河蟹螺原体非编码RNA SR01和SR05参与毒力岛致病作用的分子机制研究

Non-coding RNAs (ncRNAs) plays an important regulatory role involved in the response to bacterial metabolism, environmental adaptation, pathogenesis and the control of virulence. Spiroplasma is a new type of major epidemic pathogen of shrimp and crab. Previous studies have found that the ncRNA SR01, SR05 encoded by the 18K virulence island of Spiroplasma eriocheiris (S. eriocheiris) are associated with the pathogenicity of bacteria. However, the molecular mechanism and regulatory functions of the two ncRNAs involved in the pathogenic effect of the S. eriocheiris virulence island are still unclear. Firstly, we construct the candidate pathogenesis-related ncRNA SR01, SR05 mutants and complementary strains, subsequently, through using differential transcriptome sequencing, coding region and non-coding region arrays, real-time quantitative PCR (qRT-PCR), two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and mass spectrometry (MS), the molecular targets (target genes and proteins) of the S. eriocheiris ncRNA SR01 and SR05 will be predicted systematically; secondly, the two ncRNAs interaction with their target genes or proteins and interaction with between important target proteins in the 18K virulence island will be detected by gel mobility shift assays (EMSA) and co-immunoprecipitation (Co-IP); once again, make sure about the biological functions of the two ncRNAs and their important targets in the 18K virulence island by using bacterial CRISPR Interference (CRISPRi), antisense RNA technology, gene overexpression and antibody blocking assays, combination of cell and animal experiments; finally, make sure the molecular mechanisms of the two ncRNAs and their targets involved in the pathogenicity of the 18K virulence island by using network biology analysis, and lay the foundation for the further study of the pathogenic molecular mechanism of the disease-causing pathogens, to provide theoretical and experimental basis for the prevention and control for such bacterial diseases.

非编码RNA在细菌新陈代谢、环境适应、致病机理和毒力调控等方面发挥重要调节作用。螺原体是虾蟹重大流行病的新型致病菌，前期研究发现由河蟹螺原体18K毒力岛编码的ncRNA SR01、SR05与细菌的致病性相关，但它们参与该病原毒力岛致病作用的调控功能和分子机制至今仍不清楚。本项目拟将构建SR01、SR05突变株与回补株，利用差异转录组测序、表达芯片、荧光差异凝胶电泳、质谱技术和生物信息学方法等，系统鉴定SR01、SR05的分子靶标；利用胶迁移实验和免疫共沉淀验证SR01、SR05和靶标间的相互作用；利用细菌CRISPR干扰、反义RNA、过表达和蛋白抗体阻断等，结合虾蟹细胞与体内感染实验，确定SR01、SR05及其靶标的生物学功能；最后，结合网络生物学分析，阐明这两个ncRNA和靶标在参与毒力岛致病作用中的分子机制，以此探明ncRNA在该病原致病过程中的作用，为防控该类疫病提供理论与实验依据。

本项目：（1）利用蛋白基因组学方法，对河蟹螺原体原有的基因组进行了新的功能注释与补充；（2）利用比较基因学方法，系统解析了河蟹螺原体ncRNAs(包括SR01、SR05)、靶基因、致病岛、毒力因子和操纵子间的分子关系；（3）利用RNA-RNA/pull-down、原核链特异性转录组测序、RNA-Protein/pull-down和质谱鉴定分析，全面筛选河蟹螺原体ncRNAs ( SR01和SR05)的靶基因和靶蛋白；（4）根据多组学实验研究数据，通过系统生物学方法，对河蟹螺原体ncRNAs(包括SR01、SR05)和分子靶标进行网络生物学分析，构建了差异表达ncRNAs、差异表达靶基因（毒力基因）和差异表达靶蛋白（毒力蛋白）的共表达调控网络；（5）利用三代、二代测序、qRT-PCR技术、RNA干扰技术和生物信息学分析等，全面挖掘河蟹螺原体感染宿主虾类的免疫相关基因、ncRNAs及其靶基因，系统解析重要节点免疫基因的生物学功能和抗菌作用。. 研究结果显示：共发现新蛋白134个，筛选出毒力蛋白68个，其中与河蟹螺原体致病相关的毒力靶蛋白21个；分子互作分析得到SR01的潜在靶mRNA 241个、靶lncRNA 31个、靶sRNA 25个和靶蛋白9个，SR05的潜在靶mRNA 53个、靶lncRNA 6个、靶sRNA 17个和靶蛋白 120个；网络生物学分析总共发现6个重要的生物网络节点ncRNAs，它们分别为：SR01、SR05、SR16、SR02、S26和SR04；宿主许多miRNAs、LncRNAs和免疫靶基因在螺原体感染中表达都差异显著，对其中某些重要免疫基因（CTL4、ALF、MNK1和GILT）进行功能抑制后，螺原体的清除率降低，虾类的死亡率增加；虾类血细胞免疫相关LncRNA-miRNA-mRNA网络，参与虾类抗螺原体感染的分子调控。 . 总之，相关研究成果，将为进一步探讨河蟹螺原体ncRNAs在宿主侵染过程中和宿主虾类ncRNAs在抗螺原体感染中的分子作用机制奠定基础，为防控该疫病提供理论与实验依据，也为虾类该疫病的诊断与治疗提供新的免疫策略。 .