【摘要】：蜜蜂作為一種重要的傳粉昆蟲在全球農(nóng)作物和生態(tài)農(nóng)業(yè)中發(fā)揮著重要的作用。然而,疾病和環(huán)境因素的協(xié)同作用共同威脅著蜜蜂健康及養(yǎng)蜂行業(yè)的持續(xù)發(fā)展,并可能導(dǎo)致蜂群的大量損失。在所有蜜蜂傳染病中,白堊病一直處于上升趨勢。為了更好的探究蜜蜂應(yīng)對球囊菌Ascosphaera apis的免疫反應(yīng),我們利用臨床分離的一株蜜蜂白堊病病原真菌蜂球囊菌,對蜜蜂幼蟲進(jìn)行人工感染,進(jìn)行了基于高通量測序的蜜蜂幼蟲應(yīng)對蜂球囊菌感染的轉(zhuǎn)錄組學(xué)分析,并對部分顯著差異表達(dá)的免疫相關(guān)基因進(jìn)行克隆測序及生物信息學(xué)分析,以期為蜜蜂的抗病基因的篩選及分子標(biāo)記提供寶貴的基因資源,為今后進(jìn)一步深入研究蜜蜂免疫防御相關(guān)分子機(jī)制及基因功能提供一定的理論依據(jù)。1.蜜蜂球囊菌的分離與鑒定本研究從臨床上疑似白堊病感染的蜂群患病幼蟲中,通過分離培養(yǎng)出一株致病性真菌,根據(jù)真菌形態(tài)學(xué)及18s核糖體RNA區(qū)域的通用引物進(jìn)行分子生物學(xué)鑒定,確定該菌為蜜蜂白堊病病原真菌—蜜蜂球囊菌(Ascosphaera apis),該致病菌的分離鑒定與培養(yǎng),將為后續(xù)的實(shí)驗(yàn)研究提供基礎(chǔ)。2.基于球囊菌感染的蜜蜂轉(zhuǎn)錄組學(xué)分析通過飼喂蜂球囊菌孢子進(jìn)行人工感染,利用轉(zhuǎn)錄組學(xué)分析,基于Illumina RNA測序技術(shù)和序列拼接從實(shí)驗(yàn)組和對照組中共獲得50175666、42001818條unigenes。從文庫中篩選到2890個差異表達(dá)的基因。顯著性分析發(fā)現(xiàn),在健康的蜜蜂幼蟲和患白堊病幼蟲中共有2214個表達(dá)上調(diào)基因和676個表達(dá)下調(diào)基因。GO富集分析及Pathways富集分析在蜜蜂球囊菌病原壓力下的主要基因家族及其相關(guān)途徑,篩選在轉(zhuǎn)錄水平免疫相關(guān)基因的差異表達(dá)。在這項(xiàng)研究中,我們發(fā)現(xiàn)蜜蜂幼蟲機(jī)體中參與球囊菌反應(yīng)的幾個關(guān)鍵的免疫相關(guān)轉(zhuǎn)錄途徑JAK-STAT信號通路、NF-κB信號通路、Toll樣受體信號通路的顯著差異表達(dá)及協(xié)同激活作用,這些途徑可能導(dǎo)致抗微生物活性物質(zhì)及抗菌肽的產(chǎn)生。3.部分差異基因的克隆與序列分析根據(jù)轉(zhuǎn)錄組測序獲得顯著差異表達(dá)的基因數(shù)據(jù),設(shè)計(jì)特異性引物對蜜蜂髓樣分化因子(myeloid differentiation factor 88,MyD88),蜜蜂抗菌肽基因家族Abaecin,Hymenoptaecin,Defensin 1基因CDs區(qū)序列全長進(jìn)行擴(kuò)增并構(gòu)建克隆載體,對相關(guān)序列進(jìn)行系統(tǒng)發(fā)育分析、氨基酸組成、抗原指數(shù)、抗原表位、二級結(jié)構(gòu)及三級結(jié)構(gòu)預(yù)測的相關(guān)生物信息學(xué)分析,為今后進(jìn)一步研究相關(guān)基因及蛋白功能提供一定的理論參考。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S895

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