绿针假单胞菌yl-1抗细菌活性物质的鉴定及相关基因的功能分析

Pseudomonas chlororaphis is a good prospect of biocontrol bacteria, 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 throughout the world. However, Pseudomonas chlororaphis yl-1, isolated from the root tip of soybean, showed strong antifungal and antibacterial activities against a variety of pathogenic fungi and bacteria. To explore the biocontrol mechanism on bacterial diseases of Pseudomonas chlororaphis yl-1, a lot of research had been done. For example, using transposon insertion mutagenesis techniques, 8 mutants loss of most antibacterial activities were screened among 5000 transformers using three pathogenic bacteria (Bacillus megaterium; Burkholderia glumae; Erwinia amylovora) as indicators. Additionally, the preliminary results also showed that the antibacterial compound secreted by Pseudomonas chlororaphis yl-1 belongs to secondary metabolites, and it can only regard pathogenic bacteria as the only role of target. According to these interesting results, we speculate that maybe there are some new antibacterial compound and related genes in Pseudomonas chlororaphis yl-1. This proposal is planned to firstly identify the antibacterial compound secreted by Pseudomonas chlororaphis yl-1 after comparing the difference of secondary metabolites secreted by wild type and mutants with the help of Infrared Spectroscopy (IR), Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) and Nuclear Magnetic Resonance (NMR) together. Secondly, the inhibition activity of antibacterial compound also will be studied in this proposal. Then, use the preliminary mutants to construct the rescue clones and clone antibacterial related genes associated with inverse PCR and Tail-PCR. After that, the difference between wild type and mutants on antibacterial compound production, biofilm production, colonization and biocontrol effect will be studied in this proposal after target gene knockout. Lastly, verify some regulatory genes using functional complementation and test the transcript difference between wild type and mutants. In conclusion, results based on this proposal will be of great significance not only to broaden the biocontrol spectrum of Pseudomonas chlororaphis and develop the correlative biological bacterialcide; but also benefit on the genetic improvement of Pseudomonas chlororaphis and provide a theoretical basis on the biocontrol mechanism of Pseudomonas chlororaphis against bacterial diseases.

申请者首次报道从大豆根围分离获得的绿针假单胞菌(Pseudomonas chlororaphis)对很多病原细菌具有较强抑制能力。为探明其抑菌机制，前期工作中申请者采用转座子诱变技术，获得病原细菌抑菌能力明显下降的8株突变菌株。进一步分析发现，该菌株分泌的抗细菌活性物质具有广谱抗细菌活性，不具有抗真菌活性，因此推测其化学结构和遗传基因可能具有新颖性。本项目首先通过色谱、光谱和质谱技术，比较分析野生型和突变菌株代谢产物差异，纯化抗细菌活性物质并鉴定结构，明确其对病原细菌的毒力作用。其次采用质粒拯救、反向PCR和Tail-PCR等方法，从突变菌株中克隆鉴定活性物质相关基因。采用定向敲除、功能互补和荧光定量PCR等手段，研究上述基因在活性物质产生、生物膜形成、定殖和生防中的功能。研究结果对拓宽绿针假单胞菌生防谱，开发相关生物农药具有重要意义；同时为绿针假单胞菌防治细菌病害的生防机理提供理论依据。

绿针假单胞菌YL-1对农业上多种重要病原细菌和病原真菌有较强的抑制作用。前期研究中采用转座子插入突变技术电转化绿针假单胞菌YL-1，构建随机插入突变体库。通过平板抑菌试验从突变体库中筛选对三种病原细菌荚壳伯克霍尔德菌Burkholderia glumae、解淀粉欧文氏菌Erwinia amylovora和胡萝卜果胶杆菌Dickeya carotovora抑菌效果下降的突变体。采用质粒拯救（Rescue clones）、反向PCR（Inverse PCR）和基因步行（Gene walking）等方法，从突变菌株中克隆抗细菌活性物质的相关基因pvdA、pvdD、pvdE、pvdL、pvdP、pvdS、tatABC、secY、secG等13个。7株对三种病原细菌抑制效果明显下降的突变体，PCR和Southern Blot试验结果表明转座子成功插入到绿针假单胞菌YL-1染色体基因组上，并且突变体以单拷贝形式插入；克隆到7个突变体插入位点的基因序列并测序，生物信息学分析结果表明其中6个突变位点是嗜铁素合成基因簇，1个突变位点是蛋白分泌基因secY。本项目首次发现嗜铁素（Pyoverdine）与绿针假单胞菌YL-1抗细菌活性密切相关。在细菌中，至少有3种主要的蛋白输出系统。Sec分泌蛋白转运系统是最早也是最多研究的对象，是最重要的运输途径，主要分泌各种蛋白如毒素、水解酶以及抗菌肽等。鉴于此，我们选择secG基因进行定向敲除和功能互补实验，结果表明回复突变菌株能部分回复抑制解淀粉欧文氏菌Erwinia amylovora和菊果胶杆菌Pectobacterium chrysanthemi的能力，我们推断secG基因是绿针假单胞菌（Pseudomonas chlororaphis）YL-1和日本假单胞菌（Pseudomonas japonica）YL-23抗细菌活性所必需的。