植物根际促生菌Lyc2和XW10的鉴定及抑菌机理研究

发布时间：2018-06-10 03:22

本文选题：伯克霍尔德氏菌 + 假单胞菌　；参考：《山东农业大学》2016年博士论文

【摘要】：植物病害是影响农业高产稳产和优质的主要因素之一。生物防治作为一种绿色安全的防治措施,不仅可以有效降低病虫害对农作物造成的损失还可以促进植物生长,提高植物抗病性,增加作物产量。本研究以植物病原菌为指示菌筛选拮抗活性较高的植物根际促生菌(plant growth-promoting rhizobacteria,PGPR),并进行种属鉴定;构建PGPR菌株突变体库,克隆生防相关基因,研究PGPR菌株的拮抗机制。主要研究结果如下:1、鉴定了菌株Lyc2的系统分类地位。通过生理生化特征、16S rRNA序列分析、多位点序列分型分析(multilocus sequence typing,MLST)以及全基因组测序等方法,证明菌株Lyc2为吡咯伯克霍尔德氏菌(Burkholderia pyrrocinia)。2、获得了菌株Lyc2的全基因组草图。为寻找潜在的次生代谢产物生物合成基因簇,对Lyc2基因组序列进行了生物信息学预测分析,共发现8类,15个次生代谢产物生物合成基因簇。主要包括,Arylpolyene类(2个)、Terpene类(5个)、Phosphonate类(1个)、Hserlactone类(1个)、Nrps类(2个)、Bacteriocin类(2个)、Nrps-Type I PKS类(1个)和其它类(1个)。3、探索Lyc2的抗菌机制。构建菌株Lyc2的随机突变体库,通过测序验证得到一个55.2kb负责抗真菌物质生物合成的基因簇ocfABCDEFGHIJKLMN,该基因簇编码一个由八个氨基酸残基组成的环状抗菌多肽化合物occidiofungin。对功能未知的ocfI基因的突变及基因互补实验表明,ocfI基因直接参与抗菌多肽occidiofungin的生物合成;另外,筛选得到一个失去细菌拮抗能力的突变体。序列分析表明,突变基因与菌株Burkholderia ambifaria AMMD的谷胱甘肽合成酶基因(glutathione synthase)具有较高同源性,核苷酸序列一致率为93.3%。对突变体的功能分析及互补实验结果表明,谷胱甘肽合成酶基因与菌株Lyc2对病原细菌的拮抗活性直接相关。4、明确了假单胞菌XW10的系统分类地位。分离自大豆根际的假单胞菌XW10对多种病原菌尤其是青枯劳尔氏菌(Ralstonia solanacearum)具有显著拮抗活性。通过生理生化、16S rRNA和MLST分析发现菌株XW10与之前报道的假单胞属细菌具有显著差异,为假单胞菌属的一个新种,命名为Pseudomonas beanensis XW10。5、明确了菌株XW10对R.solanacearum的拮抗机制。构建了菌株XW10的随机突变体库,筛选得到一个失去R.solanacearum抗性的突变体。对突变体的分析表明,发生突变的基因为双精氨酸转运系统(Twin-arginine translocase secretion system,Tat)中的TatA基因。功能互补试验表明,互补TatA基因可以恢复突变体对病原菌的拮抗活性,说明Tat系统介导的转运途径对P.beanensis XW10拮抗R.solanacearum具有重要作用。
[Abstract]:Plant disease is one of the main factors affecting high and stable yield and high quality of agriculture. Biological control, as a green and safe control measure, can not only effectively reduce the losses caused by pests and diseases to crops, but also promote plant growth, improve plant disease resistance and increase crop yield. In this study, plant pathogenic bacteria were used as indicator bacteria to screen growth-promoting rhizobacteriae prostaglandin with high antagonistic activity, and to construct mutant library of PGPR strain, clone biocontrol related genes, and study the antagonistic mechanism of PGPR strain. The main results are as follows: 1. The phylogenetic status of the strain Lyc 2 was identified. The whole genome sketch of strain Lyc2 was obtained by analyzing 16s rRNA sequence of physiological and biochemical characteristics, multilocus sequence typing MLSTs and whole genome sequencing. The results showed that Lyc2 was Burkholderia pyrrocinia.2. In order to search for potential secondary metabolite biosynthesis gene clusters, the bioinformatics analysis of Lyc2 genome sequence was carried out. A total of 8 classes and 15 secondary metabolite biosynthesis gene clusters were found. It mainly includes Arylpolyene class (2 Terpene classes) (5 Phosphonate class (1 Hserlactone class) (1 Nrps class (2 Bacteriocin class) (2 Nrps-Type I PKS class (1) and other class (1. 3) to explore the antibacterial mechanism of Lyc2. A random mutant library of the strain Lyc2 was constructed, and a gene cluster ocfABCDEFGHIJKLMNwhose 55.2kb was responsible for biosynthesis of antifungal substances was obtained by sequencing. The gene cluster encodes a cyclic antibacterial peptide compound occidiofunginconsisting of eight amino acid residues. The mutation of OCI gene with unknown function and gene complementation experiment showed that OCI gene was directly involved in the biosynthesis of antibacterial polypeptide occidiofungin, and a mutant which lost the ability of bacterial antagonism was screened. Sequence analysis showed that the mutant gene had high homology with glutathione synthase gene of Burkholderia ambifaria AMMD, and the nucleotide sequence consistency rate was 93.3%. The functional analysis and complementary experiment of the mutant showed that the glutathione synthase gene was directly related to the antagonistic activity of the strain Lyc2 against pathogenic bacteria. The phylogenetic status of Pseudomonas XW10 was clarified. Pseudomonas sp. XW10 isolated from soybean rhizosphere showed significant antagonistic activity against many pathogens, especially Ralstonia solanacearum. By physiological and biochemical analysis of 16s rRNA and MLST, it was found that XW10 was a new species of Pseudomonas beanensis XW10.5. the antagonistic mechanism of XW10 against R. solanacearum was determined. A random mutant library of strain XW10 was constructed and a mutant without R. solanacearum resistance was screened. The analysis of the mutant showed that the mutant gene was Tata gene in Twin-arginine translocase secretion system. The functional complementation test showed that the complementary TatA gene could restore the antagonistic activity of the mutant against the pathogen, which indicated that the translocation pathway mediated by tat system played an important role in P.beanensis XW10 antagonism against R. solanacearum.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476

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