线虫病原细菌全基因组水平侵染分子机制研究

Understanding the mechanisms of host-pathogen interaction has always been the frontier and hot point in biological researches. The knowledge is also the most important theoretical principle for biocontrol of plant parasitic nematodes. But now, most of the investigations in this area only deal on the single gene. Therefore based on our previous study to describe the process of pathogenic bacterium Bacillus nematocida B16 infecting nematodes, we shall complete the genomic sequence of this bacterial strain. Meantime, we shall also select the different bacterial strains in the genus Bacillus, which have the different nematocidal activities or/and different strategies to kill nematodes. Then employing the methods such as comparative genomics and transcriptomics/proteomics, we try to identify the candidate genes involved in the different stages of infection. Furthermore, we shall screen a random mutant library of B. nematocida B16 for the typical mutants with attenuated nematicidal activities at the different stages after success construction of the random mutant library. Combined with these two methods above, a variety of candidate genes responsible for the infectious process will be obtained. Finally, we shall use the functional experiments, including qPCR, fluorescent localization, gene knockout or overexpression, to validate the important candidate genes. In conclusion, analyzing the data of genetics background, gene types, gene quantities, and gene expressional regulation, our investigation tries to explain the different interaction relashionships between bacteria and nematodes as well as the different strategies of infection among Bacillus, which also helps to reveal the comprehensive and systematic mechanisms of infection at genomic level.

细菌与宿主的相互关系一直是生物学研究领域的前沿热点，也是植物寄生线虫病害生物防控的重要理论基础。但目前该领域仍多为聚焦于单一基因的独立研究。本项目拟在前期对病原细菌Bacillus nematocida B16侵染线虫过程已有深入认识的基础上,完成该菌株全基因组测序。进而选取芽孢杆菌属中其它对线虫具有不同毒杀活性、以及杀线虫方式不同的近缘菌株，围绕该菌株侵染宿主的主要步骤，借助于比较基因组学等工具，寻找与侵染各阶段相关的候选基因。同时，筛选该菌株随机突变文库中侵染各阶段具有表型变化的典型突变子。综合以上两种水平获得的致病候选基因，再通过qPCR、荧光定位、基因敲除或过表达等功能实验对重要基因进行验证。本研究尝试从遗传背景、基因含量、基因种类、基因表达调控方式的不同，阐明芽孢杆菌属中不同物种之间拮抗线虫活性的差异以及杀虫方式的差异，以期在组学水平上更全面地揭示线虫病原细菌侵染宿主的分子机制。

理解和阐明病原与宿主的互作机制一直是生命科学领域的前沿和热点。本项目在完成线虫病原细菌Bacillus nematocida 16全基因组测序基础上，利用GO、COG、KEGG、SignalP、Protcomp、TargetP等数据库完成功能基因、代谢通路、分泌蛋白等注释；进而围绕病原细菌的致病过程，在基因组水平上筛选侵染线虫的毒力因子以及重要次生代谢产物的合成基因；通过与非致病近源种属的比较基因组学分析，揭示线虫病原细菌B. nematocida 16基因组适应于侵染线虫的基因获得与丢失，并在基因组水平上构建了影响病原致病过程的转录调控网络。与此同时，利用病原细菌B. nematocida B16和线虫侵染之间的互作转录组学研究，分析发掘病原细菌致病过程中的基因表达变化、以及线虫应答反应前后的基因变化；最后结合病原细菌随机突变文库的筛选，验证了部分新的致病候选基因的功能。目前该项目已经正式发表的SCI论文4篇，其中一篇发表在JOURNAL OF BIOLOGICAL CHEMISTRY (nature index 杂志之一)，另一篇发表在微生物领域JCR2区杂志上；申请国家发明专利1项。综上，项目按照计划任务书规定完成全部研究任务和考核指标。