咪唑类化合物KK-42延迟柞蚕蛹滞育解除的调控机理

Insect diapause is one of critical issues of concern in Biology. Several chemical agents have been known to be capable of manipulating the expression of insect diapause, thus not only provide insights into mechanisms regulating diapause but also potentially offer tools for diapause manipulation. Among agents known to alter diapause responses is KK-42 (1-benzyl-5-[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole), an imidazole derivative that functions as an insect growth regulator. Earlier studies demonstrated that this chemical can affect embryonic diapause, and our recent studies reveal that it also influences pupal diapause. Thus, KK-42 is rather unique in being capable of influencing diapause in both different species as well as different diapausing stages of development. The effect of KK-42 on impairment of pupal dipause termination we presented is likely the result of KK-42 modulating ecdysteroidogenesis in pupal prothotacic glands (PGs) by inhibiting ecdysteroid synthesis pathway, or restraining Torso pathway that mediates PTTH-triggered MARK pathway, thereby shunting more into diapause. .To address this hypothesis, in this project, we will chose diapausing pupae of A. pernyi as the model insect, which is not only a classic organism for studying diapause regulation but also an economically important insect used for silk production and as a human food source in China. This project aims to explore the modulatory mechanism of KK-42 on impairment of pupal dipause termination by investigating morphological and developmental changes in the PGs that is the target organ of KK-42, dynamic changing process between KK-42 and ecdysteroids of the titer, effect of KK-42 on the mRNA expression of genes related to ecdysteroid synthesis pathway (Nvd, Spo, Phm, Dib, Sad, and Shd), and effect of KK-42 on the expression of genes related to Torso pathway that mediates PTTH-triggered MARK pathway (Torso, Ras, Raf, MEK, and ERK). Our project will provide insights into understanding the modulatory mechanism of KK-42 on impairment of pupal dipause termination in insects.

昆虫滞育是备受关注的基础性生物学问题。已发现一些化学物质可以改变滞育的表达，它们可为滞育机理的阐明提供线索，也为滞育操控提供了工具。KK-42既可影响柞蚕等的蛹滞育（项目组的研究结果），又可影响家蚕等的胚胎滞育，是已知的唯一既可影响不同物种滞育又可影响不同滞育类型的化合物。KK-42延迟柞蚕等蛹滞育的解除，推测是KK-42抑制了前胸腺的蜕皮激素合成途径，或者抑制了Torso介导的PTTH-MAPK途径，从而影响了滞育解除的进程。项目以KK-42的靶器官——前胸腺为研究对象，对提出的假设进行检验，将追踪KK-42处理后滞育蛹中前胸腺的形态和发育变化；比较KK-42处理后体内KK-42与蜕皮激素动态变化过程；检测KK-42对蜕皮激素合成途径相关基因表达的影响；调查KK-42对Torso途径相关基因表达的影响。结果可为理解KK-42延迟作蚕等昆虫蛹滞育解除的调控机理提供分子生理学方面的证据。

昆虫滞育是一个重要的基础生物学问题。前人研究发现了咪唑类化合物KK-42可影响家蚕等昆虫的胚胎滞育，项目组前一个项目建立了KK-42与蛹滞育的联系。KK-42成为已知的唯一既可影响不同物种滞育又可影响不同滞育类型的化合物。本项目以前胸腺为靶标器官，从形态学、激素、分子三个水平探讨了KK-42延迟蛹滞育解除的调控机制。形态学观察发现，柞蚕蛹的前胸腺至少由120个细胞组成的，无色透明状，滞育解除后25度发育6天的蛹即很难发现；家蚕品种大造和秋白前胸腺的形态结构不同；KK-42对前胸腺细胞并不造成损伤。体外试验发现了KK-42可以结合20-E和破坏20-E的证据。转录组分析揭示了前胸腺和咽侧体的整体基因表达情况，发现蜕皮激素代谢途径相关的8个基因（Neverland、Spook、Phantom、Disembodied、Shroud、Cyp6ul、Non-molting glossy、Ecdysone receptor）和保幼激素代谢相关的5个基因（Aldehyde dehydrogenase、JHEH、cJHBP、JHAMT、JHEBP）在咽侧体和前胸腺中的均有表达，表明这两个内分泌器官存在联系。KK-42对Torso途径（Torso、MEK、ErK、Raf、Ras）的基因表达没有影响；对蜕皮激素生物合成途径（Neverland、Spook、Phantom、Disembodied、Shadow、Cyp6u1、Ecdysone receptor、CYP314A1、CYP18A1、ecdysone 20-hydroxylase）相关基因的表达也没有影响。家蚕蛹和柞蚕蛹KK-42处理组的滞育激素基因表达量均显著低于对照组，表明KK-42抑制了滞育激素基的表达。这些研究结果表明，KK-42对蜕皮激素的影响并不在基因水平，而是由于与蜕皮激素的拮抗作用造成的。重要的发现有二：一是KK-42可以结合20-E和破坏20-E；二是KK-42可以抑制滞育激素基因的表达。这是将昆虫生理学上2个重要激素KK-42与滞育激素直接关联起来的首个证据，在昆虫分子生理学研究领域具有重要意义。