绿针假单胞菌YL-1抗细菌活性物质-嗜铁素生物合成基因簇的鉴定及功能研究

Pseudomonas chlororaphis is a kind of biocontrol bacteria of good prospect, mainly used to control a variety of fungal diseases. Until now the research about biocontrol on bacterial diseases using Pseudomonas chlororaphis has not been studied through the world. Strain YL-1 was isolated from soybean root tips and identified to be Pseudomonas chlororaphis. This strain showed broad-spectrum antibacterial and antifungal activities against plant phytopathogens that are economically important in agriculture. In the preliminary study，in order to characterize the genes dedicated to antibacterial activities against microbial phytopathogens, a Tn5-mutation library of YL-1 was constructed. Plate bioassays revealed that seven mutants MT1-7 reduced their antibacterial activities against Burkholderia glumae, Erwinia amylovora and Pectobacterium carotovora as compared with its wild type strain while remained steady antifungal activities against Rhizoctonia solani. All the mutants MT1-7 were verified by the PCR and Southern Blot, the results showed that the transposon was inserted into YL-1 genomic DNA successfully by single copy. Seven genes around the insertion site were cloned and sequenced using a plasmid rescue procedure as recommended by the manufacturer of the EZ-Tn5 kit. The bioinformatic analysis results showed that six genes were related to pyoverdine synthesis and secY gene belonged to the Sec protein translocation system. Our recent results showed that pyoverdine (PVD) crude extract secreted by strain YL-1 could uniquely inhibit the growth of pathogenic bacteria Erwinia amylovora and Pectobacterium carotovora, but had no antifungal activity. Additionally, the bioinformatic analysis showed that the secondary structure of PvdJ protein was different from that of other Pseudomonas, so it was presumed that the synthetic gene cluster of pyoverdine in strain YL-1 might be novel. This proposal is planned to firstly determine PVD yield and anti-bacteria activity of each gene mutant in the gene cluster by directed mutation technology, then analyze the contribution of each gene in the gene cluster involved in the PVD mediated anti-bacterial activity, and then double conform the results of these genes by functional complementation. After that, the difference of PVD activity peak was detected between the wild type and mutant strains using HPLC and other methods, then locate the target PVD using the activity tracking method. Lastly, the product structure of PVD in the strain was identified by mass spectrometry, nuclear magnetic and other technical means. In conclusion, results based on this proposal will be of great significance not only to broaden the biocontrol spectrum of PVD and develop the correlative biological bacterialcide; but also provide a theoretical basis on the biocontrol mechanism of Pseudomonas chlororaphis against bacterial diseases.

绿针假单胞菌(Pseudomonas chlororaphis)YL-1对多种病原细菌具有较强抑制作用。前期工作中申请者发现菌株YL-1产生的荧光性嗜铁素（PVD）与以往报道不同，能抑制病原细菌Erwinia amylovora和Pectobacterium carotovora的生长。生物信息学分析发现其合成基因簇PvdJ蛋白二级结构与假单胞菌同系物有差异，因此推测其化学结构和合成基因簇可能具有新颖性。本项目首先通过定向突变技术，测定基因簇中每个基因突变株PVD产量及抗细菌活性，分析这些基因在PVD介导的抗细菌活性的贡献，功能互补后进行结果确认；利用生物活性追踪、HPLC等方法纯化靶标PVD，通过质谱、核磁等技术手段对产物PVD进行化学结构鉴定。研究结果为绿针假单胞菌YL-1防治细菌病害的作用机理提供理论依据，同时对拓宽PVD生防谱，开发相关抗细菌病害的生物农药具有重要指导意义。

绿针假单胞菌YL-1是申请者分离获得的一株对农业上多种重要病原细菌和病原真菌有较强抑制作用的生防细菌。本项目主要研究内容：① 利用生物信息学手段，预测菌株YL-1全基因组中可能负责抗细菌活性物质合成的基因簇6个：吩嗪类合成基因簇，硝吡咯菌素合成基因簇，氢氰酸合成基因簇，2套嗜铁素合成基因簇和参与杀虫的Fit毒素生物合成基因簇；② 成功构建sacB介导的绿针假单胞菌YL-1两亲交配遗传转化方法，逐个敲除菌株YL-1中重要的生防相关基因，进行功能研究；结果表明：菌株YL-1在营养环境中产生的主要抗细菌活性物质是吩嗪（PCA），而在缺铁环境中产生的主要抗细菌活性物质是嗜铁素（PVD）；③ 随后进行菌株YL-1中PCA的提取和质谱鉴定，研究其作用机制，结果表明：PCA处理靶细胞后，增加胞内活性氧ROS积累，使靶细胞中毒死亡。在此过程中，靶细胞会被激发产生氧化应激反应，激活氧化应激响应转录因子 OxyR调控的抗氧化防御基因（katB、katG和ahpCF）的表达；④ 完成菌株YL-1嗜铁素合成基因簇的鉴定与功能分析，结果表明：菌株YL-1产生可见的PVD，对测试的4种革兰氏阴性菌和革兰氏阳性菌均表现出较强的室内抑制作用，并且随着缺铁环境中外源铁含量的增加，PVD的产生、抗菌活性和PVD转录因子基因pvdS的表达均明显下降；⑤ 采用离子交换层析、液质联用（UHPLC-LTQ-ESI-Orbitrap）等技术，分离纯化PVD，解析其中一条侧链肽段序列为Asp-FOHOrn-Lys-(Thr-Ala-Ala-FOHOrn-Lys（质合比为1287）。⑥ 最后通过盆栽试验，验证PVD样品对水稻白叶枯病具有较好的防治效果。项目研究结果揭示荧光性嗜铁素在防治细菌病害中除了传统学说认为的“铁竞争作用原理”之外，还可作为抗生素直接抑制病原细菌的生长，对开发新型抗细菌病害的生物农药具有重要意义。