根际促生解淀粉芽孢杆菌SQR9组氨酸激酶KinA-E响应的根系分泌物信号鉴定

Bacillus spp. play important roles in microbial fertilizer as gram-positive beneficial rhizobacteria. Rhizosphere colonization of microbes determines the efficiency of their plant beneficial effects. Biofilm formation of Bacillus spp, which is under the regulation of phosphorylated Spo0A, is a critical factor for their root colonization. Spo0A accepts phosphoryl groups from histine kinases termed KinA, KinB, KinC, KinD and KinE. Biofilm formation of Bacillus is known to be affected by plant root exudates. However, the ligand of the each histine kinase as potential signal affecting biofilm formation in root exudates is poorly known. Bacillus amyloliquefaciens SQR9 is an efficient plant growth-promoting rhizobacteria (PGPR) isolated from cucumber rhizosphere, which has been shown to respond to root exudates of cucumber and form stronger biofilm on root as a consequence. In this research, KinA, KinB, KinC, KinD and KinE of SQR9 will be expressed and purified, and subsequently incubated with cucumber root exudates, respectively. The ligand(s) or signal(s) bind to each kinase will be separated and identified and their effects on the root colonization and biofilm formation of SQR9 will be evaluated. The molecules that enhance colonization of SQR9 will be further characterized. This work would expand knowledge on rhizosphere interaction between bacteria and plant, and theoretically support the regulation of behavior of PGPR in rhizosphere.

根际高效定殖是微生物菌剂发挥植物益生作用的关键步骤。根际促生芽孢杆菌的生物膜形成能力是影响其根际定殖的重要因素。芽孢杆菌组氨酸激酶KinA、KinB、KinC、KinD和KinE能够感受不同信号并使Spo0A磷酸化，进而调控生物膜的形成。芽孢杆菌的生物膜形成受到根系分泌物的影响，但是该5种组氨酸激酶所感受的根系分泌物中的信号分子仍缺乏研究。Bacillus amyloliquefaciens SQR9是本实验室从黄瓜根际分离到的一株根际有益细菌，能够对根系分泌物作出响应，并且能在根际高效定殖。本研究拟以B. amyloliquefaciens SQR9为研究材料，通过外源表达该5种激酶，使其特异性结合根系分泌物中的信号分子，进而鉴定每个激酶所结合的根系分泌物中的配体，并在其中寻找促进SQR9根际定殖的信号分子。研究成果有望对外源调控芽孢杆菌的根际定殖行为、增强其植物生长促进作用提供指导。

根际益生菌的根际定殖是有效发挥其益生作用的前提。植物根际益生菌能够感受植物根系分泌物中的信号分子，这对于该类微生物的根际定殖是至关重要的。磷酸化的Spo0A是芽孢杆菌中重要的全局转录调控因子，能够控制定殖和芽孢形成，而跨膜组氨酸激酶KinD是Spo0A的磷酸化供体并且负责感受胞外信号。在本研究中，我们发现在解淀粉芽孢杆菌SQR9中敲除kinD导致其在根系分泌物中的生物膜形成能力减弱，说明KinD负责感受根系分泌物中的信号。本研究通过非靶向的配体垂钓方法和靶向性的表面等离子共振方法在根系分泌物中鉴定了KinD的配体。我们一共筛选了根系分泌物中80种物质，结果发现，精氨和鸟苷能够以较强的亲和力与KinD发生结合，KD分别为213和51μΜ。另外，苏糖酸钙、N-乙酰天冬氨酸、癸酸钠、仲班酸能够以稍弱的形式与KinD发生结合。本研究进一步构建了KinD与根系分泌信号互作的三维结合模型，对接结果与表面等离子共振实验结果吻合，精氨和鸟苷与KinD的结合能力比另外四种物质更强。我们还发现外源添加的精氨能够抑制kinD突变体生物膜的褶皱的形成，这说明KinD可能参与感受根系分泌的精氨，进而在这种负面效应因子的影响下稳定生物膜形成。该研究能够为根际细菌感受器与根系分泌信号之间的互作提供更深入的认识，并为根际益生菌的农业应用提供理论基础。