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Functional characterization of two paralogous JH receptors, methoprene-tolerant 1 and 2, in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

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Abstract

Methoprene-tolerant (Met) is a bHLH-PAS transcription factor that functions as a core component of juvenile hormone (JH) receptors in insects. The genome of the silkworm, Bombyx mori (L.), encodes two Met paralogs, BmMet1 and BmMet2. We have previously proposed that BmMet2 forms a functional complex with steroid receptor co-activator (BmSRC) in the presence of JH and induces Krüppel-homolog 1 (BmKr-h1). In this study, we characterized BmMet2 domains and examined the role of BmMet1. A series of reporter and two-hybrid assays on BmMet2 revealed that the bHLH domain played a critical role in the binding to the JH response element of BmKr-h1 (kJHRE), the PAS-A in the transcriptional activation, and the PAS-B in the JH recognition. Furthermore, we confirmed that the BmMet2/BmSRC complex recognizes the core kJHRE (GGCCTCCACGTG). BmMet1 was found to interact with BmSRC and weakly activated the kJHRE reporter in a JH-dependent manner. The BmMet1 transcript level was much higher than the BmMet2 transcript level in the epidermis during the larval and pupal stages. By contrast, the BmMet2 transcript was hardly detected during the larval stage, but was abundant in the adult stage. These results suggest that BmMet1 is involved in the JH-dependent induction of BmKr-h1 in larvae, whereas BmMet2 is involved in adults.

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Acknowledgments

This study was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), JSPS KAKENHI grant nos. 25850230 and 25252059, and the NIAS Strategic Research Fund.

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  1. National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki, 305-8634, Japan

    Takumi Kayukawa & Tetsuro Shinoda

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  1. Takumi Kayukawa
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  2. Tetsuro Shinoda
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Correspondence to Tetsuro Shinoda.

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13355_2015_345_MOESM1_ESM.tif

Fig. S1. Interaction of BmMet1 and BmSRC with kJHRE in S2 cells. S2 cells were cotransfected with a kJHRE reporter carrying firefly luciferase (pGL4.14), a reference reporter plasmid carrying Renilla luciferase (pRL-TK), and an expression plasmid carrying native BmMet1 and/or BmSRC. Transfected cells were treated with 10 μM methoprene (JHA) for 24 h, and reporter activities were measured using a Dual-Luciferase reporter assay system. The activity of the firefly luciferase reporter was normalized to that of the Renilla luciferase reporter in the same samples. Data represent mean ± SD (n = 3). Data were analyzed using Student’s t tests (***p < 0.001; **p < 0.01; *p < 0.05; not indicated, p > 0.05) (TIFF 115 kb)

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Kayukawa, T., Shinoda, T. Functional characterization of two paralogous JH receptors, methoprene-tolerant 1 and 2, in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Appl Entomol Zool 50, 383–391 (2015). https://doi.org/10.1007/s13355-015-0345-8

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