拮抗芽孢杆菌在根部定殖及扩展分子机制探究

Antagonistic Bacillus has been used as a powerful weapon for the suppression of plant pathogenic organisms and the enhancement of plant development in the laboratory and greenhouse experiments. Unfortunately, however, results in the field have been less consistent because of the less competence of root colonization. The colonization of roots by inoculated bacteria is an important step in the interaction between benifical bacteria and the host plant. In the past, scientist focused on the cloning and function analysis of new genes which involved in the biofilm formation, mobility and chemotaxis after induced by root exudates. These results can't evaluate molecular mechanism of root colonization of Bacillus comprehensively. Bacillus subtilis Bs916 has excellent ability of colonization on tomato roots and antagonistic activity on Ralstonia solanacearum in our lab.This program aimed to evaluate the molecular mechanism of root colonization and expansion of Bacillus. According to the colonization laws of Bs916, the rhizosphere bacterial cells would be collected at different time points and performed RNA sequence for gene expression profile analysis. The different expression genes which relate to colonization will be screened based on the Bs916 genome sequence data. Subsequently, some key genes will be given deep investigation by Bacillus molecular method (gene knock out, over expression, promoter analysis et al). This project offer some new ideas and clues to understand the colonization molecular mechanism, and provide some theory and target genes to improve the competence of root colonization for biocontrol agent.

研究表明，生防菌的防效与其在植物根表的定殖能力密切相关。生防菌能否有效定殖已成为其大规模商品化应用的重要制约因子。已有定殖研究主要集中于根围分泌物对生防菌的影响，不能全面、准确地反映生防菌参与定殖及扩展的相关分子机制。申请人基于对拮抗芽孢杆菌Bs916在番茄根部定殖研究结果，提出本研究内容，拟采用基因表达谱技术对Bs916在番茄根部定殖及扩展过程实时分析，结合全基因组测序平台，建立Bs916定殖及扩展相关基因数据库；采用芽孢杆菌分子生物学技术（启动子研究、基因敲除等）对定殖及扩展中的关键基因进行功能研究，明确其作用机制，从而丰富生防芽孢杆菌在根部定殖和扩展的分子机制。预期研究结果将能够更加实时，全面地反映出生防菌参与定殖及扩展相关基因的表达情况，为阐释定殖分子机制提供新思路和线索，同时也为改善生防菌定殖能力、提高防效提供靶标位点和理论依据。

本项目在国家自然科学基金的资助下，经过项目负责人和江苏省农科院植保所稻病与生防研究室同事们的共同努力，就项目中所提出的科学问题，按照计划进行了相关研究，具体研究情况概述如下：1）通过GFP 荧光蛋白标记和荧光显微镜观察发现，盆栽实验条件下，芽孢杆菌Bs916 在番茄根部能够定殖并形成生物膜，平板回收检测显示，无论是否有番茄青枯病原菌的存在，生防菌在根部定殖量基本保持在106cfu/g /根以上。2）基于无菌砂石-营养液番茄栽培系统和Bs916在番茄根部的定殖规律，目前已初步建立生防菌Bs916 根部定殖扩展相关差异表达基因数据库，数据分析显示：定殖初期 (Bs916灌根后8h)，Bs916中有1502个基因发生了差异表达，其中1050(69.91%)个上调表达，452(30.09%)个下调表达；定殖早期(Bs916灌根后24h)与初期相比，Bs916中仍旧有1374个基因发生了差异表达，其中245(17.83%)个上调表达，1129(82.17%)个下调表达。3）基于所构建的芽孢杆菌在番茄根部定殖相关表达差异表达数据库，从中挑取生物膜主要组分等关键基因，采用序列分析、基因敲除和突变体表型验证等技术深入研究这些基因在定殖和扩展过程中的作用和生物学功能。以上研究结果，初步解释了生防芽孢杆菌在番茄根部定殖形成生物膜的情况及作用，丰富了芽孢杆菌定殖番茄根部的分子机制。