细菌种间群感信号AI-2调控根际促生解淀粉芽孢杆菌SQR9成膜和运动性的分子机理研究

Plant growth-promoting rhizobacteria (PGPRs) can promote plant growth and suppress soil-borne diseases, so the rational application of PGPR strains is one of the effective and promising approaches to overcome crop soil-borne diseases, reduce agricultural chemical fertilizer and support the agricultural sustainability. Bacillus amyloliquefaciens SQR9 is the PGPR strain isolated from the rhizosphere by our laboratory, it has been applied widely in agricultural practice as the microbial fertilizer. This proposal will focuse on the PGPR strain Bacillus amyloliquefaciens SQR9, our previous researches found that the inter-species quorum-sengsing signal autoinducer-2 (AI-2) was related to the biofilm formation, root coloniazation and cell motility of SQR9. However, it's not clear for the molecular regulation mechanism of AI-2 on phenotypes metioned above, and so far, the AI-2 signal pathways in the Gram-positive strains have not been elucidated, which hinder the reasonable use and control of the AI-2 signal molecule. Based on all of the reported AI-2 receptor proteins, i.e. LuxP, LsrB and RbsB are belong to Peripla BP4 protein family, we predicted 4 possible AI-2 receptor protein in the SQR9's genome, and we have identified the AI-2 target genes related to biofilm formation and cell motility through the analysis of the transcriptome data of luxS mutant and wild type of SQR9. Based on the results mentioned above, this research include : (1) To test the predicted AI-2 receptor proteins, and to identify the AI-2 uptake genes and AI-2 receptor proteins through screening the TnYLB-1 transposon mutant library of the strain SQR9 and also using other strategies; (2) To further identify the AI-2 target genes responsible for biofilm formation, root colonization and motility through the ways of comparative (phospho)-proteome and further deep analysis of the transcriptome, and also combining with the phenotypical characteristics, to elucidate the functions of the target genes; (3) To clarify the regulation mechanism of AI-2 and its receptors on target genes through the methods: EMSA, DNase I footprinting and lacZ labeling of AI-2 target genes. This research will find the AI-2 receptors and elucidate the molecular mechanisms of the effects of AI-2 and its receptors on the biofilm formation and root colonization, and also improve our understanding of the AI-2 signal transduction in the Gram-positive bacteria, and will provide the theoretical instructions for improving the in situ root colonization and agricultural application of the strain SQR9 and even other PGPR strains.

根际促生菌在根表有效成膜定殖是保障其功效的前提。我们前期研究发现群感信号AI-2调控根际促生解淀粉芽孢菌SQR9菌株的运动性和根表成膜定殖，但AI-2调控这些表型的机制未知，且目前对所有革兰氏阳性菌中AI-2的作用机理尚不清楚，这也限制了对AI-2的利用和控制。基于AI-2已知受体LuxP和LsrB均属于Peripla BP4家族，已预测到SQR9中4个可能的AI-2受体，且转录组给出了AI-2调控以上表型的靶基因。基于以上，本申请拟开展：验证已预测的AI-2受体，并筛选SQR9的TnYLB-1转座子文库，鉴定与AI-2摄取相关的基因和受体；深入挖掘转录和蛋白组，确证AI-2靶基因、调控蛋白及其功能；通过EMSA、DNase I等实验阐明AI-2通过受体对靶基因调控通路。研究结果有助阐释AI-2调控根际定殖机理和阐明革兰氏阳性菌中AI-2调控通路，为提高根际促生菌应用效果提供更多理论指导。

植物根际促生细菌（plant growth-promoting rhizobacteria，PGPR）是一类能在植物根际存活定殖并具有促进植物生长或防控土传病害能力的一类有益微生物，近年来因其环境友好以及高效的促生作用在农业生产中被制成微生物肥料而广泛应用，贝雷斯芽孢杆菌SQR9（Bacillus velezensis SQR9）便是其中的一个优秀代表。微生物肥料发挥功能的首要条件就是微生物需在植物根际有效的形成生物膜并定殖，而有研究发现群体感应信号分子AI-2（Autoinducer 2）可以影响细菌生物膜的形成。本课题对芽孢杆菌SQR9中AI-2信号的合成酶LuxS进行敲除、回补以及在野生型SQR9中添加AI-2等处理对菌株的运动性、生物膜形成、发酵液粘度、γ-聚谷氨酸（γ-PGA）合成和玉米根际定殖能力进行了检测，结果显示群体感应信号分子AI-2对SQR9的运动性、生物膜形成、发酵液粘度、γ-PGA合成和玉米根际定殖均有显著的促进作用。AI-2作为一个群感信号分子，其作用方式是由合成酶LuxS在细胞内合成后分泌到细胞外再由受体蛋白感受并转运到胞内。因此，本课题进一步深入探寻了SQR9中存在的AI-2受体蛋白，通过生物信息学预测得到4个候选蛋白，分别是：AHZ17608.1、AHZ17603.1、AHZ17476.1和AHZ17479.1。通过大肠杆菌异源表达了这4个候选蛋白并在体外进行了与AI-2结合实验的验证，验证结果显示该4个蛋白均能与AI-2发生结合。本课题首次验证了AI-2具有对根际促生芽孢杆菌SQR9的根际定殖具有促进作用，且该作用主要通过促进细菌的运动性和生物膜形成而实现。AI-2广泛存在于革兰氏阴性菌和革兰氏阳性菌中可以调控多种细菌的群体行为，具有种间交流的作用，因此明确AI-2在植物根际促生菌中的作用对于该类菌在农业生产上的应用具有重大的指导意义。