生防解淀粉芽孢杆菌CC09菌株环脂肽合成酶基因的转录调控机理研究

Cyclic lipopeptides (CLPs) that are synthesized by nonribosomal synthetic pathway are a group of the important secondary metabolites, which play a key role in control of plant diseases. Although the biochemical and operon properties of CLPs synthetases are known relatively well, the transcriptional regulation mechanism of the synthetase genes is not understood yet. At the present project, a biocontrol endophytic strain of Bacillus amyloliquefaciens CC09 that was isolated from Cinnamomum camphora is used as a start strain to prepare a seris of RNA polynerase mutated mutants, which show both increase and decrease of CLPs (iturin A and surfactin) production by using the RNA polymerizing (RNAP) mutation technique. The wild type and the mutant strains are used as materials to study the transcriptional properties of CLPs synthetase encoding genes by in vitro and in vivo transcriptional detection methods. Meanwhile, DNA chips and 2-D electrophoresis techniques are used to analyze the transcriptomics and proteomics spectra of these test strains at different growth phases. All the regulators invoving CLPs synthetase transcription and their interation will be studied.The unknown transcriptional regulation mechanisms of CLPs in B. amyloliquefaciens CC09 will be disclosed by comparing all the data between wild strain and its RNA polymerase muation strains. The obtained results also can provide new ideas and methods for studying the transcriptional regualtion mechanisms of nonribosomal synthetase genes ecoding antibitic synthetases in other microorganisms.

环脂肽(CLPs)是生防芽孢杆菌通过非核糖体肽合成酶催化合成的次生代谢物质，在防治植物病害过程中发挥着关键作用。虽然CLPs合成酶的生化性质和操纵子结构已比较清楚，但其转录调控机制至今尚未充分阐明。本项目以生防内生解淀粉芽孢杆菌CC09菌株为出发菌株，利用RNA聚合酶突变技术，制备一系列对CLPs(Iturin A和Surfactin)合成具有促进(正突变)和抑制(负突变)作用的突变菌株；以上述野生型和突变型菌株为材料，采用RNA聚合酶的体内和体外转录研究技术，研究CLPs合成酶基因的转录特性；通过芯片分析以及二维电泳等手段，分析不同生长阶段各菌株之间的转录组学和蛋白质组学差异，了解CLPs合成酶基因转录过程中存在的调控因子及其相互作用，揭示尚未知晓的解淀粉芽孢杆菌非核糖体合成CLPs的转录调控机制，并为研究其他微生物非核糖体合成抗生素的转录调控机理提供新思路和新方法。

采用全基因组测序、RNA聚合酶突变、比较转录组学技术，研究了内生解淀粉芽孢杆菌CC09菌株的生防基础及环脂肽合成酶基因的转录调控机制，取得了如下结果：⑴完成了CC09菌株全基因组测序，4.17 Mb，46.1% GC，4021个基因（Accession No. CP015443）。⑵基于核心基因组序列的同源性分析，将CC09菌株鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。⑶全面分析了CC09菌株产次生代谢产物的潜力，共鉴定出13个具有生防效果的代谢物，其中4个尚未报道。⑷获得并确认了6个抗利福平RNA聚合酶亚基单点突变菌株【CC09-RIF1（H485Y），CC09-RIF2（H485C），CC09-RIF3（H485R），CC09-RIF4（H485D），CC09-RIF5（S490L），CC09-RIF6（Q472R）】和1个双点突变菌株【CC09-RIF7（S490L/S617F）】。⑸深入研究了不同突变菌株的生长和生理学表型，发现H485突变显著影响CC09菌株的生防能力（运动性，生物膜，芽孢，抑制真菌分生孢子，iturin A产量），其中，CC09-RIF3和CC09-RIF4产iturin A的能力分别是CC09菌株的2.03倍和1.67倍，为正突变菌株；而CC09-RIF6和CC09-RIF5产iturin A能力则显著低于CC09菌株，为负突变菌株。⑹优化了CC09菌株和CC09-RIF3产iturin A的发酵条件。⑺CC09-RIF3、CC09-RIF6与CC09菌株的比较转录组学研究结果显示，CC09-RIF3有615个基因上调，379个基因下调，上调基因数是下调基因数的1.6倍；而CC09-RIF6则有386个基因上调，648个基因下调，上调基因数是下调基因数的0.6倍。⑻基于差异基因表达水平和iturin A产量，构建了CC09菌株iturin A合成途径的转录调控网络，其中至少有20多个基因参与了iturin A合成的转录调控。⑼体内转录活性证明，iturin A基因簇的转录调控十分复杂，既与转录启动子与终止子序列有关，又可能与转录后调控有关。这些研究结果为深入了解内生贝莱斯芽孢杆菌CC09菌株生防作用的化学和分子机理奠定了重要理论基础。