基于球囊菌感染的蜜蜂转录组测序及部分差异基因克隆与序列分析

发布时间：2018-08-27 12:22

【摘要】：蜜蜂作为一种重要的传粉昆虫在全球农作物和生态农业中发挥着重要的作用。然而,疾病和环境因素的协同作用共同威胁着蜜蜂健康及养蜂行业的持续发展,并可能导致蜂群的大量损失。在所有蜜蜂传染病中,白垩病一直处于上升趋势。为了更好的探究蜜蜂应对球囊菌Ascosphaera apis的免疫反应,我们利用临床分离的一株蜜蜂白垩病病原真菌蜂球囊菌,对蜜蜂幼虫进行人工感染,进行了基于高通量测序的蜜蜂幼虫应对蜂球囊菌感染的转录组学分析,并对部分显著差异表达的免疫相关基因进行克隆测序及生物信息学分析,以期为蜜蜂的抗病基因的筛选及分子标记提供宝贵的基因资源,为今后进一步深入研究蜜蜂免疫防御相关分子机制及基因功能提供一定的理论依据。1.蜜蜂球囊菌的分离与鉴定本研究从临床上疑似白垩病感染的蜂群患病幼虫中,通过分离培养出一株致病性真菌,根据真菌形态学及18s核糖体RNA区域的通用引物进行分子生物学鉴定,确定该菌为蜜蜂白垩病病原真菌—蜜蜂球囊菌(Ascosphaera apis),该致病菌的分离鉴定与培养,将为后续的实验研究提供基础。2.基于球囊菌感染的蜜蜂转录组学分析通过饲喂蜂球囊菌孢子进行人工感染,利用转录组学分析,基于Illumina RNA测序技术和序列拼接从实验组和对照组中共获得50175666、42001818条unigenes。从文库中筛选到2890个差异表达的基因。显著性分析发现,在健康的蜜蜂幼虫和患白垩病幼虫中共有2214个表达上调基因和676个表达下调基因。GO富集分析及Pathways富集分析在蜜蜂球囊菌病原压力下的主要基因家族及其相关途径,筛选在转录水平免疫相关基因的差异表达。在这项研究中,我们发现蜜蜂幼虫机体中参与球囊菌反应的几个关键的免疫相关转录途径JAK-STAT信号通路、NF-κB信号通路、Toll样受体信号通路的显著差异表达及协同激活作用,这些途径可能导致抗微生物活性物质及抗菌肽的产生。3.部分差异基因的克隆与序列分析根据转录组测序获得显著差异表达的基因数据,设计特异性引物对蜜蜂髓样分化因子(myeloid differentiation factor 88,MyD88),蜜蜂抗菌肽基因家族Abaecin,Hymenoptaecin,Defensin 1基因CDs区序列全长进行扩增并构建克隆载体,对相关序列进行系统发育分析、氨基酸组成、抗原指数、抗原表位、二级结构及三级结构预测的相关生物信息学分析,为今后进一步研究相关基因及蛋白功能提供一定的理论参考。
[Abstract]:As an important pollinating insect, honeybee plays an important role in global crops and eco-agriculture. However, the synergistic effects of disease and environmental factors threaten the health of honeybee and the sustainable development of beekeeping industry, and may lead to a large number of loss of bee colony. In order to better explore the immune response of honeybees to Bacillus spp. Ascosphaera apis, we used a clinically isolated pathogenic fungus, Bacillus honeybees, to artificially infect bee larvae, and analyzed the transcriptome of bee larvae against Bacillus spp. infection based on high-throughput sequencing. Cloning, sequencing and bioinformatics analysis of the expressed immune-related genes were carried out in order to provide valuable gene resources for screening and molecular markers of disease-resistant genes in honeybee, and provide theoretical basis for further study on molecular mechanism and gene function of immune defense in honeybee. 1. Isolation and identification of Bacillus honeybee A pathogenic fungus was isolated from the larvae suspected to be infected by chalk disease. The fungus was identified as the pathogenic fungus of chalk disease, Ascosphaera apis, according to the morphology of the fungus and the universal primers of 18S ribosomal RNA region. Based on the transcriptome analysis of bee infected by Bacillus mellifera, 50175666,42001818 unigenes were obtained from the experimental group and the control group by feeding Bacillus mellifera spores for artificial infection, transcriptome analysis, Illumina RNA sequencing and sequence splicing. 2890 differentially expressed genes were screened. Significance analysis showed that 2214 up-regulated genes and 676 down-regulated genes were found in healthy bee larvae and chalk disease larvae. GO enrichment analysis and Pathways enrichment analysis were used to analyze the major gene families and their related pathways under the pathogenic pressure of Bacillus honeybee, and transcriptional levels were screened. In this study, we found that JAK-STAT signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, which are key immune-related transcription pathways involved in the response of the bee larvae to Bacillus cerevisiae, are significantly differentially expressed and synergistically activated. These pathways may lead to antimicrobial activity. Production of substances and antimicrobial peptides. 3. Cloning and sequencing of some differentially expressed genes were obtained by transcriptome sequencing. Specific primers were designed to sequence the CDs of honeybee myeloid differentiation factor 88 (MyD88), honeybee antimicrobial peptide gene family Abaecin, Hymenoptaecin, and Defensin 1 genes. The clone vector was amplified and constructed. The phylogenetic analysis, amino acid composition, antigen index, antigen epitope, secondary structure and tertiary structure prediction were carried out. The results will provide a theoretical reference for further study of related genes and protein functions.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S895

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