Abstract
Double-stranded RNA (dsRNA) is a valuable tool for reverse genetics research and gene silencing applications. It is also an important management method for pests and diseases in agriculture. It can be synthesized both in vivo and in vitro. The latter presents the drawback of high production cost, the former is less expensive and suitable for scalable production. In general, dsRNAs are obtained in vivo from Escherichia coli heterologous systems that require the IPTG-inducible T7 RNA polymerase. In this report, we describe the construction of an RNAi system for the expression of dsRNA using the HT115 bacterial strain and the L4440 plasmid, and the extraction and identification of dsRNA.
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References
Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, Roberts J (2007) Control of coleopteran insect pests through RNA interference. Nat Biotechnol 25(11):1322–1326
Cagliari D, Dias NP, Galdeano DM, Dos Santos EÁ, Smagghe G, Zotti MJ (2019) Management of pest insects and plant diseases by non-transformative RNAi. Front Plant Sci 10:1319
Dubrovina AS, Kiselev KV (2019) Exogenous RNAs for gene regulation and plant resistance. Int J Mol Sci 20(9):2282
Ganbaatar O, Cao B, Zhang Y, Bao D, Bao W (2017) Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors. BMC Biotechnol 17(1):9
Gordon KH, Waterhouse PM (2007) RNAi for insect-proof plants. Nat Biotechnol 25(11):1231–1232
Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Ahringer J (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421(6920):231–237
Kim J, Badaloni A, Willert T, Zimber-Strobl U, Kühn R, Wurst W, Kieslinger M (2013) An RNAi-based approach to down-regulate a gene family in vivo. PLoS One 8(11):e80312
Koch A, Biedenkopf D, Furch A, Weber L, Rossbach O, Abdellatef E, Kogel KH (2016) An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery. PLoS Pathog 12(10):e1005901
Koch A, Höfle L, Werner BT, Imani J, Schmidt A, Jelonek L, Kogel KH (2019) SIGS vs HIGS: a study on the efficacy of two dsRNA delivery strategies to silence Fusarium FgCYP51 genes in infected host and non-host plants. Mol Plant Pathol 20(12):1636–1644
Li X, Zhang M, Zhang H (2011) RNA interference of four genes in adult Bactrocera dorsalis by feeding their dsRNAs. PLoS One 6(3):e17788
Liu S, Jaouannet M, Dempsey DMA, Imani J, Coustau C, Kogel KH (2020) RNA-based technologies for insect control in plant production. Biotechnol Adv 39:107463
Mamta B, Rajam MV (2017) RNAi technology: a new platform for crop pest control. Physiol Mol Biol Plants 23:487–501
Niño-Sánchez J, Chen LH, De Souza JT, Mosquera S, Stergiopoulos I (2021) Targeted delivery of gene silencing in fungi using genetically engineered bacteria. J Fungi 7(2):125
Pitino M, Coleman AD, Maffei ME, Ridout CJ, Hogenhout SA (2011) Silencing of aphid genes by dsRNA feeding from plants. PLoS One 6(10):e25709
Price DR, Gatehouse JA (2008) RNAi-mediated crop protection against insects. Trends Biotechnol 26(7):393–400
Rio DC (2013) Expression and purification of active recombinant T7 RNA polymerase from E coli. Cold Spring Harbor Protocols 2013(11)
Silva J, Chang K, Hannon GJ, Rivas FV (2004) RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age. Oncogene 23(51):8401–8409
Takahashi Y, Nishikawa M, Takakura Y (2009) Nonviral vector-mediated RNA interference: its gene silencing characteristics and important factors to achieve RNAi-based gene therapy. Adv Drug Deliv Rev 61(9):760–766
Tenllado F, Martínez-García B, Vargas M, Díaz-Ruíz JR (2003) Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnol 3:3
Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can produce specifc and potent genetic interference in Caenorhabditis elegans. Gene 263:103–112
Wang M, Thomas N, Jin H (2017) Cross-kingdom RNA trafficking and environmental RNAi for powerful innovative pre- and post-harvest plant protection. Curr Opin Plant Biol 38:133–141
Yan S, Qian J, Cai C, Ma Z, Li J, Yin M, Shen J (2020) Spray method application of transdermal dsRNA delivery system for efficient gene silencing and pest control on soybean aphid Aphis glycines. J Pest Sci 93:449–459
Zheng Y, Hu Y, Yan S, Zhou H, Song D, Yin M, Shen J (2019) A polymer/detergent formulation improves dsRNA penetration through the body wall and RNAi-induced mortality in the soybean aphid Aphis glycines. Pest Manag Sci 75(7):1993–1999
Zhu F, Xu J, Palli R, Ferguson J, Palli SR (2011) Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata. Pest Manag Sci 67(2)
Zotti MJ, Smagghe G (2015) RNAi technology for insect management and protection of beneficial insects from diseases: lessons, challenges and risk assessments. Neotrop Entomol 44:197–213
Zotti M, Dos Santos EA, Cagliari D, Christiaens O, Taning CNT, Smagghe G (2018) RNA interference technology in crop protection against arthropod pests, pathogens and nematodes. Pest Manag Sci 74(6):1239–1250
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Duan, S., Wang, G. (2024). Inducible Expression of dsRNA in Escherichia coli. In: Cheng, X., Wu, G. (eds) Double-Stranded RNA. Methods in Molecular Biology, vol 2771. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3702-9_9
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DOI: https://doi.org/10.1007/978-1-0716-3702-9_9
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