两种肠道细菌调控橘小实蝇卵黄生成的分子机制

Bactrocera dorsalis (Hendel) can cause serious damage to the fruits and vegetables, and has been listed as one invasive species of keynote management by Ministry of Agriculture of China. It has received the worldwide attention because of its increasingly severe spread and invasion. Gut bacteria is very important for insect, and their regulation on insect development and reproduction through Insulin, Juvenile hormone (JH), and Ecdysone (20E) signaling pathway has been reported. Though some genes function has been confirmed, the detailed molecular regulating mechanism still need to be further studied. Our previous research showed that two main gut bacteria (Enterbacter and Pseudomonas) of B. dorsalis can regulate its reproduction, including shortening the pre-oviposition period, and increasing the egg production, via activating the Insulin signaling pathway and promoting the yolk protein (YP) production. They can also increase the expression level of some key genes in Insulin, JH and 20E signaling pathway. Based on our previous study, this project is thus aimed to: (1) validate the regulating mechanism how Enterbacter and Pseudomonas activating the transcription and phosphorylation of FoxO; (2) demonstrate the molecular mechanism how two gut bacteria regulated the transcription of Jhamt and Shade via FoxO from host insect; (3) validate the mode of action how JH and 20E regulate Vg expression in fat body and ovary; and then reveal the molecular mechanism of two main gut bacteria in regulating the YP production in B. dorsalis. The results will provide important theoretical basis for the invasion mechanism research of B. dorsalis and the development of new control method named “killing pests via eliminating symbiotic bacteria”.

橘小实蝇严重危害果蔬生产和贸易，是我国重点防控的外来入侵物种之一，因扩散趋势日益严峻受到国内外高度重视。有研究报道，肠道细菌能通过胰岛素（insulin）、保幼激素（JH）和蜕皮激素（20E）通路调控宿主的生长发育和生殖，但其中的分子调控机制有待深入研究。我们初步研究结果表明，肠道细菌Enterbacter和Pseudomonas通过促进橘小实蝇的卵黄生成而缩短其产卵前期，增加其产卵量，并提高insulin，JH和20E信号通路关键基因的表达。本项目拟在前期研究基础上：（1）解析Enterbacter和Pseudomonas触发橘小实蝇FoxO转录和磷酸化的调控机制；（2）阐明两种肠道细菌通过宿主FoxO调控Jhamt和Shade基因转录的分子机制；（3）明确JH和20E对脂肪体和卵巢中卵黄原蛋白表达的调控模式。研究结果将为橘小实蝇入侵机制研究和“抗菌杀虫”防控技术研发提供重要理论依据。

橘小实蝇Bactrocera dorsalis (Hendel)严重危害果蔬生产和贸易，是我国重点防控的外来入侵物种之一，因扩散趋势日益严峻受到国内外高度重视。有研究报道，肠道细菌能通过胰岛素（Insulin）、保幼激素（JH）和蜕皮激素（20E）通路调控宿主的生长发育和生殖，但其中的分子调控机制有待深入研究。我们前期的实验结果显示，橘小实蝇重要肠道共生菌—阴沟肠杆菌Enterobacter cloacae对宿主相关生殖指标有显著影响。因此，本研究选定阴沟肠杆菌和橘小实蝇为研究对象，探究阴沟肠杆菌对橘小实蝇卵巢发育和卵黄生成的影响。研究证实了阴沟肠杆菌对橘小实蝇卵巢发育、卵黄原蛋白（Vg）、JH和20E合成具有显著的促进作用，且发挥该作用的靶组织为脂肪体；从转录水平筛选出阴沟肠杆菌调控的Insulin信号通路及JH和20E合成过程中的关键因子4个，分别是Insulin受体InR，叉头形转录因子FoxO，JH合成基因Jhamt，20E合成基因Shade；利用显微注射技术进一步研究了4个关键因子的功能，表明4个关键基因的干扰均会抑制宿主卵黄原蛋白基因的表达，降低卵黄原蛋白、保幼激素和蜕皮激素的浓度，进而抑制卵巢发育。我们在此基础上初步提出了阴沟肠杆菌调控橘小实蝇生殖的分子机制，即该菌通过胰岛素受体，启动转录因子FoxO，调控保幼激素、蜕皮激素和卵黄原蛋白的合成，进而调控宿主实蝇的卵黄发生和卵巢发育。.本研究筛选出参与橘小实蝇卵黄生成调控的关键肠道细菌，及该肠道细菌促发的调控宿主实蝇生殖发育的关键基因、重要通路和调控网络，初步提出橘小实蝇关键肠道细菌阴沟肠杆菌调控宿主生殖发育的分子机制，能够丰富入侵昆虫的入侵机制研究。这对于揭示肠道细菌调控宿主昆虫生殖发育的分子机制有重要意义，也将为我们揭示橘小实蝇和其共生菌的协同入侵机制，以及在此基础上提出基于“抗菌杀虫”的新防治技术奠定基础，在实蝇精准防控领域具有广阔的应用前景。