固氮基因的表达调控严谨而高效，受到外界氧、氮及能量等多个因素的影响。具有调控功能的非编码RNA在细菌中被大量发现，广泛参与了物质代谢、胁迫反应等重要生理过程，但在固氮微生物中鲜有报道。在上一个基金项目的支持下，我们通过转录组分析发现施氏假单胞菌A1501在固氮条件下高表达的17个非编码RNA，其中非编码RNA nfiS可特异响应外界的氧信号，而且其表达依赖于固氮代谢调控sigma因子RpoN，nfiS的突变降低了固氮酶活性和固氮结构基因的表达水平，暗示该基因可能以某种机制参与了固氮调控。本研究拟通过：1）功能互补、启动子活性测定等方法明确nfiS的功能特征及在固氮调控网络中的定位；2）采用转录组、蛋白质组技术研究nfiS的调控网络并寻找其作用靶标；3）通过qRT-PCR、凝胶阻滞技术研究nfiS与靶标基因互作的分子机制。研究结果将为深入解析固氮基因表达调控机理奠定重要的理论基础。

To respond to multiple environmental cues, nitrogen-fixing bacteria have evolved sophisticated regulatory networks to adjust expression of nitrogen fixation genes at the transcriptional level. The ammonium ion, oxygen concentration and energy supply are the most three important environmental signal that affect the nif gene expression. Small non-coding RNAs (ncRNAs) that play important regulatory roles exist in numerous organisms but few were functionally characterized in nitrogen-fixing bacteria. Supported by the former NSFC, we identified 17 ncRNAs that were upregulated under nitrogen fixation conditions compared with nitrogen excess conditions in the root-associated nitrogen-fixing bacterium Pseudomonas stutzeri A1501 using a deep sequencing approach. Among those ncRNAs, nfiS can specifically respond to oxygen signals and its expression was under the control of RpoN, a well-studied nitrogen regulator. Mutation of nfiS not only impaires the nitrogenase activity but also decreases the expression of the nif genes, implies that nfiS might be involved in the nitrogen fixation regulation process. In order to investigate the regulatory mechanism of nfiS involved in the nitrogen fixation gene regulation and the mode of its response to external signals, the nfiS disruption mutant and overexpression strain will be constructed in this study; qRT-PCR and EMSA will be performed to research the expression characteristics of nfiS and its location in the regulatory system of nitrogen fixation; the proteome will also be employed to analyze and verify the target genes of nfiS. Ultimately, the roles of nfiS in the nitrogen fixation regulation will be determined. This work will lay an important theoretical foundation for the in-depth analysis of the expression regulation mechanism of nif genes.