Molecular Cloning and Characterization of Ecdysone oxidase and 3-dehydroecdysone-3α-reductase Involved in the Ecdysone Inactivation Pathway of Silkworm, Bombyx mori

Sun, Wei; Shen, Yihong; Qi, Deng-Wei; Zhang, Xiang; Zhang, Ze

doi:10.7150/ijbs.8.125

Cited by 39 publications

(38 citation statements)

References 37 publications

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“…The polyclonal Ab against 3DE-3b-reductase (at a dilution of 1:8000) shown above was used to perform this assay. The method was also described previously (33).…”

Section: Western Blotting Analysesmentioning

confidence: 99%

“…The procedure was shown in our previous study (33). All the amplified products were purified with an agarose gel purification kit (Omega Bio-tek, Norcross, GA) and then cloned into pEASY-T1-Clone vectors (TransGen Biotech, Beijing, China).…”

Section: Cdna Cloning and Racementioning

confidence: 99%

“…Two hundred-microliter cell suspensions were moved on the slides for 20 min and then fixed with 4% paraformaldehyde for 20 min at room temperature. Immunohistochemical staining was performed following the previous study (33).…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…The 3DE-3b-reductase gene was induced by isopropyl b-D-1-thiogalactopyranoside, and the recombinant protein with the GST tag was purified using a GSTrap FF column (GE Healthcare, Uppsala, Sweden) according to the manufacturer's instruction. The polyclonal Abs against 3DE-3b-reductase were produced by immunizing mice with the purified proteins as described (33).…”

Section: Preparation Of Antiserummentioning

confidence: 99%

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Ecdysone Titer Determined by 3DE-3β-Reductase Enhances the Immune Response in the Silkworm

Sun

Shen

Zhou

et al. 2016

The Journal of Immunology

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Although recent studies have demonstrated that 20-hydroxyecdysone (20E), one of the two most important hormones for development, could promote the insect innate immune response, how insects regulate 20E titer to affect the immunity after suffering pathogen attack remains unknown. In this study, to our knowledge, we first found that 20E titer was significantly elevated after bacterial infection in the domesticated silkworm, Bombyx mori. Furthermore, the elevated 20E enhanced the silkworm innate immune system against invading bacteria via ecdysone receptor. During immune response, the expression of the silkworm 3-dehydroecdysone-3β-reductase (3DE-3β-reductase) that converts 3DE released from prothoracic glands into ecdysone was induced. RNA interference experiments suggested that 3DE-3β-reductase is essential to upregulate the 20E titer after bacterial infection. The rescue experiments showed that injection with the recombinant 3DE-3β-reductase protein can significantly elevate the 20E concentration and modulate the expressions of the silkworm immune-related genes. Taken together, 20E titer determined by 3DE-3β-reductase enhances the silkworm defense against the bacterial infection. Thus, our findings reveal an important role of the 20E synthesis pathway from 3DE in enhancing the silkworm immune response and have profound implications for the understanding of interaction mechanisms between insect hormone and immunity.

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“…The polyclonal Ab against 3DE-3b-reductase (at a dilution of 1:8000) shown above was used to perform this assay. The method was also described previously (33).…”

Section: Western Blotting Analysesmentioning

confidence: 99%

Section: Cdna Cloning and Racementioning

confidence: 99%

Section: Immunohistochemistrymentioning

confidence: 99%

Section: Preparation Of Antiserummentioning

confidence: 99%

See 2 more Smart Citations

Ecdysone Titer Determined by 3DE-3β-Reductase Enhances the Immune Response in the Silkworm

Sun

Shen

Zhou

et al. 2016

The Journal of Immunology

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“…Another major inactivation route of steroid hormone is through 3-epimerization [10][11][12]. 20E conversion to 3-dehydroecdysone (3DE) can be catalysed by ecdysone oxidase (EO), which could be reversed by 3-dehydroecdysone 3b-reductase [13].…”

Section: Introductionmentioning

confidence: 99%

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori

You

Zeng

et al. 2015

Proc. R. Soc. B.

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Metamorphosis in insects includes a series of programmed tissue histolysis and remolding processes that are controlled by two major classes of hormones, juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase (EO), a 20E inactivation enzyme, expresses predominantly in the midgut during the early pupal stage in the lepidopteran model insect, Bombyx mori. Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system extended the duration of the final instar larval stage. Ubiquitous transgenic overexpression of BmEO using the Gal4/UAS system induced lethality during the larval-pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset of metamorphosis was blocked. Transmission electron microscope and LysoTracker analyses showed that the autophagy pathway in MSG is inhibited by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the genes involved in autophagic cell death and the mTOR signal pathway are affected by overexpression of BmEO. Taken together, BmEO functional studies reported here provide insights into ecdysone regulation of tissue degeneration during metamorphosis.

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Selection of reference genes for tissue/organ samples on day 3 fifth‐instar larvae in silkworm, Bombyx mori

Wang

Chen

Zhang

et al. 2018

Arch Insect Biochem Physiol

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The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth-instar B. mori larvae using geNorm and NormFinder. The results revealed that, using the combination of the expression of BGIBMGA003186 and BGIBMGA008209 was the optimum choice for normalizing the expression data of the B. mori tissue/organ samples. The most stable gene, BGIBMGA003186, is recommended if just one reference gene is used. Moreover, the commonly used reference gene encoding cytoplasmic actin was the least appropriate reference gene of the samples investigated. The reliability of the selected reference genes was further confirmed by evaluating the expression profiles of two cathepsin genes. Our results may be useful for future studies involving the quantification of relative gene expression levels of different tissue/organ samples in B. mori.

show abstract

Molecular Cloning and Characterization of Ecdysone oxidase and 3-dehydroecdysone-3α-reductase Involved in the Ecdysone Inactivation Pathway of Silkworm, Bombyx mori

Cited by 39 publications

References 37 publications

Ecdysone Titer Determined by 3DE-3β-Reductase Enhances the Immune Response in the Silkworm

Ecdysone Titer Determined by 3DE-3β-Reductase Enhances the Immune Response in the Silkworm

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori

Selection of reference genes for tissue/organ samples on day 3 fifth‐instar larvae in silkworm, Bombyx mori

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