基于RNA-seq的蜜蜂幼虫肠道响应球囊菌胁迫的免疫应答研究

发布时间：2018-05-16 03:10

本文选题：意大利蜜蜂 + 中华蜜蜂　；参考：《福建农林大学》2017年硕士论文

【摘要】：白垩病是一种最具代表性的蜜蜂真菌病,该病由蜜蜂球囊菌(Ascosphaeraapis,简称球囊菌)特异性侵染蜜蜂幼虫而导致,严重危害养蜂生产。意大利蜜蜂(意蜂)幼虫极易被球囊菌侵染而罹患白垩病,而中华蜜蜂(中蜂)幼虫的球囊菌抗性较强。前期球囊菌的研究主要涉及分类鉴定、培养方法、病原检测、流行病学、侵染过程以及免疫防御等方面,由于参考基因组信息的缺失,球囊菌的分子研究进展缓慢。相关研究主要集中在球囊菌侵染意蜂幼虫的诸多方面,有关球囊菌侵染中蜂幼虫的研究非常滞后。目前,白垩病的组学研究极其有限,有关蜜蜂幼虫响应球囊菌胁迫的转录组学研究未见报道。因此,本研究用107孢子/mL的球囊菌纯化孢子饲喂感染意蜂及中蜂幼虫,利用RNA-seq技术对对照组和处理组蜜蜂幼虫肠道进行深度测序,首先组装并注释中蜂幼虫肠道的参考转录组,进而对两种蜜蜂幼虫肠道响应球囊菌胁迫的免疫应答进行深入分析,获得的主要结果如下:(1)首次通过形态学和分子生物学手段证实从自然蜂群中的中蜂白垩状幼虫尸体上分离的病原为球囊菌,且中蜂幼虫可在实验室条件下通过饲喂接种球囊菌而发生白垩病。(2)成功组装并注释中蜂幼虫肠道参考转录组,可为中蜂及其幼虫的分子生物学及组学研究提供重要的参考信息;基于此转录组数据开发出21个中蜂幼虫的SSR分子标记,可应用于中蜂及其幼虫的基因图谱构建、基因多样性分析以及基因定位等研究,同时说明利用转录组数据大规模开发非模式生物的SSR是一种高效快速的可行途径。(3)通过中蜂幼虫肠道在球囊菌胁迫过程中的差异表达基因(DEGs)分析和趋势分析,发现富集在细胞免疫通路的DEGs中表现为上调趋势的基因数远多于下调趋势,富集在体液免疫通路的DEGs均表现为上调趋势,表明宿主的细胞和体液免疫通路均被球囊菌显著激活,全面解析了中蜂幼虫肠道响应球囊菌胁迫的免疫应答。(4)通过意蜂幼虫在球囊菌胁迫过程中的DEGs分析和趋势分析,发现富集在细胞免疫通路上的部分DEGs表现为上调趋势,而更多DEGs表现为下调趋势,富集在体液免疫通路上的绝大多数DEGs表现为上调趋势,表明宿主的细胞免疫通路被部分激活,而体液免疫通路被显著激活,全面解析了意蜂幼虫肠道响应球囊菌胁迫的免疫应答。
[Abstract]:Chalky disease is one of the most representative fungal diseases of bees. It is caused by the specific infection of bee larvae by Ascosphaeraapis, which seriously endangers the production of beekeeping. The larvae of Italian honeybee (Italian bee) are susceptible to chalkiness, but the larvae of Chinese bee (Chinese bee) are more resistant to Balloon. The previous studies of balloon bacteria mainly involved in classification and identification, culture methods, pathogen detection, epidemiology, infection process and immune defense, etc. Due to the lack of reference genome information, the molecular research progress of the balloon bacteria was slow. The related studies mainly focused on many aspects of the infecting of Italian wasp larvae by Balloon bacteria, and the study on the larvae of Spinel wasps infected by Balloonella spp was very late. At present, the study of chalky disease is very limited, and there is no report on the transcriptome of bee larvae responding to the stress of Balloon. In this study, the purified spores of 107spore / mL spore were fed to infected bee and Chinese bee larvae. The intestinal tract of the control and treated groups were further sequenced by RNA-seq technique. First, the reference transcription group of bee larval intestine was assembled and annotated. Then the immune responses of the intestinal tract of two honeybee larvae in response to the stress of Balloon bacteria were analyzed in depth. The main results obtained are as follows: (1) for the first time, the pathogen isolated from the corpses of the Chinese bee chalkiness larvae in the natural bee colony was proved to be a balloon bacterium by morphological and molecular biological means. And the Chinese bee larvae can be successfully assembled and annotated by inoculation of the chalkiness disease by inoculating Balloonella spp. under laboratory conditions, which can provide important reference information for the molecular biology and stratigraphic study of the Chinese bee larvae and their larvae. Based on the transcriptional data, the SSR markers of 21 larvae were developed, which can be applied to the construction of gene map, gene diversity analysis and gene mapping of Chinese wasp and its larvae. At the same time, using transcriptome data to develop SSR of non-model organisms on a large scale is a feasible and efficient and rapid approach. It can be used to analyze the differentially expressed genes in the intestinal tract of wasp larvae during Balloon stress. It was found that the number of up-regulation genes in DEGs enriched in cellular immune pathway was much more than that in down-regulation, and that DEGs enriched in humoral immune pathway was up-regulated, indicating that the cellular and humoral immune pathways of host were significantly activated by Balloon. The immune response of the intestinal tract of Chinese bee larvae to Balloon stress was analyzed. Through the DEGs analysis and trend analysis, it was found that some of the DEGs enriched in the cellular immune pathway showed an up-regulation trend, through the DEGs analysis and the trend analysis of the sputum larvae during the stress process of the sputum. However, more DEGs showed down-regulation, and most of DEGs enriched in humoral immune pathway were up-regulated, indicating that the cellular immune pathway of host was partially activated, while humoral immune pathway was significantly activated. The immune response of Italian bee larvae to balloon stress was analyzed.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S895

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