BcSinR-BcSinI蛋白互作在蜡质芽孢杆菌AR156防治蔬菜根结线虫病过程中的生物学功能研究

Root-knot nematode disease caused by the root-knot nematodes (Meloidogyne spp.) is one of the most severe vegetable diseases in China. It causes huge economic losses, because of the reduction of vegetable yield and quality. Biological control with antagonistic microorganisms provides an eco-friendly strategy to reduce this damage. Bacillus cereus AR156 isolated from rhizosphere soil showed a good biocontrol efficacy against root-knot nematode disease under greenhouse conditions and in the field in our previous study. However, the exact biocontrol mechanism of B. cereus AR156 is still unknown. Our recent results indicate that the transcription factor BcSinR plays an important role during biofilm formation of AR156 by transcriptome. Meanwhile, the insertion mutant of BcSinI, a predicted BcSinR interacting protein in AR156, presented a strong biofilm defect and a significant reduction of the biocontrol efficacy. In this proposal, we will investigate the molecular details of BcSinR-BcSinI interaction and function of BcSinR regulon during the biofilm formation in AR156. By comparing the biocontrol efficacy of wild type and biofilm mutants, we will reveal the role of biofilm during biocontrol of root-knot nematode disease with B. cereus AR156. The proposed investigation will also help us understand the biocontrol mechanism of B. cereus AR156 against Meloidogyne spp., and accelerate the commercial application of AR156 as a biocontrol nematicide.

由根结线虫(Meloidogyne spp.) 引起的蔬菜根结线虫病为蔬菜生产中的重要病害之一，严重制约着蔬菜的产量及品质。蜡质芽孢杆菌（Bacillus cereus）AR156为本课题组筛选得到的一株能高效防治蔬菜根结线虫病害的生防菌株，但其生防机理还不清晰。前期研究通过转录组比较发现AR156的BcSinR转录因子对该菌株的生物膜形成起重要调控作用。同时，其可能的互作蛋白BcSinI的缺失突变体（生物膜形成能力缺失）降低了AR156生防根结线虫病效果。本项目将主要研究AR156菌株的BcSinR-BcSinI蛋白互作及其对生物膜的调控机理；通过离体和温室防治蔬菜（番茄）根结线虫病害试验，明确BcSinR-BcSinI互作调控的生物膜在AR156生防蔬菜根结线虫病害过程中的生物学功能。研究结果将进一步揭示AR156菌株防治蔬菜根结线虫病害的机理，为该菌株的产业化应用提供理论基础。

本项目建立了生防蜡质芽孢杆菌AR156的遗传操作体系，并完成生物被膜形成条件的优化。发现AR156菌株在LBGM条件下能在空气-液体界面形成生物被膜。对AR156基因组进行分析，查找到芽孢杆菌生物被膜合成关键调控关键基因“Spo0A-SinR-SinI”，利用同源重组技术构建了Bcspo0A和BcsinI的敲除突变体并进行了生物被膜、产孢、游动等生物学表型测定。结果发现BcSpo0A和BcSinI对AR156菌株生物被膜的形成至关重要。在枯草芽孢杆菌中异源表达Bcspo0A、BcsinR和BcsinI能完全恢复枯草芽孢杆菌同源基因的缺少表型，可见蜡质芽孢杆菌AR156中关于生物被膜的形成调控与枯草芽孢杆菌中存在类似的调控机制。利用转录组技术，分析了野生型AR156、ΔBcspo0A和ΔBcsinI菌株在生物被膜形成条件下的转录水平差异，发现了一批可能参与蜡质芽孢杆菌生物被膜形成的基因。且发现，与枯草芽孢杆菌的生物被膜基质成分的合成途径相比，蜡质芽孢杆菌的胞外多糖、蛋白等有显著差异。离体和温室试验结果发现，AR156的生物被膜突变后，菌株在线虫体表的定殖能力显著下降；突变体防治根结线虫病的能力也显著降低。由此可见，AR156生物膜形成能力对其防控病害起着重要作用。此外，研究还发现乙酸、γ-聚谷氨酸等物质参与芽孢杆菌的生物被膜形成。