<meta charset="utf8"></head><body>纳米颗粒介导的 dsRNA 喂养用于家蚕幼虫基因沉默

<ol>
	<li>Xia, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+draft+sequence+for+the+genome+of+the+domesticated+silkworm+(bombyx+mori).">A draft sequence for the genome of the domesticated silkworm (bombyx mori).</a> Science. 306 (5703), 1937-1940 (2004).</li><li>Kawamoto, M., Kiuchi, T., Katsuma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silkbase:+An+integrated+transcriptomic+and+genomic+database+for+bombyx+mori+and+related+species.">Silkbase: An integrated transcriptomic and genomic database for bombyx mori and related species.</a> Database (Oxford). 2022, (2022).</li><li>Yokoi, K., Tsubota, T., Jouraku, A., Sezutsu, H., Bono, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+transcriptome+data+in+silkworm+bombyx+mori.">Reference transcriptome data in silkworm bombyx mori.</a> Insects. 12 (6), (2021).</li><li>Mita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+construction+of+an+est+database+for+bombyx+mori+and+its+application.">The construction of an est database for bombyx mori and its application.</a> Proc Natl Acad Sci U S A. 100 (24), 14121-14126 (2003).</li><li>Zhou, Q. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rna-based+antiviral+immunity.">Rna-based antiviral immunity.</a> Nat Rev Immunol. 10 (9), 632-644 (2010).</li><li>Yu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+dsRNA+for+RNAi+in+insects:+An+overview+and+future+directions.">Delivery of dsRNA for RNAi in insects: An overview and future directions.</a> Insect Sci. 20 (1), 4-14 (2013).</li><li>Liang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+the+immune+gene+bmpgrp-l4+in+the+midgut+affects+the+growth+of+silkworm+(bombyx+mori)+larvae.">Silencing of the immune gene bmpgrp-l4 in the midgut affects the growth of silkworm (bombyx mori) larvae.</a> Insect Mol Biol. 32 (4), 340-351 (2023).</li><li>Tian, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+control+of+a+lepidopteran+pest+Spodoptera+exigua+by+ingestion+of+bacteria+expressing+dsrna+of+a+non-midgut+gene.">Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsrna of a non-midgut gene.</a> PLoS One. 4 (7), e6225 (2009).</li><li>Kamath, R. 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G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+specific+rna-mediated+interference+through+ingested+double-stranded+rna+in+Caenorhabditis+elegans.">Effectiveness of specific rna-mediated interference through ingested double-stranded rna in Caenorhabditis elegans.</a> Genome Biol. 2 (1), RESEARCH0002 (2001).</li><li>Arimatsu, Y., Kotani, E., Sugimura, Y., Furusawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+a+cdna+encoding+extracellular+dsrnase+and+its+expression+in+the+silkworm+Bombyx+Mori.">Molecular characterization of a cdna encoding extracellular dsrnase and its expression in the silkworm Bombyx Mori.</a> Insect Biochem Mol Biol. 37 (2), 176-183 (2007).</li><li>Liu, J., Swevers, L., Iatrou, K., Huvenne, H., Smagghe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bombyx+mori+DNA/rna+non-specific+nuclease:+Expression+of+isoforms+in+insect+culture+cells,+subcellular+localization+and+functional+assays.">Bombyx mori DNA/rna non-specific nuclease: Expression of isoforms in insect culture cells, subcellular localization and functional assays.</a> J Insect Physiol. 58 (8), 1166-1176 (2012).</li><li>Christiaens, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+rnai+efficacy+in+Spodoptera+exigua+via+the+formulation+of+dsrna+with+guanylated+polymers.">Increased rnai efficacy in Spodoptera exigua via the formulation of dsrna with guanylated polymers.</a> Front Physiol. 9, 316 (2018).</li><li>Dass, C. 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<table><tbody><tr><td>1.5 mL centrifuge tube</td><td>Sangon</td><td>F601620</td><td>for dsRNA or nanoparticles reaction</td></tr><tr><td>10 &amp;mu;l pipette</td><td>Eppendorf</td><td>P13473G</td><td>to aspirate or resuspend liquid</td></tr><tr><td>100 &amp;mu;l pipette</td><td>Eppendorf</td><td>Q12115G</td><td>to aspirate or resuspend liquid</td></tr><tr><td>2.5 &amp;mu;l pipette</td><td>Eppendorf</td><td>P20777G</td><td>to aspirate or resuspend liquid</td></tr><tr><td>20 &amp;mu;l pipette</td><td>Eppendorf</td><td>H19229E</td><td>to aspirate or resuspend liquid</td></tr><tr><td>200 &amp;mu;l pipette</td><td>Eppendorf</td><td>H20588E</td><td>to aspirate or resuspend liquid</td></tr><tr><td>6-well Clear TC-treated Multiple Well Plates</td><td>Costar</td><td>3516</td><td>for silkworm rearing individually</td></tr><tr><td>Acetic acid</td><td>Aladdin</td><td>A116165</td><td>to make TAE</td></tr><tr><td>Agarose M</td><td>BBI Life Sciences</td><td>A610013</td><td>for agarose gel electrophosis</td></tr><tr><td>Analytical balance</td><td>Sartorius</td><td>BSA224S</td><td>to weight ingredients</td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Sartorius</td><td>Centrisart A-14C</td><td>to centrifuge to form dsRNA or nanoparticles</td></tr><tr><td>Chitosan</td><td>Sigma-Aldrich</td><td>C3646</td><td>to combine with dsRNA for preparation of nanoparticles</td></tr><tr><td>EDTA</td><td>Sangon</td><td>A500895</td><td>to make TAE</td></tr><tr><td>Ethanol</td><td>Aladdin</td><td>E130059</td><td>to make TAE, or for dsRNA precipitation</td></tr><tr><td>Freezer</td><td>Siemens</td><td>iQ300</td><td>to store dsRNA or nanoparticles</td></tr><tr><td>GoTaq Green Master Mix</td><td>Promega</td><td>M712</td><td>for PCR reaction</td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6002</td><td>for qRT-PCR reaction</td></tr><tr><td>Isopropanol&amp;nbsp;</td><td>Aladdin</td><td>I112011</td><td>for dsRNA precipitation</td></tr><tr><td>NanoDrop Microvolume UV-Vis Spectrophotometer</td><td>ThermoFisher</td><td>One</td><td>to determine the concentration of dsRNA</td></tr><tr><td>ph meter</td><td>Sartorius</td><td>&amp;nbsp;PB-10</td><td>to prepare buffers</td></tr><tr><td>SanPrep Column PCR Product Purification Kit</td><td>Sangon</td><td>B518141</td><td>for PCR product purification</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td>to make 100 mM sodium acetate buffer</td></tr><tr><td>Sodium sulfate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>239313</td><td>to make 100 mM sodium sulfate buffer</td></tr><tr><td>T7 RiboMAX Express RNAi System</td><td>Promega</td><td>P1700</td><td>for dsRNA synthesis</td></tr><tr><td>ThermoMixer</td><td>Eppendorf</td><td>C</td><td>for dsRNA generation or nanoparticles heating</td></tr><tr><td>Tris</td><td>Sangon</td><td>A501492</td><td>to make TAE</td></tr><tr><td>Vortex&amp;nbsp;</td><td>IKA</td><td>Vortex 3</td><td>to prepare chitosan/dsRNA nanoparticles</td></tr></tbody></table>

larval rna interference in silkworm bombyx mori through chitosan/dsrna nanoparticle delivery

<meta charset="utf8"></head><body>作者聚焦：通过饲喂壳聚糖纳米颗粒推进 dsRNA 递送家蚕的基因沉默研究

幼虫 RNA 通过壳聚糖/dsRNA 纳米颗粒递送对 家蚕 Bombyx 的 干扰

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of ...

larval-rna-interference-silkworm-bombyx-mori-through-chitosandsrna

Research

JoVE Journal

Biology

Larval RNA Interference in Silkworm Bombyx mori through Chitosan/dsRNA Nanoparticle Delivery

1.7K 次观看.  Guangzhou University. 本研究的范围是开发一种方法，用 dsRNA 喂养家蚕 Bombyx mori 的幼虫，从而产生基因沉默效果。这将有助于基因调控研究和高通量基因筛选。壳聚糖是一种廉价、无毒且可生物降解的聚合物。壳聚糖/dsRNA 纳米颗粒被发现通过喂养蚊子来有效引起 RNAi 效应。在家蚕中，到目前为止，RNAi 只能通过注射 dsRNA 来实现。但是，注射需要足够的经过身份验证...

视频: 作者聚焦：通过饲喂壳聚糖纳米颗粒推进 dsRNA 递送家蚕的基因沉默研究

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

环境科学

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

果蝇幼虫NMJ免疫

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

幼虫RNA干扰在赤拟谷盗，<em&gt;赤拟谷盗</em

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing &#946;-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

加载编码RNA核酸适体和核糖开关的DNA质粒的多功能丝基微胶囊的制备

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

RNA干扰在松林线虫、木犀牛线虫中的应用

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

推进树 突毛 虫 RNAi 技术用于基因功能分析

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

作者聚焦:研究 Wolbachia 诱导的 Thelytokous 孤雌生殖和通过 RNA 干扰的遗传工具包开发

The manipulation of RNA interference (RNAi) presents a formidable challenge in many parasitoid species with diminutive size, such as Trichogramma wasps. This study delineated an efficient RNAi method in Trichogramma denrolimi. The present methodology provides a robust model for investigating gene regulation in Trichogramma wasps.

幼虫 RNA 通过壳聚糖/dsRNA 纳米颗粒递送对家蚕 Bombyx 的干扰

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在果蝇幼虫中枢神经系统的剖析

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

果蝇幼虫NMJ免疫

可视化的RNA中的本地化爪蟾卵母细胞

幼虫RNA干扰在赤拟谷盗，

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

加载编码RNA核酸适体和核糖开关的DNA质粒的多功能丝基微胶囊的制备

RNA干扰在松林线虫、木犀牛线虫中的应用

卵寄生虫中的RNA干扰， Trichogramma dendrolimi Matsumura

幼虫 RNA 通过壳聚糖/dsRNA 纳米颗粒递送对 家蚕 Bombyx 的 干扰

副本

摘要

探索更多视频

此视频中的章节

在果蝇幼虫中枢神经系统的剖析

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

果蝇幼虫NMJ免疫

可视化的RNA中的本地化爪蟾卵母细胞

幼虫RNA干扰在赤拟谷盗，

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

加载编码RNA核酸适体和核糖开关的DNA质粒的多功能丝基微胶囊的制备

RNA干扰在松林线虫、木犀牛线虫中的应用

卵寄生虫中的RNA干扰， Trichogramma dendrolimi Matsumura

幼虫 RNA 通过壳聚糖/dsRNA 纳米颗粒递送对家蚕 Bombyx 的干扰