植物促生贝莱斯芽胞杆菌反式乌头酸代谢途径的解析及其对根际定殖的影响研究

Poor colonization ability is an important factor that usually causes the unsatisfactory application effects of microbial agents nowadays. trans-Aconitic acid (TAA) can be highly produced and secreted by plants, however, its physiological function has been unknown. Previously, we found that the ability of utilizing TAA as the sole carbon source by different bacteria is closely related to their origins of plant-related environments, and deleting tcrA, the key gene which functions in TAA assimilation in plant-growth-promoting bacterium Bacillus velezensis FZB42, could lead to significantly impaired growth ability in TAA-containing tomato root environment, suggesting that TAA carbon assimilation is very likely a novel pathway that functioning in bacterial colonization. In view of all the above, we next plan to conduct the following experiments in FZB42: 1) identification of the complete metabolic and regulatory pathways of TAA assimilation; 2) determination of the differences between the original strain FZB42 and the mutant strain ΔtcrA in their abilities in alone-colonization and mixed-colonization, respectively in TAA producing and non-producing sterile tomato root systems ; 3) sequencing the transcriptome of FZB42 and ΔtcrA respectively, when both strains are colonized lonely, and identifying the key colonization-related genes and pathways whose expression levels are significantly altered, to reveal the molecular mechanism of the effect of TAA assimilation on bacterial colonization. This project is believed to have important theoretical significance to the development of microbial agents with higher stability and efficiency.

定殖能力不足是导致当今微生物制剂应用效果不理想的重要因素，深入挖掘定殖机制是解决问题的关键。反式乌头酸(TAA)可被植物大量合成与分泌，但生理功能一直未知。我们前期发现不同细菌将TAA用作碳源的能力与菌株的植物环境来源密切相关，且在植物促生贝莱斯芽胞杆菌FZB42中缺失TAA同化关键基因tcrA能明显削弱菌株在含有TAA的番茄根系环境中的生长能力，暗示TAA碳源同化极可能是参与细菌定殖的新功能途径。鉴于此，拟在FZB42中开展以下研究：1）解析完整的细胞同化TAA的代谢与调控途径；2）分别在有TAA和无TAA的番茄无菌根系中，比较出发菌株FZB42与突变株ΔtcrA单独定殖与混合定殖能力的差异；3）分别测定FZB42和ΔtcrA单独定殖时的转录组，比较和分析定殖关键基因及代谢途径的表达变化，揭示TAA同化影响定殖的分子机制。本研究的开展对研制更加稳定有效的微生物制剂具有重要理论指导意义。

当前，基于植物益生微生物开发生物肥料与生物农药已经成为我国发展绿色农业的重要手段。然而，这些制剂在实际使用过程中经常面临效果不稳定、不持久等严峻问题。制剂中功能微生物的根际定殖能力不足是造成这一现象的重要原因。而碳代谢能力是细菌适应植物营养环境进而完成定殖的必需功能，但少有从这一角度关注并改善细菌定殖表现的工作。我们基于“细菌能够代谢反式乌头酸——一种非主流类型碳源营养”的科学发现，通过遗传学、生物化学、细胞生物学水平实验，首次揭示了细菌代谢TAA碳源的同化分子机制。又继而阐明了细菌对自身该功能的调控机制，呈现出了完整的TAA碳源适应系统。然后，通过blast分析发现了不同细菌种属中都具有TAA同化功能同源基因，并通过从自然环境中分离到TAA同化细菌及鉴定到乌头酸异构酶活性，证实了TAA同化系统在细菌界和自然界中广泛分布与存在的事实，揭示了TAA代谢是细菌普遍利用的碳代谢本领，丰富了对细菌营养代谢多样性的科学认识。接着，我们利用无菌土壤体系证明了TAA同化功能能够为细菌提供生长优势与竞争性定殖优势，最后通过比较转录组学揭示了该功能主要以影响碳代谢途径的方式影响定殖。本项目从代谢调控途径到生理效应层面，再到效应分子机制，系统地揭示了TAA同化途径在细菌定殖中的作用，对细菌环境适应性机制及具有更强稳定性微生物制剂的研发应用具有重要意义。