小麦内生生防芽孢杆菌B3-7 菌株受转录因子PlcR 调控的生防相关基因的克隆及功能分析

Soil-borne diseases of wheat ,such as take-all and sharp eyespots, are serious root diseases worldwide. No efficient control measures against these diseases are available at present. Biocontrol by rhizobacteria,as an alternative measure, was intensively studied . Bacillus cereus which are able to form endospores that are tolerant to heat, offer advantages over other bacteria. Endophytic bacteria may not be affected by detrimental environmental factors against soil-borne pathogens have gained increasing scientific interest. . PlcR is a B.cereus transcriptional regulator that positively regulates the expression of several genes, during the late vegetative growth.This regulator activates gene expression by binding to a nucleotidic sequence called the 'PlcR box' located upstream from controlled genes, and at various distances ahead of the -35 box of the sigma A promoter to governs the expression of a large regulon encoding extracellular proteins including degradative enzymes and cell-surface proteins..B.cereus B3-7 is an endophytic bacteria, isolated from wheat roots, which have biocontrol capacity against take-all of wheat. we have constructed a transposon inserted mutants library contained 12000 strains. After screening in vivo, a biocontrol capacity deficiency mutant acquired which was demonstrated plcR gene disrupted by transposon insertion. To elucidate the function of plcR in the process of biocontrol. We constructed plcR deletion mutant（ΔplcR）. After testing bicontrol activity of wild type B3-7, B3-7 (ΔplcR) and B3-7(ΔplcR::TnYLB-1), we reveal plcR is essential for biocontrol activities of B3-7. we have showed several secretion protein accumulated in ΔplcR deletion mutant based on 2D electrophoresis . .Based on research previously, we would characterize the secretion proteins accumulated with MALDI-TOF-MS and characterize PlcR box upstream from controlled genes which encode secretion protein. We then would carry out gene knock and compare the biocontrol activity against taka-all among mutants and its wild type to ascertain biocontrol related genes regulated by PlcR. Next ,we would analyze the metabolic circuits of biocontrol related genes involved and carry out multigene deletion to disclose temporal and spatial expression patterns of genes in the process of biocontrol. All the research findings will lay a foundation of development biocontrol agents againat take-all based B3-7 in the future.

利用植物内生细菌进行植物病害尤其是植物土传病害的生物防治，可以克服利用根际微生物进行生物防治容易出现的防治效果不稳定的现象。研究内生芽孢杆菌生防作用发挥的调控网络，有助于深入理解其生防作用机制。全局性转录调控因子PlcR 调控芽孢杆菌的自我保护、环境适应和营养获取等基本生物学过程，但是受PlcR 调控生防因子的类型和调控作用方式有待阐明。本研究利用一株经过安全性评价的生防芽孢杆菌B3-7 菌株plcR 基因缺失导致对于两种小麦土传病害生防能力降低的突变体ΔplcR，通过蛋白质组学方法确定受PlcR 调控的蛋白因子，分离编码蛋白因子的生防相关基因，利用基因敲除、基因互补和基因超表达等方法确定各个基因及其编码产物的功能，解析受PlcR 调控的生防因子的多样性和PlcR 因子调控对于生防菌B3-7 菌株生防作用发挥的影响。为进一步开发利用该生防菌株提供依据。

小麦纹枯病（sharp eyespot of wheat）是由禾谷丝核菌（Rhizoctonia cerealis）侵染小麦引起的一种重要土壤传播病害，广泛分布于各个小麦产区。目前，生产上尚缺乏小麦纹枯病抗病品种，化学防治又存在防效低且容易导致环境污染等不足。利用植物根际微生物及其代谢产物进行小麦纹枯病的生物防治具有重要价值。蜡样芽孢杆菌（Bacillus cereus）B3-7是一株小麦内生细菌，对于小麦纹枯病和小麦全蚀病具有较好的防治效果。前期研究中，我们发现B3-7菌株中plcR基因突变后，突变体对于小麦纹枯病的生防能力下降。PlcR是普遍存在于蜡样芽孢杆菌中的一个多效转录调节因子，参与稳定期细菌基因表达的调控。为了揭示多效转录因子PlcR在蜡样芽孢杆菌B3-7生物防治小麦纹枯病中的作用，我们综合利用蛋白质组学、分子遗传学和植物病理学等多学科研究技术，系统分析了PlcR参与调控的基因种类，发现PlcR可以直接调控27个基因表达，其中13个基因的编码产物分泌到细胞外，11个基因的编码产物位于细胞表面，3个基因的编码产物位于细胞内。对这些基因的功能分析表明，受PlcR调控的基因主要参与获取营养、保护细胞、感受环境条件变化和提高竞争能力等作用。通过基因敲除、互补和表型测定，发现4个基因参与B3-7对于小麦纹枯病的生物防治。这4个基因分别是inhA2，dgcC，nprB，flgB，它们分别编码免疫抑制子A金属蛋白酶（immune inhibitor A metalloprotease）、sensory box / GGDEF 家族蛋白、中性蛋白酶(Bacillolysin) 和鞭毛基体杆蛋白（flagellar basal-body rod protein）。上述4个基因表达均受PlcR正调控。通过分析不同基因及其生物在B3-7菌株生物防治中的作用，发现flgB影响细菌在植物根内定殖，inhA2和nprB影响细菌在小麦根际的定殖，而dgcC作为第二信使c-di-GMP结合蛋白，通过影响细菌的生物膜形成和对于病原真菌的拮抗能力多方位影响细菌在病害系统中的生存能力。至于4个基因在生防过程中是否存在直接互作，成为下一步研究的重点。