BdDUOX和BdRelish在橘小实蝇肠道微生物群落稳态维持中的作用机理

It is well known that huge quantity of microbes inhabit in the digestive tract of vertebrates and invertebrates where they establish a symbiotic relationship with the host. Literatures suggested that an important function for the commensal microbiota in supplying essential nutrients, metabolizing indigestible compounds, defending against colonization by opportunistic pathogens and promoting the development and regulation of the mucosal immune system. Therefore, the gut epithelium seemly tolerates the proliferation of resident bacteria to a certain extent to allow beneficial interactions to take place. Because of the conservative cell wall of commensal bacteria and pathogenic bacteria, it is seemly very difficult for host gut cells to discriminate and mount the immune defenses to eliminate the pathogenic bacteria. To the best of our knowledge, it is still known little about how the gut epithelium tolerates and regulates the commensal bacteria while retaining the ability to trigger an efficient immune response once infected with pathogens. The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most destructive pests of agricultural fruit in many South East Asian and the Pacific countries, with the larval stages feeding on a wide range of fruits and vegetables. Previous study discovered there was a very complex bacterial community habored by the gut of B. dorsalis. In the current study, we firstly cloned the full length of two key regulating genes such as BdDUOX and BdRelish on the production of vital effectors containing reactive oxygen species and antimicrobial peptides, and then elucidated the molecular mechanisms underlying the maintaining of gut microbiota homeostasis in B. dorsalis by RNAi experiments coupled with fluorescent-labeled quantifying techniques. In arthropods, the alteration of the gut microbial climax community significantly has been documented to be linked to health and fitness disequilibrium. Thus the address of the scientific questions in the study will provide novel targets to develop new ways of integrative management on this pest agent.

在多细胞动物的肠道中栖居着丰富且复杂的微生物群落。而且，该肠道微生物群落参与了宿主诸如生长发育、免疫和繁殖等许多关键的生理过程。但是，这些微生物如何在宿主肠道中定殖以及宿主肠道如何调控该微生物群落以和谐共存的机理目前还不清楚。橘小实蝇Bactrocera dorsalis Hendel是我国的一种毁灭性果蔬害虫。前期研究发现，在橘小实蝇肠道中生活着复杂的细菌群落。本项目拟首先采用分子克隆方法获取与橘小实蝇肠道活性氧、抗菌肽产生相关的两个关键调控基因BdDUOX和BdRelish，随后整合RNA干扰方法和荧光定量检测等技术手段从基因表达水平、生理生化层次以及肠道菌群变化三个层次阐明上述基因在橘小实蝇肠道微生物稳态维持中的作用机理。肠道微生物群落稳态的维持是多细胞动物正常生存的关键前提。因此，该研究能够为该害虫的综合治理新技术开发提供新的科学思路和作用靶标。

橘小实蝇Bactrocera dorsalis Hendel是一种严重危害我国果蔬安全生产的毁灭性害虫。前期研究证实，橘小实蝇的肠道中栖居着复杂的细菌群落。已有证据发现肠道菌群在参与了宿主许多关键的生理过程，例如宿主的生长发育、免疫防御和繁殖等。然而，宿主如何耐受和调控其肠道菌群的机理尚不清楚。紧密围绕该科学问题，我们首先克隆了可能与肠道菌群调控相关的基因BdDuox和BdRelish全长cDNA序列，然后采用RNA干扰技术分析了它们在该过程中所扮演的角色。研究发现，当橘小实蝇被饲喂非肠道细菌后，其肠道的BdDuox基因表达量明显增加，同时肠道内的活性氧ROS含量也显著地增高；当给饲喂抗氧化剂维生素C时，肠道内的菌群数量显著增多；而且当采用RNAi手段降低表达量后，橘小实蝇肠道菌群的数量明显增加。这些数据说明BdDuox基因介导的ROS在橘小实蝇肠道菌群稳态的维持和调控中扮演关键角色。当橘小实蝇被注射感染细菌和喂食感染非肠道细菌后，其脂肪体和肠道中的BdRelish基因表达丰度均呈现显著的增加；而且当BdRelish的表达量被RNAi干扰降低后，橘小实蝇体内的抗菌肽表达量显著降低，而且注射感染细菌后，试虫的死亡率明显高于对照组。这些结果说明橘小实蝇BdRelish介导的免疫反应在其系统免疫和肠道菌群稳态的维持和调控中具有重要作用。另外，我们的研究还发现随着橘小实蝇从幼虫到蛹的发育，其体内的免疫活性水平明显增加，但是到成虫阶段后，其体内的免疫活性显著降低。同时，交配后雌虫和雄虫的体液免疫活性明显高于处女雌虫和雄虫的体液免疫活性。这表明，交配能够显著地上调橘小实蝇成虫的免疫活性。本项目研究结果证实BdDuox介导的ROS和核转录因子BdRelish介导的免疫反应在橘小实蝇肠道菌群稳态的维持和调控中具有重要作用而且橘小实蝇还具有一种灵活的免疫投资策略，同时这些结果的获取也为靶向橘小实蝇肠道菌群稳态调控的新防控策略的研发提供了关键的科学依据。