【作者】 陈龙；

【导师】 张克云；

【作者基本信息】 南京农业大学 ， 动物学， 2015， 硕士

【摘要】 昆虫病原线虫(EPNs)作为一类特异的寄生昆虫病原体,因其具有主动搜索寄主能力、侵染致死效率高、便于大规模生产且对人类和环境无危害等特点而作为生物防治因子被广泛用于农业害虫的防治,具有极高的应用潜力和研究价值。崇明拟异小杆线虫(Heterorhabaitidoides chongmingensis)DZ0503CMFT(DZ)是一种与嗜线虫沙雷氏菌(Serratianematodephila DZ0503SBS1T(S1)特异性共生,并对昆虫具有高致病力的EPN。在前期的研究中发现,DZ线虫与非自身细菌菌株S.nematodephila DR186(186)共培养时,单菌线虫组合DZ/186第一代的发育率和性别比、第二代IJs幼虫产量及带菌率都显著低于DZ/S1。为了深入解析共生菌对线虫生长发育及繁殖的影响,我们基于IlluminaHiSeqTM2000高通量测序平台,构建了 2组单菌线虫组合DZ/S1、DZ/186的数字基因表达谱(DGE)文库并进行了测序和相关分析。所用的关键技术方法和获得的主要研究结果如下:将DGE测序获得的clean reads比对到DZ参考转录组数据库中并对每一基因的表达量进行统计,共筛选到10,043个差异表达基因(DEGs),其中1,246个基因上调,8,797个基因下调。通过GO、KEGG pathway功能显著性富集分析,对每一 DEG进行功能注释和分类。GO功能分类结果显示,DEGs主要富集在50个GO分类子条目中的单一生物过程、细胞过程、多细胞生物过程、发育过程和代谢过程等生物过程;细胞、细胞器、膜及其成分等细胞中所处位置;以及催化反应、结合和转运活性等分子功能。KEGG pathway功能显著性富集分析结果显示,DEGs显著性富集在Tuberculosis、Small cell lung cancer、Focal adhesion、ECM-receptor interaction、Amoebiasis 和 Basal transcription factors 等 23 条 pathways 中。5个上调DEGs和5个下调DEGs被挑选并进行定量PCR以验证DGE测序及分析结果的准确性。这10个差异表达基因的荧光定量PCR结果与DGE数据呈现相同的上下调局势,线性相关性分析结果显示2者具有显著的正相关性,其中相关系数R2= 0.80;这一结果证明了 DGE数据的准确性。GO功能分析中一部分DEGs归类于在发育、生长及繁殖的生物过程中,表明单菌线虫组合DZ/186的生长发育及繁殖等生物过程受到抑制。基于KEGGpathway显著性富集分析所筛选得到的23条DEGs显著性富集pathways中的ECM-receptor interaction、focal adhesion 及 regulation of actin cytoskeleton 这三条通路参与调节线虫的繁殖过程;还有很大一部分差异表达基因富集在线虫的初级代谢途径上且表现下调趋势,与DZ/186的退化的性状相一致,这些初级代谢途径可能与线虫的生长发育相关。更多还原

【Abstract】 Entomopathogenic nematodes(EPNs)are obligate parasitism insect pathogens,which are widely used to control agricultural pests,and serve as a kind of important biological control agents.There are many traits of EPNs includeing the active searching host ability,high efficiency and pathogenicity for killing pests,easily to mass production and being safe to human and environment,and so forth.Heterorhabditidoides chongmingensis DZ0503CMFT(DZ)is a novel member of entomopathogenic nematodes that has evolved a mutualism relationship with Serratia nematodiphila DZ0503SBS1T(S1)bacteria to serve as a highly virulent insect pathogen.Our previous study showed that when associated with non native bacterial strain S.nematodiphila DR186(186),the developmental rate,sex ratio of its first generation adults,bacterial carriage rate and output for its next generation IJs of the monoxenic nematode-bacterium complex DZ/186,were significantly lower than those of the wild combination DZ/S1.In order to deep understanding the impact of different symbiotic bacteria strain on growth;development and reproduction of the nematode,digital gene expression profiling(DGE)sequencing libraries of the monoxenie nematode-bacterium complexes DZ/S1 and DZ/186 were constructed and sequencing.The key methods and main results are as follows:After mapping and statistics analysis of the DGE sequencing reads with reference transcriptome data of Heterorhabditidoides chongmingensis,10,043 differentially expressed genes(DEGs)were identified,1,246 of which showed up-regulated trends and 8,797 of them showed down-regulated trends.Then the DEGs were functional annotated by GO and KEGG pathway significantly enriched analysis.The GO functional classification showed that the DEGs were assigned to 50 sub-GO terms:single-organism process,cellular process,multicellular organism process,development process,metabolic process and in other biological process;cell,organelle,membrane,their components and other cell component;and catalytic activity,binding,transporter activity and other molecular function.KEGG pathway enriched analysis showed that the DEGs were significantly enriched in 23 pathways,such as Tuberculosis,Small cell lung cancer,Focal adhesion,ECM-receptor interaction,Amoebiasis and Basal transcription factors.Five up-regulated genes and five down-regulate genes were selected for quantitative PCR analysis to validate the accuracy of DEG data.QRT-PCR results of these genes showed the same up/down trends as DGE data with significantly positive correlation(correlation coefficient R2= 0.80)between them.These results confirmed the accuracy of DGE data in this study.Quit a few parts of the DEGs were assigned to growth,development and reproduction biological processes,indicating that the growth,development and reproduction processes were inhibited in the recombined monoxenic nematode-bacterium complex DZ/186.Three pathways,ECM-receptor interaction,focal adhesion and regulation of actin cytoskeleton,were involved in regulating reproduction of H.chongmingensis.A considerable part of DEGs were enriched in primary metabolic processes and showed down-regulated trends,which were negatively to the development rate and reproduction of DZ/186,indicating the primary metabolic processes may play an important role in the growth and development of H.chongmingensis.更多还原