c-di-AMP候选受体蛋白TerD的结构功能及其信号途径研究

c-di-AMP 是2008年新发现的细菌第二信使分子。目前，只报道了c-di-AMP参与调控细菌的芽胞形成与肽聚糖的合成两种生理功能。而其信号调节途径相关受体与靶标完全未知。我们利用苏云金芽胞杆菌（Bt）中的DisA合成了c-di-AMP，构建了以c-di-AMP为配基的亲和色谱体系，捕获并鉴定了一种特异结合的蛋白TerD。为了证明TerD是c-di-AMP的受体，并解析c-di-AMP的信号通路，我们拟进行以下几个方面的研究：1）解析TerD的结构及TerD与c-di-AMP的复合结构；2）构建敲除与过表达terD的Bt菌株，并观察不同条件下的表型变化及测定转录表达谱差异；3）构建以TerD为配基的亲和色谱体系，分别垂钓能与TerD特异结合的DNA片段或蛋白质。在这些实验基础上，推测TerD调控的靶基因及调控方式，构建一条从结合受体到作用靶标的完整的c-di-AMP信号通路。

Cyclic diadenosine monophosphate (c-di-AMP) is a new member of bacterial cyclic nucleotide messengers which is firstly discovered in 2008. For now it is reported that c-di-AMP is involved in two physiological functions in bacteria: i) to regulate the formation of endospore in Bacillus subtilis, and ii) to control the synthesis of peptidoglycan in both Staphyloccocus aureus and Bacillus subtilis. However, the receptors and targets along with these c-di-AMP modulating signaling pathways are is still unknown. ..In our previous research, we have expressed and purified the c-di-AMP synthetase DisA from Bacillus thuringiensis, and obtained a large amount of chromatographically pure c-di-AMP. Then we successfully established an affinity chromatography system concerning c-di-AMP as ligand. As a result, a probably receptor protein named TerD annotated as a stress response protein were gained...To confirm that TerD is a candidate acceptor of c-di-AMP and reveal its signaling pathway, we will focus on the following experiment: .1) To solve the structure of TerD and the complex structure of TerD together with c-di-AMP;.2) To construct a series of bacterial strains including the terD knock-out strain, the complementary terD knock-out strain and the overexpression terD strain, and observe the phenotype and determine the transcriptomic differences under various conditions, including culture conditions and stimulus conditions, together with mother strain Bacillus thuringiensis BMB171;.3) To set up a affinity chromatography system with TerD as the ligand and fish DNA fragments and proteins having specially affinity with TerD, respectively...Based on the foundation of these tests, we try to find out the kind of target genes and reveal the intact signaling pathway from the receptors to the targets mediated by c-di-AMP as well as its physiological function in bacteria.