Knock down of target genes by RNA interference in the embryos of lepidopteran insect, Bombyx mori

Xu, Guanfeng; Tian, Yu‐Lin; Peng, Yuling; Zheng, Sichun

doi:10.1016/j.xpro.2022.101219

Cited by 7 publications

(4 citation statements)

References 6 publications

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“…Then, dsRNA was injected into the eggs using a glass needle attached to a capillary puller (PC-100; NARISHIGE, Solana Beach, CA, USA) and FemtoJet (Eppendorf, Oldenburg, Germany). The eggs injected with dsRNA were incubated at 25 • C in darkness with a relative humidity of 80%, and after hatching, the larvae were fed fresh mulberry leaves at 25 • C ± 1 • C. The control group was replaced with an equal amount of dsGFP [28].…”

Section: Embryonic Rna Interferencementioning

confidence: 99%

Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori)

Wang,

Zhu,

Zhang

et al. 2024

Insects

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Genetic mutations leading to premature termination codons are known to have detrimental effects. Using the Lepidoptera model insect, the silkworm (Bombyx mori), we explored the genetic compensatory response triggered by mutations with premature termination codons. Additionally, we delved into the molecular mechanisms associated with the nonsense-mediated mRNA degradation pathway. CRISPR/Cas9 technology was utilized to generate a homozygous bivoltine silkworm line BmTrpA1−/− with a premature termination. Transcript levels were assessed for the BmTrpA paralogs, BmPyrexia and BmPainless as well as for the essential factors Upf1, Upf2, and Upf3a involved in the nonsense-mediated mRNA degradation (NMD) pathway. Upf2 was specifically knocked down via RNA interference at the embryonic stage. The results comfirmed that the BmTrpA1 transcripts with a 2-base deletion generating a premature termination codon in the BmTrpA1−/− line. From day 6 of embryonic development, the mRNA levels of BmPyrexia, BmPainless, Upf1, and Upf2 were significantly elevated in the gene-edited line. Embryonic knockdown of Upf2 resulted in the suppression of the genetic compensation response in the mutant. As a result, the offspring silkworm eggs were able to hatch normally after 10 days of incubation, displaying a non-diapause phenotype. It was observed that a genetic compensation response does exist in BmTrpA1−/− B. mori. This study presents a novel discovery of the NMD-mediated genetic compensation response in B. mori. The findings offer new insights into understanding the genetic compensation response and exploring the gene functions in lepidopteran insects, such as silkworms.

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Section: Embryonic Rna Interferencementioning

confidence: 99%

Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori)

Wang,

Zhu,

Zhang

et al. 2024

Insects

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“…dsRNA (20−50 ng) was microinjected into embryos within 2 h of oviposition. The method of B. mori embryo microinjection was described in detail in our previous study (Xu et al, 2022c), and 3 replicates of 100−150 embryos per replicate were included. To measure the knockdown efficiency, qPCR was performed using the specific primers listed in Table S2.…”

Section: Rnai In Embryosmentioning

confidence: 99%

Homeobox protein A1‐like and DNA methylation regulate embryo‐specific Zinc finger protein 615 gene expression and embryonic development in the silkworm Bombyx mori

Tian

Zhong

et al. 2023

Insect Science

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DNA methylation and transcription factors play roles in gene expression and animal development. In insects, DNA methylation modifies gene bodies, but how DNA methylation and transcription factors regulate gene expression is unclear. In this study, we investigated the mechanism that regulates the expression of Bombyx mori Zinc finger protein 615 (ZnF 615), which is a downstream gene of DNA methyltransferase 1 (Dnmt1), and its effects on the regulation of embryonic development. By progressively truncating the ZnF 615 promoter, it was found that the −223 and −190 nt region, which contains homeobox (Hox) protein cis-regulatory elements (CREs), had the greatest impact on the transcription of ZnF 615. RNA interference (RNAi)-mediated knockdown and overexpression of Hox family genes showed that Hox A1-like can enhance the messenger RNA level of ZnF 615. Further studies showed that Hox A1-like regulates ZnF 615 expression by directly binding to the −223 and −190 nt region of its promoter. Simultaneous RNAimediated knockdown or overexpression of Hox A1-like and Dnmt1 significantly inhibited or enhanced the regulatory effect of either gene alone on ZnF 615 expression, suggesting that both DNA methylation of gene bodies and binding of transcription factors to promoters are essential for gene expression. RNAi-mediated knockdown of Hox A1-like and Dnmt1 showed that the embryonic development was retarded and the hatching rate was decreased. Taken together, these data suggest that Hox A1-like and DNA methylation enhance the expression of ZnF 615, thereby affecting the development of B. mori embryos.

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“…As an autoimmune mechanism, it has an important role in suppressing viral infestation and in antiviral immunity. RNAi is a specific degradation of messenger RNA (mRNA) mediated by double‐stranded non‐coding RNA (dsRNA), which is widely present in various organisms, and is an important mechanism by which organisms to respond to external environmental stress and regulate their development 3–5 . Because of its specificity, stability, high efficiency, and selectivity, RNAi technology has an important role in virus resistance research.…”

Section: Principles Of Strategies In Silkworm Disease Resistance Bree...mentioning

confidence: 99%

Common strategies in silkworm disease resistance breeding research

Dong

Pan

2023

Pest Management Science

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The silkworm, which is considered a model invertebrate organism, was the first insect used for silk production in human history and has been utilized extensively throughout its domestication. However, sericulture has been plagued by various pathogens that have caused significant economic losses. To enhance the resistance of a host to its pathogens,numerous strategies have been developed. For instance, gene-editing techniques have been applied to a wide range of organisms, effectively solving a variety of experimental problems. This review focuses on several common silkworm pests and their pathogenic mechanisms, with a particular emphasis on breeding for disease resistance to control multiple types of silkworm diseases. The review also compares the advantages and disadvantages of transgenic technology and gene-editing systems. Finally, the paper provides a brief summary of current strategies used in breeding silkworm disease resistance, along with a discussion of the establishment of existing technologies and their future application prospects.

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Knock down of target genes by RNA interference in the embryos of lepidopteran insect, Bombyx mori

Cited by 7 publications

References 6 publications

Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori)

Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori)

Homeobox protein A1‐like and DNA methylation regulate embryo‐specific Zinc finger protein 615 gene expression and embryonic development in the silkworm Bombyx mori

Common strategies in silkworm disease resistance breeding research

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