Inducible Expression of dsRNA in Escherichia coli

Duan, Saiya; Wang, Guangjun

doi:10.1007/978-1-0716-3702-9_9

Cited by 2 publications

(2 citation statements)

References 26 publications

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“…Positive clones were selected and incubated at 37 °C and 250 rpm for 6 h, followed by 1:100 inoculation in LB liquid medium and incubation until the OD 600 was approximately 0.8. Then, dsRNA expression was induced using isopropyl β- d -1-thiogalactopyranoside (IPTG; final concentration, 0.4 mM) for 4 h. The culture was then centrifuged at 7500 × g for 2 min, and the resulting pellet used for total RNA isolation . To detect dsRNA produced in Escherichia coli HT115 cells after IPTG induction, the total bacterial RNA was extracted using TRIzol reagent (Monad Biotech Co., Ltd., Wuhan, China) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Then, dsRNA expression was induced using isopropyl β-D-1-thiogalactopyranoside (IPTG; final concentration, 0.4 mM) for 4 h. The culture was then centrifuged at 7500 × g for 2 min, and the resulting pellet used for total RNA isolation. 31 To detect dsRNA produced in Escherichia coli HT115 cells after IPTG induction, the total bacterial RNA was extracted using TRIzol reagent (Monad Biotech Co., Ltd., Wuhan, China) according to the manufacturer's instructions. The isolated RNA was treated with RNase (Promega, Madison, WI, USA) at 37 °C for 1 h to remove the single-stranded RNA.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Gustatory Receptor 206 Participates in the Foraging Behavior of Larvae of Polyphagous Pest Spodoptera litura

Li,

Chen,

Wang

et al. 2024

J. Agric. Food Chem.

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Insect gustatory receptors (GRs) aid in the precise identification of deterrent or stimulant compounds associated with food, mating, and egg-laying. Thus, they are promising targets for developing efficient insecticides. Here, 61 GRs in the chemosensory organs of Spodoptera litura larvae and adults were identified. Among them, SlitGR206 exhibited larval labium (LL)-specific expression characteristics. To explore the role of SlitGR206, a bacterial expression system was established to produce high-quality double-stranded RNA (dsRNA) and suppress SlitGR206 expression in LL. Subsequent behavioral assessments revealed that SlitGR206 silencing influenced larval feeding preferences and absorption. Moreover, it was found to reduce the ability of larvae to forage the five crucial host odorants. These findings demonstrate that SlitGR206 likely plays an indirect regulatory role in host recognition, consequently affecting foraging behavior. This provides a crucial foundation for the analysis of functional diversity among insect GRs and the precise development of nucleic acid pesticides in the future.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Gustatory Receptor 206 Participates in the Foraging Behavior of Larvae of Polyphagous Pest Spodoptera litura

Li,

Chen,

Wang

et al. 2024

J. Agric. Food Chem.

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The salivary gland transcriptome of Varroa destructor reveals suitable targets for RNAi‐based mite control

Becchimanzi,

Cacace,

Parziale

et al. 2024

Insect Molecular Biology

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The mite Varroa destructor Anderson and Trueman (Mesostigmata: Varroidae) has a dramatic impact on beekeeping and is one of the main causes of honey bee colony losses. This ectoparasite feeds on honey bees' liquid tissues, through a wound created on the host integument, determining weight loss and a reduction of lifespan, as well as the transmission of viral pathogens. However, despite its importance, the mite feeding strategy and the host regulation role by the salivary secretions have been poorly explored. Here, we contribute to fill this gap by identifying the salivary components of V. destructor, to study their functional importance for mite feeding and survival. The differential expression analysis identified 30 salivary gland genes encoding putatively secreted proteins, among which only 15 were found to be functionally annotated. These latter include proteins with putative anti‐bacterial, anti‐fungal, cytolytic, digestive and immunosuppressive function. The three most highly transcribed genes, coding for a chitin‐binding domain protein, a Kazal domain serine protease inhibitor and a papain‐like cysteine protease were selected to study their functional importance by reverse genetics. Knockdown (90%–99%) by RNA interference (RNAi) of the transcript of a chitin‐binding domain protein, likely interfering with the immune reaction to facilitate mite feeding, was associated with a 40%–50% decrease of mite survival. This work expands our knowledge of the host regulation and nutritional exploitation strategies adopted by ectoparasites of arthropods and allows the identification of potential targets for RNAi, paving the way towards the development of new strategies for Varroa mite control.

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Inducible Expression of dsRNA in Escherichia coli

Cited by 2 publications

References 26 publications

Gustatory Receptor 206 Participates in the Foraging Behavior of Larvae of Polyphagous Pest Spodoptera litura

Gustatory Receptor 206 Participates in the Foraging Behavior of Larvae of Polyphagous Pest Spodoptera litura

The salivary gland transcriptome of Varroa destructor reveals suitable targets for RNAi‐based mite control

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