【作者】 刘磊；

【导师】 计山明；

【作者基本信息】 安徽农业大学 ， 微生物学， 2021， 硕士

【摘要】 果蝇先天免疫系统能够对不同病原体的感染做出快速有效的应答,其中Toll信号通路在进化上高度保守,且和哺乳动物TLR途径具有较高的相似性,对果蝇的早期胚胎发育和免疫至关重要。在果蝇脂肪体中,Toll信号通路能够激活并诱导抗菌肽的产生,是果蝇抵抗革兰氏阳性菌和真菌感染的重要途径,对维持果蝇的体液免疫稳态至关重要。本研究以模式生物果蝇为研究对象,分别从分子、细胞以及个体的水平探究了E3泛素连接酶相互作用蛋白3(Sip3)对Toll信号通路的调控作用,主要研究结果如下:1.通过大通量筛选在S2细胞中确定sip3可能是Toll通路的正调控因子,对sip3结构域以及表达谱进行分析,确定其含有TM、RING、LC三个较经典的结构域以及在果蝇脂肪体中表达量较高。2.通过双荧光素酶报告系统以及荧光定量PCR实验表明:过表达sip3以及特异性敲低sip3在体外S2细胞中均能正调控果蝇Toll信号通路。3.通过遗传杂交手段获得脂肪体组织特异性敲低sip3转基因果蝇,双荧光素酶报告系统以及荧光定量PCR实验表明:特异性敲低sip3在果蝇体内正调控Toll信号通路。4.果蝇寿命实验表明:敲低sip3转基因果蝇显著抑制果蝇感染藤黄微球菌后抗菌肽的表达,并且能够降低果蝇感染藤黄微球菌后的存活率。更多还原

【Abstract】 The innate immune system of Drosophila can respond quickly and effectively to the infection of different pathogens,in which Toll signaling pathways are highly conserved in evolution and have high similarity with mammalian TLR pathways.It is essential for early embryonic development and immunity of Drosophila.Toll signaling pathway can activate and induce the production of antimicrobial peptides in drosophila adiposomes,which is an important way for drosophila to resist gram-positive bacteria and fungal infections,and is essential to maintain the humoral immune homeostasis of drosophila.This study investigated the effects of E3 ubiquitin ligase Sip3 on Toll signaling pathway from molecular,cellular and individual levels.The results are as follows:1.To determine in S2 cells sip3 may be a positive regulator of Toll pathways.Through the analysis of sip3 domain and expression spectrum,which has three classical domains TM、RING、LC biological functions were determined.2.The results of double luciferase reporting system and fluorescence quantitative PCR showed that overexpression sip3 and specific knockout sip3 could positively regulate the signal pathway of Drosophila Toll in vitro.3.Through genetic hybridization,adipose tissue specific knockout sip3 transgenic Drosophila,double luciferase reporting system and fluorescence quantitative PCR experiments showed that specific knockout sip3 positively regulated Toll signaling pathway in Drosophila.4.Drosophila longevity experiments showed that sip3 transgenic Drosophila significantly inhibited the expression of antimicrobial peptides after Drosophila infection with Trichoderma lucidum,and could reduce the survival rate of Drosophila infection with Trichoderma lucidum.更多还原