Li Zhiqian, You Lang, Zeng Baosheng, Ling Lin, Xu Jun, Chen Xu, Zhang Zhongjie, Palli Subba Reddy, Huang Yongping, Tan Anjiang
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China.
Department of Entomology, College of Agriculture, University of Kentucky, S-225 Agriculture Science Center North, Lexington, KY 40546, USA.
Proc Biol Sci. 2015 Jun 22;282(1809):20150513. doi: 10.1098/rspb.2015.0513.
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.
昆虫的变态包括一系列由两类主要激素（保幼激素和蜕皮甾体）控制的程序性组织溶解和重塑过程。蜕皮甾体的精确脉冲（最活跃的蜕皮甾体是20-羟基蜕皮酮，20E）受生物合成和代谢的调节。在本研究中，我们发现蜕皮酮氧化酶（EO），一种20E失活酶，在鳞翅目模式昆虫家蚕的蛹早期主要在中肠表达。使用转基因CRISPR/Cas9（成簇规律间隔短回文重复序列/RNA引导的Cas9核酸酶）系统敲除BmEO延长了末龄幼虫阶段的持续时间。使用Gal4/UAS系统在全身转基因过表达BmEO会在幼虫-蛹转变期间导致死亡。当BmEO在中部丝腺（MSG）中特异性过表达时，变态开始时MSG的退化被阻断。透射电子显微镜和LysoTracker分析表明，MSG中的自噬途径受到BmEO异位表达的抑制。此外，RNA测序分析显示，参与自噬性细胞死亡和mTOR信号通路的基因受到BmEO过表达的影响。综上所述，本文报道的BmEO功能研究为变态过程中蜕皮酮对组织退化的调节提供了见解。
