Expression, subcellular localization and protein‑protein interaction of four isoforms of EcR/USP in the common cutworm
Expression, subcellular localization and protein‑protein interaction of four isoforms of EcR/USP in the common cutworm
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DOI：
10.1111/1744-7917.12101
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发表时间：
2015-02
期刊：
影响因子：
4
通讯作者：
Li‐Xia Huang;Yan-Jun Gong;J. Gu;Baojuan Zeng;Li-Hua Huang;Q. Feng
中科院分区：
文献类型：
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作者：
Li‐Xia Huang;Yan-Jun Gong;J. Gu;Baojuan Zeng;Li-Hua Huang;Q. Feng
蜕皮激素受体（EcR）和超气门（USP）形成异二聚体，在蜕皮和变态过程中介导蜕皮激素信号。已经报道了各种EcR/USP异二聚体。然而，目前尚不清楚鳞翅目昆虫在幼虫-蛹变态期间采用何种EcR/USP组合，以及EcR/USP异二聚体是否在不同组织中存在差异。为了解决这些问题，每个EcR和USP的两个异构体从普通地老虎克隆，其信使RNA的表达模式进行了研究，通过真实的实时定量聚合酶链反应在不同组织中的幼虫蛹变态和中肠响应激素诱导。此外，它们的亚细胞定位和蛋白质-蛋白质相互作用分别通过瞬时表达和远蛋白质印迹法进行了探索。所有四种基因在包皮和/或包皮中均显著上调。EcRB 1和USP 1在表皮、脂肪体和中肠中的表达谱几乎相同，EcRA和USP 2也存在类似的情况。除USP 2外，这三个基因对20-羟基蜕皮激素（20 E）诱导有反应，USP 1可被20 E和保幼激素上调。这4种蛋白主要定位于细胞核内，20 E促进了细胞核定位。在体外发现每个EcR和USP之间的蛋白质-蛋白质相互作用。这些结果表明，地老虎体内可能同时存在两种EcR/USP异二聚体（EcRA/USP 2和EcRB 1/USP 1），后者在幼虫-蛹变态过程中可能发挥更重要的作用。此外，EcR/USP异二聚体的类型在变态过程中经历组织溶解和再生的组织中没有变化。
Ecdysone receptor (EcR) and ultraspiracle (USP) form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval‑pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real‐time quantitative polymerase chain reaction in different tissues during the larval–pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein‑protein interaction were explored by transient expression and far‐western blotting, respectively. All the four genes were significantly up‐regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20‐hydroxyecdysone (20E) induction except for USP2, and USP1 could be up‐regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein‑protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer (EcRA/USP2 and EcRB1/USP1) may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval‑pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.