昆虫变态蜕皮发生中几丁质蛋白复合体降解的分子机制

Insect molting is an important part of metamorphosis during their life-cycle. Reveal the molecular mechanism of procedural degradation of chitin-protein complex in the cuticle; turn to the core issue of understanding the molting of insect in metamorphosis. This project, resulting from the long-term accumulation of research on the molecular biology of insect development, is based on that molting may depend primarily on the degradation of the proteins around the chitin, rather than the results of chitin hydrolysis. This study intends to focus on the molting concerning Choristoneura fumiferana serine protease (CfSPase) and larval cuticle protein (CfLCP) obtained from the epidermis by using proteomics technology and cDNA library screening methods.The method of immunohistochemical redyeing and multidyeing will be used to: detect the distribution of CfLCPs in the cuticle during molting; determine the CfLCPs perform in the degradation of the chitin-protein complex; to use protein expression, biochemistry, and molecular biology techniques to examine the physiological and molecular biological relationship between CfLCPs and CfSPases in different stages during cuticle degradation. Thus proving the procedure of the CfLCP mediated CfSPase digesting to expose chitin fibers, which cause the degradation of cuticle during insect molting. This study will not only establish the important theoretical basis for recognizing the molting mechanism of insect metamorphosis, but also to provide the scientific foundation for genetically engineering intelligent insecticides.

昆虫变态发育中蜕皮是其完成生活史的重要环节。表皮几丁质蛋白复合体程序性降解及分子机制，成为认识昆虫变态发育蜕皮发生的核心问题。本研究立足于长期对昆虫发育分子生物学的研究积累，基于前期揭示的蜕皮发生可能首先依赖蛋白酶对包被几丁质蛋白的降解，而非几丁质被酶解的研究结果，针对以蛋白质组学技术与文库筛选方法获得的蜕皮相关Choristoneura fumiferana丝氨酸蛋白酶（CfSPase）与幼虫表皮蛋白（CfLCP），拟利用免疫组化复染及套染等方法，研究蜕皮期表皮中CfLCPs分布，确定几丁质蛋白复合体程序降解相关CfLCPs；利用蛋白表达和生化与分子生物学等技术方法，确定不同降解阶段酶解CfLCPs的相关CfSPases；进而探明蜕皮发生中几丁质和CfLCP降解及其CfSPase作用的时序性，为认识昆虫变态中蜕皮发生机制奠定重要的理论基础，并可为基因工程创建智能生物杀虫剂提供科学的依据。

本项为期一年的研究其主旨是通过分析六种云杉夜蛾（Choristoneura fumiferana）幼虫表皮蛋白（LCP）的发育表达及结构性分布，确定幼虫蜕皮过程中启动离层（Apolysis）发生的节点蛋白，为逆向探索表皮生物降解相关的关键蛋白酶提供必要的理论基础。LCP分布的调查主要通过表达的蛋白制作抗体探针并利用免疫组化的方法来进行。利用构建的六种重组表达载体对目标蛋白基进行了重组表达，表达的检测结果表明 LCP-12、LCP-14、LCP-17、LCP-18、LCP-19 的重组蛋白表达量较高，通过对表达的蛋白进一步纯化，CFLCP14 和CFLCP19 已经获得了制作抗体所需要量的纯化蛋白。其中，CFLCP16表达不稳定。目前，LCP-16蛋白的表达和抗体的制作工作正在进行中。