表皮形成关键基因对飞蝗前肠内膜结构及功能的影响机制研究

Insect cuticle and foregut intima are vital barriers against microorganisms from the environment. Both cuticle and foregut intima originate from the ectoderm, and therefore have similar components and functions. Our previous study showed highest expression of the key genes involved in cuticle formation in the foregut, suggesting their important roles in the formation of foregut intima. However, the molecular mechanism controlling foregut intimal formation has virtually not been studied. In this project, we will focus on three key genes known to contribute to the cuticle formation and showed the highest expression in the foregut intima in migratory locust. We will: 1) reveal the formation and structural dynamic changes of the foregut intima during the locust development by using optical microscope and transmission electron microscope; 2) determine the dynamic changes of chitin content in the foregut intima using the combined strategies of chitin staining and optical microscope; 3) localize the proteins encoded by the key genes in the foregut by using immunohistochemistry; and 4) determine the roles of the key genes in the formation of the foregut intima using RNAi coupled with microscopic or ultramicroscopic technology. We will also examine the changes of food intake behavior and food consumption after RNAi. This project is expected to elucidate the functions of the key genes in the formation of the foregut intima, and enhance our knowledge in the development of cuticular structures in the foregut. Results from this study are expected to help develop new strategies for insect management by interrupting insect feeding as the foregut plays critical roles in food passage, storage and initial digestion in insects.

昆虫表皮和前肠内膜是抵御环境微生物等侵害的重要屏障。昆虫前肠内膜和表皮均来源于外胚层，两者具有相似的结构和组成成分。本课题组前期研究发现，表皮形成关键基因在前肠显著高表达，提示其在前肠内膜形成中发挥重要作用。然而，国内外有关昆虫前肠内膜代谢的分子机制尚未见研究报道。本项目拟选择飞蝗前肠高表达的3个表皮形成关键基因开展如下研究工作：1）采用透射电镜观察发育过程中前肠内膜的结构及动态更替；2）几丁质染色结合光学显微镜观察明确内膜几丁质含量的动态变化；3）采用免疫组化的方法，研究表皮形成关键基因在前肠内膜发育过程中的定位；4）采用RNAi结合显微和超微技术研究靶基因对前肠内膜结构影响，同时观察飞蝗取食行为和取食量的变化。通过上述研究，阐明表皮形成关键基因对前肠内膜结构和功能的影响机制，补充和完善表皮发育生物学的研究内容，同时筛选调控取食行为的靶基因，为飞蝗绿色防控提供新的策略和手段。

昆虫表皮和前肠内膜是抵御环境微生物等侵害的重要屏障。昆虫前肠和表皮均来源于外胚层，两者具有相似的结构和组成成分。本课题组前期研究发现，表皮形成关键基因在前肠显著高表达，提示其在前肠中发挥重要作用。本项目选取飞蝗前肠高表达的3类表皮形成关键基因，研究其在飞蝗前肠的分子特性和生物学功能。研究结果如下：1）昆虫发育过程中，前肠内膜经历加厚、新旧内膜溶离、旧内膜降解和新内膜形成的动态发育过程；2）RT-qPCR结果显示关键基因在前肠3个部位稳定表达，且在蜕皮前、后显著高表达；3）免疫组化结果显示关键蛋白定位具有特异性，如LmCDA1定位于内膜原表皮的下部分，LmCDA2定位于内膜原表皮上部分，LmKnk和LmRtv定位于内膜原表皮上部分和肠细胞中；4）RNAi结合显微和超微结果显示LmKnk和LmRtv参与表皮内膜结构的维持。LmKnk和LmRtv沉默后，前肠内膜厚度显著变薄，几丁质含量显著降低，几丁质片层结构消失，但不影响前肠内膜内表面的棘结构，而沉默LmCDA1和LmCDA2后，与对照相比，前肠内膜没有明显变化；5）生物学功能分析显示沉默LmKnk和LmRtv后，飞蝗前肠显著变细，食物残渣显著变少，同时取食量显著降低。通过上述研究，阐明表皮形成关键基因对前肠内膜结构和功能的影响机制，补充和完善表皮发育生物学的研究内容，同时筛选调控取食行为的靶基因，为飞蝗绿色防控提供新的策略和手段。