《环境昆虫学报》

作者	单位
许亚婷，杨荣蓉，吴洪鑫，陆永跃，金丰良，许小霞	华南农业大学植物保护学院昆虫学系，广东省生物农药创制与应用重点实验室，“一带一路”农业有害生物绿色防控科技产业创新院，广州 510642

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中文摘要:

为探究红火蚁Solenopsis invicta Buren Toll受体家族基因如何免疫响应绿僵菌Metarhizium的侵染。本研究采用浸渍法测定了不同浓度下绿僵菌对红火蚁大中小3种不同大小工蚁的致病力，并用显微镜观察了绿僵菌侵染后红火蚁3种工蚁的表型变化。利用生物信息学筛选Toll受体基因，并对Toll受体家族基因的理化性质、结构域、染色体位置、系统进化进行分析鉴定。运用实时荧光定量PCR（RT-qPCR）检测了红火蚁大中小型工蚁中Toll受体家族的发育历期和绿僵菌侵染后的表达模式。结果表明，绿僵菌在96 h对红火蚁大中小型工蚁的致死中浓度（LC50）分别为5.8×10^7孢子/mL、3.1×10^7孢子/mL、1.5×10^7孢子/mL；绿僵菌侵染红火蚁第3天，显微镜下观察到红火蚁体壁细小的菌丝，虫体僵硬，寄主死亡后菌丝迅速蔓延并逐渐长成橄榄绿色分生孢子；RT-qPCR结果表明Toll受体基因在红火蚁不同发育阶段中的mRNA表达水平存在显著差异，且Toll受体基因在雌性生殖蚁表达水平显著高于雄性生殖蚁；RT-qPCR结果表明红火蚁大中小型工蚁Toll受体免疫响应绿僵菌不一样。在大型工蚁中，绿僵菌侵染后，Toll受体家族基因能显著上调表达，6 h是一个诱导高峰期，Toll2-1和LRR转录水平最高，响应绿僵菌最强；在中型工蚁中，绿僵菌不能刺激Toll2-2基因的表达，却能强烈诱导Toll1，Toll2-1，Toll6，Toll7和LRR基因的上调表达，Toll1和Toll2-1响应绿僵菌最强。在小型工蚁中，绿僵菌能显著诱导Toll受体家族基因基因的上调表达，在24 h时，LRR基因表达量最高，相比于对照，LRR基因表达提高25倍，LRR在6 h和24 h响应绿僵菌最强。以上研究表明Toll受体可以免疫响应入侵的绿僵菌，且不同的Toll对绿僵菌可能具有不同的响应机制。本研究为进一步阐明Toll受体的功能奠定基础，为进一步利用绿僵菌控制红火蚁提供技术指导。

英文摘要:

To investigate the immune response of Toll receptor family gene of Solenopsis invicta to Metarhizium anisopliae infection. In this study, the impregnation method was used to determine the pathogenicity of M. anisopliae infection three different grades of worker S. invicta, and the phenotypic changes of S. invicta after M. anisopliae infection were photographed with the microscope. Based on the genome of S. invicta, the Toll receptor gene family sequence were screened by bioinformatics analysis, the physical and the chemical properties, structural domain, chromosomal location and phylogenetic evolution of the Toll receptor family gene were analyzed.Real time quantitative PCR (RT-qPCR) was used to analyze the expression patterns of the Toll receptor family of developmental stage in major, medium and minor-sized worker ants and the induced expression patterns of the Toll receptor family in ants infected by M. anisopliae. The results showed that the lethal medium concentration (LC50) at 96 h of M. anisopliae in major, medium and minor-sized worker ants. LC50 were 5.8×10^7 spores /mL, 3.1×10^7 spores /mL and 1.5×10^7 spores /mL, respectively. On the third day of M. anisopliae infection, small mycelia could be observed on the body of S. invicta. The mycelia spread rapidly and gradually grew into olive green conidia after the death of the host. The results of RT-qPCR indicated that the mRNA level of Toll receptor gene was significantly different in developmental stages of S. invicta, especially, the expression level of Toll receptor family in female were much higher than in male S. Invicta. The RT-qPCR results also showed that the immune response of Toll receptors to M.anisopliae was different. In major-sized worker ants, the expression level of Toll receptor family genes were significantly upregulated after M. anisopliae infection, and the induction peak was 6 h. The transcription levels of Toll2-1 and LRR were the highest, and the response to M. anisopliae was the strongest. In medium-sized worker ants, M. anisopliae could not stimulate the expression of Toll2-2 gene, but could strongly induce the up-regulated expression of Toll1, Toll2-1, Toll6, Toll7 and LRR genes, and Toll1 and Toll2-1 were the most responsive to M. anisopliae. In the minor-sized worker ants, M. anisopliae could significantly induce the up-regulated expression of Toll receptor family genes. The LRR gene was the highest expression level with 25 times higher than the control at 24 h post infection. The LRR was the most responsive to M. anisopliae at 6 h and 24 h. These studies indicated that Toll receptors could be immune to invading M. anisopliae, and different Toll might have different mechanism response against M. anisopliae. This study laid the foundation for further elucidate the function of Toll receptor and provided technical guidance for further control of S. invicta by M. anisopliae.

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