昆虫学报 ›› 2019, Vol. 62 ›› Issue (5): 527-534.doi: 10.16380/j.kcxb.2019.05.001

• 研究论文 •    下一篇

蜕皮激素对家蚕脂肪体中BmATG8蛋白亚细胞定位与细胞核皱缩的调控

张淑尧, 戴易晨, 吴坤钟, 李暑燕, 曹阳, 邓小娟*, 田铃*   

  1.  (华南农业大学动物科学学院, 广东省农业动物功能基因组学与分子育种重点实验室/广东省蚕桑工程技术研究中心, 广州 510642)
  • 出版日期:2019-05-20 发布日期:2019-05-16

Regulation of ATG8 subcellular localization and nuclear shrinkage by 20-hydroxyecdysone in the fat body of Bombyx mori

ZHANG Shu-Yao, DAI Yi-Chen, WU Kun-Zhong, LI Shu-Yan, CAO Yang, DENG Xiao-Juan*, TIAN Ling*   

  1.  (Key Laboratory of Agroanimal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Science, South China Agricultural University, Guangzhou 510642, China)
  • Online:2019-05-20 Published:2019-05-16

摘要:

【目的】昆虫脂肪体是物质合成代谢、先天免疫的重要器官。ATG8蛋白的亚细胞定位是细胞自噬的主要指标之一,细胞核皱缩是细胞凋亡的形态标记之一,目前家蚕Bombyx mori中尚未在蜕皮和变态发育进程中对BmATG8蛋白的细胞生物学变化进行观察。本研究旨在同时检测家蚕脂肪体细胞中BmATG8蛋白亚细胞定位和细胞核皱缩的时空变化,研究蜕皮激素(20E)信号对两者的调控作用。【方法】利用免疫荧光和Hoechst染色方法,分别在家蚕幼虫4龄第2天至预蛹第2天、5龄第2天幼虫注射20E (10 μg/头)后以及对游走期幼虫脂肪体中20E受体基因usp进行RNAi后,检测家蚕脂肪体中BmATG8蛋白定位和细胞核形态变化。【结果】在家蚕幼虫蜕皮和幼虫-蛹变态发育时期,BmATG8蛋白高水平存在于脂肪体细胞中,同时细胞核发生皱缩。在正常摄食时期,20E处理(10 μg/头)能够诱导细胞中大量出现BmATG8蛋白且存在于细胞质中并诱导细胞核皱缩。对usp基因进行RNAi后,脂肪体细胞内的BmATG8蛋白显著减少,同时细胞核皱缩减弱。【结论】家蚕BmATG8蛋白不仅在幼虫-蛹变态时期细胞质中大量存在,而且在幼虫蜕皮时期也大量表达,与细胞核的皱缩同时出现,BmATG8蛋白在细胞质中的定位与细胞核皱缩两者均受到20E信号通路的调控。本研究为BmATG8蛋白功能及其调控机制的深入研究提供了重要的科学依据。

关键词: 家蚕, BmATG8蛋白, 细胞核形态, 脂肪体, 发育变化, 蜕皮激素

Abstract: 【Aim】 The fat body is the major site for metabolism and innate immunity in insects. ATG8 protein subcellular localization is one of the key biomarkers for autophagy, and nuclear shrinkage is one morphological characteristic of apoptosis. However, there is a lack of cellular observation of BmATG8 protein during molting and metamorphosis of Bombyx mori. This work aims to determine the spatiotemporal changes in BmATG8 subcellular localization and nuclear morphology in the fat body of B. mori and to explore their regulation by 20-hydroxyecdysone (20E). 【Methods】 By using immunofluorescent staining and Hoechst staining, the changes in BmATG8 localization and nuclear morphology in the fat body of B. mori during day 2 of the 4th instar larva to day 2 of the prepupa, after injection of 20E (10 μg/larva) in the fat body of the day 2 5th instar larvae, and in larvae at the wandering stage after RNAi of 20E receptor gene usp were detected, respectively. 【Results】 During larval molting and larval-pupal metamorphosis of B. mori, BmATG8 protein was highly expressed in the fat body, and meanwhile nuclear shrinkage occurred. The 20E treatment (10 μg/larva) induced BmATG8 expression and its cytoplastic localization as well as nuclear shrinkage at the normal feeding larval stage. RNAi of usp in B. mori larva at the wandering stage decreased the content of cytoplastic BmATG8 and alleviated the nuclear shrinkage in fat body. 【Conclusion】 BmATG8 not only exists in the cytosol of B. mori during the larval-pupal metamorphosis, but also is highly expressed at the larval molting stage, occurring simultaneously with the nuclear shrinkage. Moreover, cytoplastic localization of BmATG8 and nuclear shrinkage are regulated by 20E signaling. This study provides a foundation for further studying the function and regulatory mechanism of BmATG8 in the future.

Key words: Bombyx mori, BmATG8 protein, nuclear morphology, fat body, developmental change, 20-hydroxyecdysone