【目的】本研究旨在通过差异表达基因(differentially expressed gene,DEG)分析以及毒力因子和其他侵染相关因子分析,在转录组水平揭示东方蜜蜂微孢子虫Nosema ceranae侵染意大利蜜蜂Apis mellifera ligustica的分子机制。【方法】基于...【目的】本研究旨在通过差异表达基因(differentially expressed gene,DEG)分析以及毒力因子和其他侵染相关因子分析,在转录组水平揭示东方蜜蜂微孢子虫Nosema ceranae侵染意大利蜜蜂Apis mellifera ligustica的分子机制。【方法】基于前期已获得高质量的东方蜜蜂微孢子虫纯化孢子(NcCK)及侵染意大利蜜蜂工蜂7和10 d的东方蜜蜂微孢子虫(分别为NcT1和NcT2)转录组数据,根据P≤0.05且|log 2(Fold change)|≥1的标准,通过比较分析筛选出NcCK vs NcT1,NcCK vs NcT2和NcT1 vs NcT2比较组的DEG。通过相关生物信息学软件对上述DEG进行Venn分析、GO分类和KEGG代谢通路富集分析。根据Nr和KEGG数据库注释信息和相关文献进行对东方蜜蜂微孢子虫的毒力因子和侵染相关因子的统计和分析。通过RT-qPCR验证转录组数据及DEG表达趋势。【结果】从NcCK vs NcT1,NcCK vs NcT2和NcT1 vs NcT2比较组分别鉴定出1397,1497和52个DEG。Venn分析结果显示各比较组共有的上调和下调基因分别为10和1个。GO分类结果显示,NcCK vs NcT1和NcCK vs NcT2中DEG富集数最多的功能条目为代谢进程、细胞进程、单组织进程、细胞、细胞组件、细胞器、催化活性和结合,而NcT1 vs NcT2中DEG富集数最多的是代谢进程、细胞进程、单组织进程、催化活性和结合。KEGG代谢通路富集分析结果显示,NcCK vs NcT1和NcCK vs NcT2中DEG分别富集到80和79条通路;富集在糖酵解/糖异生和MAPK信号通路的上调基因数量多于下调基因。毒力因子分析结果显示,孢壁蛋白9基因和孢壁蛋白12基因在NcCK vs NcT1和NcCK vs NcT2中均下调表达,孢壁蛋白8基因仅在NcCK vs NcT1中表达量下调;此外孢壁蛋白前体基因、孢壁和锚定盘复合蛋白基因、几丁质合酶基因、极管蛋白基因、蓖麻毒素B凝集素基因的表达水平在NcCK vs NcT1和NcCK vs NcT2中表现为上调。侵染相关因子分析结果表明,糖酵解途径的3个关键酶基因在NcCK vs NcT1和NcCK vs NcT2中上调表达;3个涉及ATP/ADP移位酶的基因在NcCK vs NcT1和NcCK vs NcT2中上调表达,但有1个表达量下调;2个涉及ABC转运蛋白的基因在NcCK vs NcT1和NcCK vs NcT2中上调表达,另有4个下调表达。RT-qPCR结果证实了本研究中转录组数据及DEG表达趋势的真实可靠性。【结论】本研究通过比较分析解析东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程的转录组动态,揭示了孢壁蛋白、孢壁和锚定盘复合蛋白、几丁质酶、极管蛋白和蓖麻毒素B凝集素等毒力因子编码基因,以及己糖激酶、丙酮酸激酶、6-磷酸果糖激酶、ATP/ADP移位酶和ABC转运蛋白等侵染相关因子编码基因在病原增殖中扮演重要角色,为阐明东方蜜蜂微孢子虫的侵染机制提供了基础。展开更多
Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack o...Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.展开更多
文摘【目的】本研究旨在通过差异表达基因(differentially expressed gene,DEG)分析以及毒力因子和其他侵染相关因子分析,在转录组水平揭示东方蜜蜂微孢子虫Nosema ceranae侵染意大利蜜蜂Apis mellifera ligustica的分子机制。【方法】基于前期已获得高质量的东方蜜蜂微孢子虫纯化孢子(NcCK)及侵染意大利蜜蜂工蜂7和10 d的东方蜜蜂微孢子虫(分别为NcT1和NcT2)转录组数据,根据P≤0.05且|log 2(Fold change)|≥1的标准,通过比较分析筛选出NcCK vs NcT1,NcCK vs NcT2和NcT1 vs NcT2比较组的DEG。通过相关生物信息学软件对上述DEG进行Venn分析、GO分类和KEGG代谢通路富集分析。根据Nr和KEGG数据库注释信息和相关文献进行对东方蜜蜂微孢子虫的毒力因子和侵染相关因子的统计和分析。通过RT-qPCR验证转录组数据及DEG表达趋势。【结果】从NcCK vs NcT1,NcCK vs NcT2和NcT1 vs NcT2比较组分别鉴定出1397,1497和52个DEG。Venn分析结果显示各比较组共有的上调和下调基因分别为10和1个。GO分类结果显示,NcCK vs NcT1和NcCK vs NcT2中DEG富集数最多的功能条目为代谢进程、细胞进程、单组织进程、细胞、细胞组件、细胞器、催化活性和结合,而NcT1 vs NcT2中DEG富集数最多的是代谢进程、细胞进程、单组织进程、催化活性和结合。KEGG代谢通路富集分析结果显示,NcCK vs NcT1和NcCK vs NcT2中DEG分别富集到80和79条通路;富集在糖酵解/糖异生和MAPK信号通路的上调基因数量多于下调基因。毒力因子分析结果显示,孢壁蛋白9基因和孢壁蛋白12基因在NcCK vs NcT1和NcCK vs NcT2中均下调表达,孢壁蛋白8基因仅在NcCK vs NcT1中表达量下调;此外孢壁蛋白前体基因、孢壁和锚定盘复合蛋白基因、几丁质合酶基因、极管蛋白基因、蓖麻毒素B凝集素基因的表达水平在NcCK vs NcT1和NcCK vs NcT2中表现为上调。侵染相关因子分析结果表明,糖酵解途径的3个关键酶基因在NcCK vs NcT1和NcCK vs NcT2中上调表达;3个涉及ATP/ADP移位酶的基因在NcCK vs NcT1和NcCK vs NcT2中上调表达,但有1个表达量下调;2个涉及ABC转运蛋白的基因在NcCK vs NcT1和NcCK vs NcT2中上调表达,另有4个下调表达。RT-qPCR结果证实了本研究中转录组数据及DEG表达趋势的真实可靠性。【结论】本研究通过比较分析解析东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程的转录组动态,揭示了孢壁蛋白、孢壁和锚定盘复合蛋白、几丁质酶、极管蛋白和蓖麻毒素B凝集素等毒力因子编码基因,以及己糖激酶、丙酮酸激酶、6-磷酸果糖激酶、ATP/ADP移位酶和ABC转运蛋白等侵染相关因子编码基因在病原增殖中扮演重要角色,为阐明东方蜜蜂微孢子虫的侵染机制提供了基础。
基金National Nature Science Foundations ofChina (30325002, 30470075)National Basic ResearchPriorities Program of China (2003CB1140).
文摘Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.
