白假丝酵母菌侵染家蝇幼虫中肠转录组及围食膜蛋白质组学研究

发布时间：2018-03-12 22:24

  本文选题：家蝇　切入点：白假丝酵母菌　出处：《贵州医科大学》2016年博士论文　论文类型：学位论文

【摘要】：目的:利用家蝇与人类机会性致病真菌白假丝酵母菌(Candida albicans)的互作探讨宿主与病原体的相互作用,筛选家蝇(Musca domestica)幼虫中肠应对白假丝酵母菌侵染的应答基因,研究家蝇3日龄幼虫中肠围食膜蛋白质组成成分以及在应对白假丝酵母菌侵染后的变化,进一步对其中筛选的围食膜蛋白基因(Md Pt1)进行研究,为深入研究家蝇幼虫肠道抵御真菌侵染的分子机制提供理论基础。方法:1.采用新一代高通量测序技术对经口感染白假丝酵母菌和空白对照的家蝇幼虫中肠进行测序分析,并筛选差异表达基因;利用生物信息学工具对转录组测序得到的基因进行了功能注释、分类以及参与的信号通路展示。应用荧光定量PCR技术验证选定的20个差异表达基因。2.解剖健康家蝇3日龄幼虫中肠围食膜,并解剖白假丝酵母菌侵染24h后(MD-T)和PBS对照组(MD-C)的围食膜,通过聚丙烯酰胺凝胶电泳检测分离提取的围食膜蛋白,将整条泳道上的蛋白从顶到底切割,胶在酶解消化后做液相色谱-串联质谱分析鉴定。3.用RT-PCR方法从家蝇cDNA中获取Md Pt1基因,利用生物信息学分析工具预测、分析基因编码的蛋白质的结构与生物学功能。将Md Pt1基因克隆到原核表达质粒pET-28a(+)中,在大肠杆菌BL-21/DE3中诱导表达,表达产物经纯化后用以免疫SD大鼠制备免疫血清。用蛋白印迹(Western Blotting)方法检测重组蛋白的几丁质结合特性。结果:1.在白假丝酵母菌侵染24h后,中肠上皮组织通过转录组测序和生物信息学分析,共获得3121个差异表达基因,其中表达上调基因1501个,下调基因1620个;通过对差异表达基因的GO分析,有2531个差异表达基因能够GO富集,在生物过程本体显著富集的是代谢过程、蛋白质水解和氨基聚糖的代谢过程;在细胞组分本体显著富集的是蛋白酶体核心复合体、蛋白酶体复合体以及细胞外区域;在分子功能本体中显著富集的是催化活性、溶菌酶活性以及水解酶活性。KEGG Pathway分析发现,共有1809个DEGs能够富集到115个代谢通路,差异基因主要参与到营养物质代谢、氧化磷酸化以及免疫调节等通路。通过对选取的20个DEGs进行qRT-PCR分析验证结果表明,所选基因表达模式与高通量测序结果完全一致。2.从健康的3日龄幼虫围食膜共鉴定到374个蛋白质,分子量分布在8.225 kDa至996.065 kDa之间,等电点为3.83至11.24之间。家蝇幼虫饲喂白假丝酵母菌24h后解剖PM并提取蛋白质进行质谱鉴定,在MD-T与MD-C组中分别有335、302个蛋白被成功鉴定,共有409个不重复蛋白质,其中MD-T组单独鉴定蛋白质221个,MD-C组单独鉴定蛋白质188个,两组同时表达114个,与对照相比,两组中分子量小于30kDa的条带差异变化明显,显示这些蛋白在家蝇幼虫对白假丝酵母菌的免疫过程中发挥了重要作用。鉴定到的大多数差异蛋白蛋白质主要涉及免疫、消化与营养代谢以及PM结构有关。根据GO注释分析显示,这些蛋白质主要参与模式识别结合、多聚糖的结合,围食膜的结构组成和几丁质结合等相关功能。3.成功获得Md Pt1基因,cDNA全长1063bp,其开放阅读框ORF长711bp,编码236个氨基酸,N端有20个氨基酸的信号肽序列;其蛋白质理论分子量为26503.7 Da,等电点为4.65;结构预测分析显示其包含3个ChtBD2结构域。RT-PCR检测显示Md Pt1只在家蝇幼虫表皮、中肠和气管中有表达。Md Pt1蛋白具有几丁质结合活性,并只能够被强变性剂6M尿素和2%SDS+5%β-巯基乙醇洗脱,属于PM第三类蛋白。结论:1.成功建立白假丝酵母菌侵染家蝇幼虫的肠道感染,比较转录组学分析获得参与免疫应答相关基因;2.374个PM蛋白在健康的家蝇3日龄幼虫被鉴定,白假丝酵母菌侵染24h后,在MD-T和MD-C分别鉴定到335、302个PM蛋白,白假丝酵母菌侵染后围食膜的变化比较大;3.成功克隆Md Pt1基因,验证其具有几丁质结合活性。总之,本研究结果从宿主与白假丝酵母菌之间的相互作用,揭示了家蝇幼虫中肠上皮组织和围食膜在应对白假丝酵母菌侵染过程中的作用,为深入理解家蝇幼虫肠道免疫提供理论基础。
[Abstract]:Objective: using the housefly and human opportunistic pathogenic fungus Candida albicans (Candida albicans) the interaction of interaction between host and pathogen, screening of housefly (Musca domestica) larvae midgut response gene of Candida albicans infection, changes of 3 day old larvae of Musca domestica peritrophic membrane protein composition and in response to the white Candida infection, further screening of the peritrophic membrane protein gene (Md Pt1) were studied to provide theoretical basis for further research on the molecular mechanism of intestinal fungal infection against housefly larvae. Methods: 1. using a new generation of high-throughput sequencing were sequenced by oral infection of housefly larvae midgut and blank yeast the control of Candida, and screening of differentially expressed genes; using bioinformatics tools for transcriptome sequencing of gene functional annotation, classification and participation The signal pathway of.2. gene display. Anatomical health 3 day old larvae of Musca domestica peritrophic membrane of 20 differential expression by fluorescence quantitative PCR technology to verify the selected, and anatomy of Candida albicans infection after 24h (MD-T) and PBS control group (MD-C) of the peritrophic membrane, extraction of peritrophic membrane proteins by polyacrylamide gel the electrophoresis separation, the lanes on the protein from top to bottom cut, glue in enzymatic digestion after tandem mass spectrometry analysis and identification of.3. using RT-PCR method to obtain the Md Pt1 gene from housefly cDNA in liquid chromatography, using bioinformatics analysis tools to predict, structure and biological function of protein analysis of gene encoding. The Md Pt1 gene was cloned into prokaryotic expression plasmid pET-28a (+), induced expression in Escherichia coli BL-21/DE3. The expression products were purified by preparation of immune serum to immune SD rats. By Western blotting (Western Blotting) detection method Chitin binding properties of the recombinant protein. Results: 1. in Candida albicans infection after 24h, midgut epithelium by transcriptome sequencing and bioinformatics analysis, a total of 3121 differentially expressed genes, including 1501 up-regulated genes and 1620 down regulated genes; gene expression analysis by GO of difference gene can GO enrichment 2531 differentially expressed, in the biological process ontology is significantly enriched the metabolic process, metabolic process of protein hydrolysis and glycosaminoglycan; proteasome core complex is in the cellular component ontology significantly enriched the proteasome complex and extracellular region; significantly enriched in molecular function ontology is the catalytic activity, lysozyme activity.KEGG Pathway and hydrolase activity analysis showed that a total of 1809 DEGs can be enriched to 115 metabolic pathways, these genes mainly involved in metabolism, oxidative phosphorylation And the immune regulatory pathway. Through the 20 selected DEGs qRT-PCR analysis results showed that the selected gene expression patterns and high-throughput sequencing results in.2. from 3 instar larvae of healthy peritrophic membrane of 374 proteins were identified, the molecular weight distribution between 8.225 kDa and 996.065 kDa, etc for 3.83 to 11.24. Housefly larvae feeding of Candida albicans 24h dissection after extraction of protein PM and identified in MD-T and MD-C group respectively, 335302 proteins were identified successfully, a total of 409 not repeat protein, MD-T protein identification alone group 221, MD-C group identified 188 separate protein at the same time, two groups of 114, compared with the control group, two groups in the change of molecular weight less than 30kDa with significant differences, showed that these proteins in the immune process of housefly larvae on Candida albicans has played an important role. Most of these proteins identified proteins mainly related to immune, digestive and nutritional metabolism and structure of PM. According to GO annotation analysis showed that these proteins were mainly involved in pattern recognition with combination of polysaccharide, peritrophic membrane structure and chitin binding and other related functions.3. Md succeeded Pt1 cDNA gene, full-length 1063bp, its open ORF long reading frame 711bp, encoding 236 amino acids N terminal signal peptide sequence of 20 amino acids; the theory of protein molecular weight of 26503.7 Da, isoelectric point was 4.65; structure prediction analysis shows that it contains 3 ChtBD2 domain Md Pt1.RT-PCR detected only in the epidermis of housefly larvae, the expression of.Md protein with Pt1 chitin binding activity in the midgut and trachea, and can only be strong denaturant urea 6M and 2%SDS+5% beta mercaptoethanol elution, belonging to the PM third protein. Conclusion: 1. the successful establishment of Candida albicans The infection of housefly larvae intestinal infection, comparative transcriptome analysis to get involved in the immune response related genes; 2.374 PM protein in healthy 3 day old larvae of housefly were identified, Candida albicans infection after 24h in MD-T and MD-C were identified to 335302 PM proteins, changes of Candida albicans infection after peritrophic the film is relatively large; 3. successful cloning of Md Pt1 gene, verifies the chitin binding activity. In conclusion, the results of this study from the host with Candida albicans interaction, reveals the housefly larvae midgut epithelium and peritrophic membrane in response to Candida albicans infection in effect, provide a theoretical basis for further understanding of housefly larvae gut immunity.

【学位授予单位】：贵州医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R379
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本文编号：1603548