• Horticultural Science & Technology
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• v.35 no.3
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• pp.323-332
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• 2017

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpin-based double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops.

• Parasites, Hosts and Diseases
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• v.46 no.1
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• pp.1-15
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• 2008

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi.

• BMB Reports
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• v.41 no.5
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• pp.358-362
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• 2008

RNA interference (RNAi) is the process of sequence-specific gene silencing. However, RNAi efficiency still needs to be improved for effective inhibition of target genes. We have developed an effective strategy to express multiple shRNAs (small hairpin RNA) simultaneously using multiple RNA Polymerase III (Pol III) promoters in a single vector. Our data demonstrate that multiple shRNAs expressed from Pol III promoters have a synergistic effect in repressing the target gene. Silencing of endogenous cyclophilin A (CypA) or key HIV viral genes by multiple shRNAs results in significant inhibition of the target gene.

• Korean Journal of Agricultural Science
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• v.42 no.1
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• pp.7-13
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• 2015

Ninety genes randomly selected from tobacco whitefly (Bemisia tabaci) cDNA library was studied for selecting target gene in order to control of tobacco whitefly using TRV-VIGS vector (tobacco rattle virus-virus induced gene silencing vector) with RNAi. First of all, the occurrence of B. tabaci adult according to agro-infiltration of TRV was no significant difference. And that of TRV inserted tobacco whitefly cDNA showed a significant difference in each sample. P CV and N CV sample were more than 80% could be confirmed in 5 samples, for example, wh11, wh36, wh46, wh50 and wh71. Lastly, the occurrence of nymph and egg also showed a significant difference in each sample. That could be confirmed in 11 samples, for example, wh01, wh09, wh10, wh15, wh16, wh23, wh24, wh48, wh64 and wh66. In case of wh46, wh50 and wh71 sample could be confirmed that occurrence of B. tabaci adult was many, but occurrence of B. tabaci nymph and egg was a little. So sample showed a physioecological good effect to control of whitefly need to be investigated variation of gene expression in whitefly body using qRT-PCR through individual test.

• Korean Journal of Agricultural Science
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• v.42 no.2
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• pp.81-86
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• 2015

Three genes selected from cDNA library of tobacco whitefly, Bemisia tabaci, were checked whether these genes expressed in plant or not, and confirmed the change of gene expression using qRT-PCR in the tobacco whitefly. First of all, three genes were inserted in Tobacco rattle virus (TRV) RNA2 vector using Sac I and Xho I restriction enzymes, and conducted agro-infiltration in tobacco plants (Nicotiana benthamianana). And then, it was confirmed that TRV RNA2 vector and genes inserted in TRV RNA2 vector were expressed in plant. So, after feeding the tobacco whitefly the plants inoculated the genes and induced RNAi of the genes, we plan to confirm the RNAi in the whitefly and investigate the changes of gene expression through the qRT-PCR.

• Animal cells and systems
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• v.8
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• pp.50-50
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• 2004

• Korean Journal of Breeding Science
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• v.42 no.5
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• pp.515-524
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• 2010

The amylose content of starch is a major factor in the texture of cooked cereal grains. Therefore, down-regulation of amylose synthesis is one of the alternative method to improve eating quality of rice. We developed transgenic rice plants designed to suppress granule-bound starch synthase I(GBSSI) gene using RNA interference(RNAi) technology. Transgenic plants with RNAi vector containing the 3'-UTR region of GBSSI showed a lower amylose content in rice endosperm than that of wild-type. The range of amylose content was 5.9~9.0% in the transgenic plants, whereas that of wild-type was 17.7~18.0%. Transgenic rices showed the decrease of short chain and the increase of long chain by analyzing chain length distribution of amylopectin in the endosperm. In the SEM micrographs, we found that compound starch granules in whole grains of the wild-type rice were readily split during fracturing, while the starch granules in RNAi-transgenic lines showed small voluminous, non-angular rounded bodies.

• The Plant Pathology Journal
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• v.36 no.3
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• pp.280-288
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• 2020

RNA interference (RNAi) has attracted attention as a promising approach to control plant viruses in their insect vectors. In the present study, to suppress replication of the rice stripe virus (RSV) in its vector, Laodelphax striatellus, using RNAi, dsRNAs against L. striatellus genes that are strongly upregulated upon RSV infection were delivered through a rice leaf-mediated method. RNAi-based silencing of peroxiredoxin, cathepsin B, and cytochrome P450 resulted in significant down regulation of the NS3 gene of RSV, achieving a transcriptional reduction greater than 73.6% at a concentration of 100 ng/μl and, possibly compromising viral replication. L. striatellus genes might play crucial roles in the transmission of RSV; transcriptional silencing of these genes could suppress viral replication in L. striatellus. These results suggest effective RNAi-based approaches for controlling RSV and provide insight into RSV-L. striatellus interactions.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10079-10083
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• 2015

Background: Endothelin-1 and Endostar are both significant for the progression, proliferation, metastasis and invasion of cancer. In this paper, we studied the effect of ET-1 RNAi and Endostar in PC-3 prostatic cancer cells. Materials and Methods: The lentiviral vector was used in the establishment of ET-1 knockdown PC-3 cells. Progression and apoptosis were assessed by CKK-8 and flow cytometry, respectively. Transwell assay was used to estimate invasion and signaling pathways were studied by Western blotting. Results: ET-1 mRNA and protein in ET-1 knockdown PC-3 cells were reduced to 26.4% and 22.4% compared with control group, respectively. ET-1 RNAi and Endostar both were effective for the suppression of progression and invasion of PC-3 cells. From Western blotting results, the effects of ET-1 regulation and Endostar on PC-3 cells were at least related to some signaling pathways involving PI3K/Akt/Caspase-3, Erk1/2/Bcl-2/Caspase-3 and MMPs (MMP-2 and MMP-9). Furthermore, combined treatment of ET-1RNAi and Endostar was found to be more effective than single treatment. Conclusions: Both ET-1 RNAi and Endostar can inhibit the progression and invasion of PC-3 cells, but combined treatment might be a better therapeutic schedule.

• Proceedings of the Korean Society of Grassland Science Conference
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• 2005.06a
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• pp.178-179
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• 2005