c-di-GMP调控蓝细菌Anabaena sp. PCC7120异形胞发育的分子机制研究

c-di-GMP as a second messenger regulates a variety of important physiological functions in bacteria, which enable them to adapt quickly to the environmental changes. But, till now, the molecular regulatory mechanisms of c-di-GMP remain poorly understood. Although c-di-GMP has been reported to control cell motility, cell aggregation and heterocyst differentiation in cyanobacteria, the specific molecular mechanisms are still not clear. In this project, we will focus on the studies of molecular mechanism of heterocyst development regulated by c-di-GMP in the cyanobacteria Anabaena sp. PCC7120. To achieve this, specific relationship between intracellular c-di-GMP level and heterocyst differentiation was firstly established by analyzing the phenotype of strains which make either a higher, or lower, than normal levels of c-di-GMP, combined with the introduction of a c-di-GMP biosensor which based on fluorescence resonance energy transfer. Secondly, Pull-Down assay, Mass spectrometry technology, ITC analysis and phenotype analysis of the deletion mutant strains will be used to screen c-di-GMP receptor related to heterocyst development. Finally, the regulator targets of c-di-GMP receptors in the signaling pathways will be identified by transcriptomics and proteomics analysis, and the relationship between c-di-GMP, c-di-GMP receptors and their targets will be defined by ITC, EMSA and protein cross-linking and other technological means. Based on these findings, we will elucidate the molecular mechanisms on c-di-GMP signaling in heterocyst development, which will deepen the understanding of the regulatory model of heterocyst differentiation in cyanobacteria, and expand the scope of c-di-GMP regulation on bacterial physiology.

第二信使c-di-GMP参与调节细菌多种重要生理功能，但关于c-di-GMP调控机制的报道不多。已有的研究表明c-di-GMP参与调控蓝细菌细胞的运动力、细胞聚集和异形胞分化，但是调控的分子机制尚不清楚。本项目将围绕c-di-GMP调控Anabaena sp. PCC7120异形胞发育的分子机制展开研究。首先通过构建超表达菌株及导入基于FRET的c-di-GMP生物传感器确定c-di-GMP水平与异形胞分化的关系；其次通过Pull-Down、质谱、ITC和缺失突变体表型分析等手段，筛选与异形胞发育有关的c-di-GMP受体；最后通过转录组学和蛋白组学分析找到c-di-GMP信号通路中受c-di-GMP受体调控的靶蛋白，并运用ITC、EMSA、蛋白交联等技术研究调控的分子机制。该研究成果将加深对蓝细菌异形胞分化发育调控模式的认识，扩展c-di-GMP调控细菌生理功能的范围。

第二信使c-di-GMP参与调控细菌多种重要生理活动，但关于c-di-GMP调控机制的报道不多。已有研究表明c-di-GMP参与调控蓝细菌细胞的运动力、细胞聚集、异形胞分化等，但是分子机制尚不清楚。蓝细菌Anabaena sp. PCC7120（Anabaena）中有16个负责c-di-GMP代谢的基因，本项目从c-di-GMP代谢基因突变株∆cdgSH和∆cdgS缺氮条件下异形胞发育异常这一表型出发，综合多学科手段开展了c-di-GMP调控Anabaena异形胞发育分子机制的研究。研究结果表明：首先，Anabaena胞内c-di-GMP 稳态平衡是缺氮时异形胞正常发育的关键；其次，Anabaena中参与异形胞发育的c-di-GMP水平主要由c-di-GMP代谢酶CdgSH和CdgS维持；CdgSH为双功能酶，但主要表现为降解酶活性，CdgS为合成酶，两者在异形胞分化的不同阶段调控胞内c-di-GMP水平。最后，c-di-GMP信号通过不同的途径调控异形胞发育，c-di-GMP水平降低时信号通过新筛选到的c-di-GMP受体CdgR首先调控细胞尺寸变化，进而影响了异形胞发育。该项目确定了c-di-GMP水平变化与Anabaena异形胞发育的关系；找到了胞内调控c-di-GMP水平进而影响异形胞发育的关键基因；发现Anabaena胞内c-di-GMP水平通过不同的信号途径调控异形胞发育；并初步阐明胞内c-di-GMP水平调控异形胞发育的分子机制；加深了对蓝细菌异形胞分化发育调控模式的认识；扩展了对 c-di-GMP信号传导途径领域的了解；同时也确立了一个在微生物领域具有普遍意义的新的研究方向，即蓝细菌细胞尺寸调控。

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