Stable transformation of fluorescent proteins into Nosema bombycis by electroporation

Dong, Zhanqi; Gao, Na; Deng, Boyuan; Huang, Xin; Hu, Congwu; Chen, Peng; Wu, Qin; Lü, Cheng; Pan, Min‐Hui

doi:10.1186/s13071-022-05236-4

Cited by 6 publications

(3 citation statements)

References 52 publications

(53 reference statements)

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“…In addition, transformation techniques for delivering genetic material to these obligate intracellular eukaryotes must be established. Some critical progress has been made in culturing sporoplasm in Nosema bombycis for this purpose ( 53 , 54 ), in delivering genetic material ( 55 ), and in generating stable transformants ( 56 ). Thus, the identification of bleomycin as having potent activity against this microsporidia species at doses that can be tolerated by host cells during selection would represent an important step in the efforts to develop tools for transforming microsporidia.…”

Section: Discussionmentioning

confidence: 99%

Bleomycin reduces Vairimorpha (Nosema) ceranae infection in honey bees with some evident host toxicity

Parrella,

Elikan,

Kogan

et al. 2024

Microbiol Spectr

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Microsporidia cause disease in many beneficial insects, including honey bees, yet few pathogen control tools are available for protecting these important organisms against infection. Some evidence suggests that microsporidia possess a reduced number of genes encoding DNA repair proteins. We hypothesized that microsporidia would thus be susceptible to treatment with DNA-damaging agents and tested this hypothesis using a novel, rapid method for achieving robust and homogenous experimental infection of large numbers of newly emerged honey bees with one of its microsporidia pathogens, Vairimorpha ( Nosema ) ceranae . In carrying out these experiments, we found this novel V. ceranae inoculation method to have similar efficacy as other traditional methods. We show that the DNA-damaging agent bleomycin reduces V. ceranae levels, with minimal but measurable effects on honey bee survival and increased expression of midgut cellular stress genes, including those encoding SHSP. Increased expression of UpdlC suggests the occurrence of epithelial regeneration, which may contribute to host resistance to bleomycin treatment. While bleomycin does reduce infection levels, host toxicity issues may preclude its use in the field. However, with further work, bleomycin may provide a useful tool in the research setting as a potential selection agent for genetic modification of microsporidia. IMPORTANCE Microsporidia cause disease in many beneficial insects, yet there are few tools available for control in the field or laboratory. Based on the reported paucity of DNA repair enzymes found in microsporidia genomes, we hypothesized that these obligate intracellular parasites would be sensitive to DNA damage. In support of this, we observed that the well-characterized DNA damage agent bleomycin can reduce levels of the microsporidia Vairimorpha ( Nosema ) ceranae in experimental infections in honey bees. Observation of slightly reduced honey bee survival and evidence of sublethal toxicity likely preclude the use of bleomycin in the field. However, this work identifies bleomycin as a compound that merits further exploration for use in research laboratories as a potential selection agent for generating genetically modified microsporidia.

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Section: Discussionmentioning

confidence: 99%

Bleomycin reduces Vairimorpha (Nosema) ceranae infection in honey bees with some evident host toxicity

Parrella,

Elikan,

Kogan

et al. 2024

Microbiol Spectr

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“…In this procedure, A. tumefaciens strain GV3101 was used. Follow standardized methods for electroporation techniques 25 .…”

Section: A Tumefaciens Transformation With Plasmidmentioning

confidence: 99%

<em>Agrobacterium tumefaciens</em>-Mediated Genetic Transformation of Narrowleaf Plantain

Levengood¹,

Yan-xia²,

Fan³

et al. 2023

JoVE

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Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species.Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium.The plants were then acclimated to a soil environment and tested for the presence of a transgene using the β-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

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“…Various genetic transformation systems for filamentous fungi [25] and mushrooms [26] have been described during recent decades. Approaches used for fungi include PEG-CaCl 2 -mediated protoplast transformation [27], Agrobacterium tumefaciens-mediated transformation [28], electroporation [29], the gene gun method [30], and restriction enzyme-mediated integration [31]. Few studies have addressed genetic transformation systems for AC.…”

Section: Introductionmentioning

confidence: 99%

Enhancement of Triterpenoid Synthesis in Antrodia cinnamomea through Homologous Expression of the Key Synthetic Pathway Genes AcLSS and AcERG4

Zheng,

Fang,

Huang

et al. 2023

Fermentation

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Antrodia cinnamomea (AC), a rare fungus endemic to Taiwan, contains high levels of various secondary metabolites, notably triterpenoids, having useful medicinal and pharmacological properties. Techniques for increasing the production of AC triterpenoids (ACT) for medicinal purposes are a high research priority. We measured and compared the biomass and ACT content of AC mycelia under various liquid fermentation culture conditions. Relative gene expression levels of ten enzymes involved in the mevalonate (MVA) pathway and “subsequent group modification pathway” were determined, and correlation analysis was performed to evaluate the roles of these enzyme genes in ACT synthesis. Two representative genes encoding the enzymes lanosterol synthase (AcLSS) and sterol C-24 reductase (AcERG4), whose activity is closely associated with ACT content, were selected for homologous expression. AcLSS and AcERG4 were separately linked to plasmid pCT74, and transformed into prepared AC protoplasts to obtain two recombinant strains, termed RpLSS and RpERG4, by polyethylene glycol (PEG)-CaCl2-mediated protoplast transformation. Upregulated expression levels of AcLSS and AcERG4 (1.78- and 1.41-fold, respectively) were associated with significantly higher (1.82- and 1.37-fold, respectively) ACT content in the recombinant strains in comparison with the wild-type. Our findings provide a theoretical and practical basis for the enhancement of ACT production using homologous expression techniques.

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Stable transformation of fluorescent proteins into Nosema bombycis by electroporation

Cited by 6 publications

References 52 publications

Bleomycin reduces Vairimorpha (Nosema) ceranae infection in honey bees with some evident host toxicity

Bleomycin reduces Vairimorpha (Nosema) ceranae infection in honey bees with some evident host toxicity

<em>Agrobacterium tumefaciens</em>-Mediated Genetic Transformation of Narrowleaf Plantain

Enhancement of Triterpenoid Synthesis in Antrodia cinnamomea through Homologous Expression of the Key Synthetic Pathway Genes AcLSS and AcERG4

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