棉铃虫滞育过程中ROS对Hsp70的调控及其机制研究

Diapause is a physiological response for insects to adapt to harsh environmental conditions. Diapausing insects possess a prominent feature of increased stress resistance. Hsp70, one of the main molecular chaperone proteins, plays an important role in enhancing stress resistance through assisting in protein folding. Previous researches indicate that Hsp70 is commonly up-regulated during diapause processing. However the regulatory mechanism is unknown. In our previous work, we found that in the cotton bollworm, Helicoverpa armigera, both the levels of Hsp70 and ROS (reactive oxygen species) were higher in diapause pupal brains than in non-diapause ones. Furthermore, raising the ROS level of HzAm1 cells can up-regulate the expression of Hsp70 and influence the activity of the up-stream regulatory protein of the transcription factor HSF1. Given these, we believe that it is possible for ROS to regulate the expression of Hsp70 through activating HSF1. Based on these findings, we will try to confirm the relevance between the ROS level and the expression of Hsp70 in the diapause pupal brains of Helicoverpa armigera in vivo, to reveal how ROS regulates the activity of HSF1, so as to illuminate the regulatory mechanism of Hsp70 expression in diapause. This study will contribute to revealing the molecular mechanism of diapause and provide guidance for developing new agricultural chemicals or strategies for prevention and control of the cotton bollworm, Helicoverpa armigera.

滞育是昆虫对外界环境的一种适应性生理反应，滞育昆虫的一个突出特征是抗逆性强。Hsp70是主要的分子伴侣蛋白，可以促进蛋白质在不利条件下的正确折叠，对提高滞育昆虫的抗逆性起重要作用。以往研究发现昆虫滞育过程中Hsp70的表达普遍上调，但Hsp70响应滞育进程的分子机制尚不明了。我们以棉铃虫为研究对象，发现滞育蛹脑中Hsp70的表达及ROS含量均明显高于非滞育蛹脑，提高棉铃虫细胞ROS含量可促进Hsp70的表达并影响其转录因子HSF1上游调节蛋白的活性。据此，我们推测ROS有可能通过活化HSF1来调节Hsp70的表达。本项目拟在前期工作的基础上，从活体水平进一步证实滞育棉铃虫蛹脑ROS含量与Hsp70表达之间的关联，明确ROS对HSF1活性的调节作用及机理，从而阐明ROS调节Hsp70的具体分子机制。本项目研究有助于从分子水平阐述滞育的机制，为今后开发新型农药和提出新的棉铃虫防治策略提供借鉴。

滞育作为一种特殊的生长发育方式，是昆虫适应恶劣环境条件的重要策略之一。昆虫滞育过程中普遍存在Hsp70表达上调的现象。然而，引起滞育昆虫Hsp70 表达上调的生理条件以及具体的调节机制未见报道。本项目以棉铃虫为研究对象，系统地研究Hsp70 在滞育昆虫中的表达调控机制，从分子水平阐明Hsp70表达与昆虫滞育之间的切实联系。. （1）克隆了棉铃虫HSF1基因，在棉铃虫蛹脑中发现了4个HSF1选择性剪接体，在其它组织中发现了7个HSF1的选择性剪接体。所发现的剪接体均定位在细胞核。. （2）调查了棉铃虫蛹脑中ROS的含量，发现滞育蛹脑中ROS明显高于非滞育蛹脑，提升细胞ROS水平不仅可以上调HSF1的表达，同时还可以促进HSF1的转录活性。通过Real-Time PCR检测了蛹脑中hsp70以及四个HSF1选择性剪接体的表达，它们在滞育蛹脑中的表达均高于非滞育蛹脑，且表达趋势基本一致。. （3）通过启动子活性分析实验，发现HSF1的四个选择性剪接体均可促进hsp70启动子的转录。在hsp70启动子的-101至+56区发现了HSE元件，EMSA实验表明四个HSF1剪接体可以与该元件结合。此外，从蛋白水平进一步证实四个选择性剪接体可以上调hsp70的表达。. 本研究有助于从分子水平阐述滞育的机制，为今后开发新型农药和提出新的棉铃虫防治策略提供借鉴。项目资助已发表和接受文章3篇。