本文選題：伯克霍爾德氏菌 + 假單胞菌�。� 參考：《山東農(nóng)業(yè)大學》2016年博士論文

【摘要】：植物病害是影響農(nóng)業(yè)高產(chǎn)穩(wěn)產(chǎn)和優(yōu)質(zhì)的主要因素之一。生物防治作為一種綠色安全的防治措施,不僅可以有效降低病蟲害對農(nóng)作物造成的損失還可以促進植物生長,提高植物抗病性,增加作物產(chǎn)量。本研究以植物病原菌為指示菌篩選拮抗活性較高的植物根際促生菌(plant growth-promoting rhizobacteria,PGPR),并進行種屬鑒定;構(gòu)建PGPR菌株突變體庫,克隆生防相關(guān)基因,研究PGPR菌株的拮抗機制。主要研究結(jié)果如下:1、鑒定了菌株Lyc2的系統(tǒng)分類地位。通過生理生化特征、16S rRNA序列分析、多位點序列分型分析(multilocus sequence typing,MLST)以及全基因組測序等方法,證明菌株Lyc2為吡咯伯克霍爾德氏菌(Burkholderia pyrrocinia)。2、獲得了菌株Lyc2的全基因組草圖。為尋找潛在的次生代謝產(chǎn)物生物合成基因簇,對Lyc2基因組序列進行了生物信息學預測分析,共發(fā)現(xiàn)8類,15個次生代謝產(chǎn)物生物合成基因簇。主要包括,Arylpolyene類(2個)、Terpene類(5個)、Phosphonate類(1個)、Hserlactone類(1個)、Nrps類(2個)、Bacteriocin類(2個)、Nrps-Type I PKS類(1個)和其它類(1個)。3、探索Lyc2的抗菌機制。構(gòu)建菌株Lyc2的隨機突變體庫,通過測序驗證得到一個55.2kb負責抗真菌物質(zhì)生物合成的基因簇ocfABCDEFGHIJKLMN,該基因簇編碼一個由八個氨基酸殘基組成的環(huán)狀抗菌多肽化合物occidiofungin。對功能未知的ocfI基因的突變及基因互補實驗表明,ocfI基因直接參與抗菌多肽occidiofungin的生物合成;另外,篩選得到一個失去細菌拮抗能力的突變體。序列分析表明,突變基因與菌株Burkholderia ambifaria AMMD的谷胱甘肽合成酶基因(glutathione synthase)具有較高同源性,核苷酸序列一致率為93.3%。對突變體的功能分析及互補實驗結(jié)果表明,谷胱甘肽合成酶基因與菌株Lyc2對病原細菌的拮抗活性直接相關(guān)。4、明確了假單胞菌XW10的系統(tǒng)分類地位。分離自大豆根際的假單胞菌XW10對多種病原菌尤其是青枯勞爾氏菌(Ralstonia solanacearum)具有顯著拮抗活性。通過生理生化、16S rRNA和MLST分析發(fā)現(xiàn)菌株XW10與之前報道的假單胞屬細菌具有顯著差異,為假單胞菌屬的一個新種,命名為Pseudomonas beanensis XW10。5、明確了菌株XW10對R.solanacearum的拮抗機制。構(gòu)建了菌株XW10的隨機突變體庫,篩選得到一個失去R.solanacearum抗性的突變體。對突變體的分析表明,發(fā)生突變的基因為雙精氨酸轉(zhuǎn)運系統(tǒng)(Twin-arginine translocase secretion system,Tat)中的TatA基因。功能互補試驗表明,互補TatA基因可以恢復突變體對病原菌的拮抗活性,說明Tat系統(tǒng)介導的轉(zhuǎn)運途徑對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.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S476

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