly bent with small caps on the stipe of fruit body. Thirty nm isometric viruses with three dsRNA segments (approximately 2.0, 1.84 and 1.82 kb in sizes) were isolated by ultracentrifugation in sucrose gradients. Western analysis of protein extracted purified viruses with anti-virus polyclonal antibody confirmed that viruses have two specific proteins (36 and 68 kDa). Computer analysis of 2.0 kb segment shows that high. sequence identity with RNA-dependent RNA polymerase (RdRp) of partitiviruses, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis, OMDV was most related to Pleurotus ostreatus virus 1.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.4
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• pp.299-307
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• 2003

Objectives: The present study was conducted to evaluate the mobile transposon-like element, clone MTi7 (MTi7) expression in the mouse ovary and to determine its role(s) in the mouse oocytes by RNA interference (RNAi). Methods: MTi7 mRNA expression was localized by in situ hybridization in day5 and adult ovaries. Double stranded RNA (dsRNA) was prepared for c-mos, a gene with known function as control, and the MTi7. Each dsRNA was microinjected into the germinal vesicle (GV) stage oocytes then oocyte maturation and intracellular changes were evaluated. Results: In situ hybridization analysis revealed that MTi7 mRNA localized to the oocyte cytoplasm from primordial to preovulatory follicles. After dsRNA injection, we found 43-54% GV arrest of microinjected GV oocytes with 68%-90% decrease in targeted c-mos or MTi7 mRNA. Conclusions: This is the first report of the oocyte-specific expression of the MTi7 mRNA. From results of RNAi for MTi7, we concluded that the MTi7 is involved in the germinal vesicle breakdown in GV oocytes, and MTi7 may be implicated with c-mos for its function. We report here that RNAi provides an outstanding approach to study the function of a gene with unknown functions.