【摘要】 【目的】Toll信号通路是昆虫天然免疫系统的重要组分,其中Toll受体在激活昆虫病原菌侵染免疫应答方面发挥了关键作用。本研究旨在探究斯氏按蚊Anopheles stephensi Toll受体基因在抵抗微生物侵染和维持肠道菌群稳态过程中的功能。【方法】根据冈比亚按蚊Anopheles gambiae Toll受体家族的蛋白氨基酸序列,通过序列同源比对鉴定斯氏按蚊中相应的Toll受体基因;运用荧光定量PCR检测Toll受体基因在未感染病原菌的斯氏按蚊脂肪体中的相对表达量,以及在真菌球孢白僵菌Beauveria bassiana和革兰氏阴性细菌胡萝卜软腐欧文氏菌Erwinia carotovora subsp.carotovora侵染斯氏按蚊过程中的表达变化;最后,在斯氏按蚊雌成蚊胸部显微注射AsToll1A和AsToll5A的双链RNA进行RNA干扰后,检测RNAi处理的斯氏按蚊受真菌侵染后的存活率、肠道细菌含量变化以及抗菌肽基因表达变化。【结果】在斯氏按蚊中共鉴定到8个Toll受体基因,即AsToll1A,AsToll5A,AsToll6,AsToll7,AsToll8,AsToll9,AsToll10和AsToll11。通过荧光定量PCR检测发现,未感染病原菌的斯氏按蚊雌成蚊脂肪体中AsToll5A表达量最高,AsToll1A表达量次之,其余Toll受体基因表达量极低。在球孢白僵菌和胡萝卜软腐欧文氏菌侵染过程中,与对照(注射PBS)比较,AsToll1A和AsToll5A在斯氏按蚊中的表达量显著升高,其余Toll受体基因表达变化不显著或降低。RNA干扰结果表明,AsToll1A或AsToll5A的表达受到抑制后,斯氏按蚊对球孢白僵菌的抵抗能力显著降低,肠道细菌总量与对照(dsGFP)比较显著增多。而且,抑制AsToll1A后抗菌肽基因DEF1和GAM1的表达受到显著抑制;抑制AsToll5A后仅有GAM1表达量下调。【结论】斯氏按蚊Toll受体在结构和功能上具有高度的保守性,其中AsToll1A和AsToll5A能响应病原真菌和革兰氏阴性细菌侵染并且影响肠道菌稳态。更多还原

【Abstract】 【Aim】 Toll signaling pathway is an important component of insect innate immunity. Toll receptors play a key role in activating the immune response to pathogen infection. This study aims to explore the function of Toll receptor genes in resistance to microbial infection and maintaining the homeostasis of gut microbiota in Anopheles stephensi. 【Methods】 The Toll receptor genes in An. stephensi were identified by BLAST search using the published Toll sequences in Anopheles gambiae. The relative expression levels of Toll receptor genes in the fat body of healthy adults of An. stephensi and the adults infected with Beauveria bassiana and Erwinia carotovora subsp. carotovora(Ecc) for different time were detected by real-time quantitative PCR. After the double-stranded RNA of AsToll1 A or AsToll5 A was microinjected into the thorax of female adults of An. stephensi, the survival rates of the RNAi-treated mosquitoes after fungal infection and the total gut bacterial load and the expression levels of antimicrobial peptide genes in the RNAi-treated mosquitoes were detected. 【Results】 Eight Toll receptor genes, i.e., AsToll1 A, AsToll5 A, AsToll6, AsToll7, AsToll8, AsToll9, AsToll10 and AsToll11, were identified in An. stephensi. The real-time quantitative PCR analysis showed that AsToll5 A had the highest expression level in the fat body of healthy female adults of the mosquito, followed by AsToll1 A, and the expression levels of other Toll receptor genes in the fat body were extremely low. Compared to the control(injecting PBS), B. bassiana or Ecc infection significantly induced the transcription of AsToll1 A and AsToll5 A, whereas the expression levels of other Toll receptor genes during pathogen infection showed no significant change. Silencing AsToll1 A or AsToll5 A significantly reduced the resistance of An. stephensi adults to B. bassiana infection, and extremely significantly increased the total gut bacterial load in the adult midgut as compared to the dsGFP-treated control. Moreover, silencing AsToll1 A remarkably inhibited the expression of antimicrobial peptide genes DEF1 and GAM1, while only the expression of GAM1 was down-regulated in the dsAsToll5 A-injected mosquitoes. 【Conclusion】 The Toll receptors in An. stephensi are conserved in structure and function. AsToll1 A and AsToll5 A are highly induced in response to both fungal and Gram-negative bacterial infections, and also regulate the homeostasis of gut microbiota in An. stephensi.更多还原