埃勒斯致病杆菌SN22次生代谢产物分离和抑菌活性研究
发布时间：2018-11-12 19:49
【摘要】：昆虫病原线虫共生菌(Entomopathogenic bacteria)以其独特的作用方式和能够产生大量的抗菌、杀虫活性物质,越来越得到国内外专家学者的重视。目前,我国对昆虫病原线虫共生菌的研究主要集中在发酵液的活性和杀虫蛋白的活性研究,而对于发酵液中含有的小分子化合物的抑菌活性的研究较少,因此本文的研究重点放在以下几个方面:昆虫病原线虫共生菌SN22分子生物学鉴定、分离纯化次生代谢产物中的小分子单体化合物、鉴定小分子化合物的化学结构并测定小分子化合物的抑菌活性,结果如下:1.SN22菌株的鉴定:对SN22菌株进行生理生化特性测定和16S rRNA序列分析。PCR扩增SN22菌株的16S rRNA序列,选择同源序列进行Clustal比对,采用Mega6.0软件构建系统发育树,结果显示SN22菌株与Xenorhabdus ehlersii DSM 16337T/AJ810294同源相似性为100%,将其编号为Xenorhabdus ehlersii SN22.2.SN22菌株次生代谢产物的分离纯化:对埃勒斯致病杆菌SN22进行大批量发酵,采用活性追踪法对其活性成分进行追踪,发现BC组分抑菌活性较好。利用硅胶柱层析结合凝胶柱层析技术从埃勒斯致病杆菌SN22(Xenorhabdus ehlersii SN22)发酵液中分离得到5个单体化合物,并分别命名为化合物1,化合物2,化合物3,化合物4和化合物5。通过核磁共振波谱、质谱及X-Ray单晶衍射技术分析,鉴定化合物1为3-(环己-2-烯)丙烯酸,文献调研发现该化合物为新的天然产物,命名为Xenorhabic acid。化合物2、化合物3、化合物4和化合物5均为已知化合物,分别鉴定为:Xenorhabdins4、Xenorhabdins5、Xenorhabdins2 和 Xenorhabdins1。3.SN22菌株次生代谢产物生物活性测定:采用菌丝生长速率法测定了该化合物对植物病原真菌的抑菌活性;采用微量稀释法测定了其对病原细菌的最小抑制浓度。结果表明:其对向日葵菌核病原菌Sclerotinia sclerotiorum和瓜果腐霉病原菌Pythium aphanidermatum菌丝生长具有明显的抑制作用,其EC50值分别为27.99和29.55 μg/mL;对大肠埃希杆菌Escherichia coli 的最小抑制浓度(MIC)为62.5 μg/mL。采用微量稀释法测定二硫吡咯酮类化合物(化合物2、化合物3、化合物4和化合物5)对病原细菌的活性。其结果表明:Xenorhabdins 4对枯草芽孢杆菌和金黄色葡萄球菌的最小抑制浓度(MIC)活性仅为4 μg/mL和2 μg/mL。Xenorhabdins 5对枯草芽孢杆菌的活性最好为32μg/mL。Xenorhabdins 2对枯草芽孢杆菌的活性表现最好,其最小抑制浓度为2 μg/mL,其次为金黄色葡萄球菌的最小抑制浓度为32 μg/mL;Xenorhabdins 1对金黄色葡萄球菌的活性最好,其最小抑制浓度为16 μg/mL。本文发现的二硫吡咯酮类化合物对柞蚕链球菌没有表现为良好的生物活性。Xenorhabdins 4和Xenorhabdins 2浓度为256 μg/mL对柞蚕链球菌表现有微弱的活性。
[Abstract]:(Entomopathogenic bacteria), a symbiotic bacterium of nematodes, has been paid more and more attention by experts and scholars at home and abroad for its unique way of action and its ability to produce a large number of antibacterial and insecticidal substances. At present, the studies on the symbiotic bacteria of insect nematodes mainly focus on the activity of fermentation broth and insecticidal protein, but less on the bacteriostatic activity of the small molecular compounds contained in the fermentation broth. Therefore, this paper focuses on the following aspects: SN22 molecular biological identification, isolation and purification of small molecular monomers from secondary metabolites of nematode symbiotic bacteria. The chemical structure and bacteriostatic activity of small molecular compounds were identified. The results were as follows: identification of 1.SN22 strain: physiological and biochemical characteristics of SN22 strain and analysis of 16s rRNA sequence. 16s rRNA sequence of SN22 strain was amplified by PCR. Homologous sequences were selected for Clustal alignment, and phylogenetic tree was constructed by Mega6.0 software. The results showed that the similarity between SN22 strain and Xenorhabdus ehlersii DSM 16337T/AJ810294 was 100%. The isolated and purified secondary metabolites of Xenorhabdus ehlersii SN22.2.SN22 strain were separated and purified. The SN22 of pathogenic bacillus Ehlers was fermented in large quantities and its active components were traced by activity tracing method. It was found that the bacteriostatic activity of BC component was better. Five monomers were isolated from the fermentation broth of pathogenic bacterium SN22 (Xenorhabdus ehlersii SN22 by silica gel column chromatography and gel column chromatography. They were named compound 1, compound 2, compound 3, compound 4 and compound 5 respectively. The compound 1 was identified as 3- (cyclohexene-2-ene) acrylic acid by NMR, mass spectrometry and X-Ray single crystal diffraction technique. It was found that the compound was a new natural product named Xenorhabic acid.. Compound 2, compound 3, compound 4 and compound 5 are known compounds, identified as Xenorhabdins4,Xenorhabdins5, respectively. Biological activity of secondary metabolites of Xenorhabdins2 and Xenorhabdins1.3.SN22 strains: the bacteriostatic activity of the compound against plant pathogenic fungi was determined by hyphal growth rate method. The minimal inhibitory concentration to pathogenic bacteria was determined by microdilution method. The results showed that it could inhibit the growth of sunflower sclerotia pathogen Sclerotinia sclerotiorum and Pythium aphanidermatum mycelium, and its EC50 values were 27.99 渭 g / mL and 29.55 渭 g / mL, respectively. The minimum inhibitory concentration (MIC) of Escherichia coli was 62.5 渭 g / mL. The activity of dithiopyrrolidone compounds (compound 2, compound 3, compound 4 and compound 5) against pathogenic bacteria was determined by microdilution method. The results showed that the minimum inhibitory concentration of (MIC) activity of Xenorhabdins 4 on Bacillus subtilis and Staphylococcus aureus was only 4 渭 g/mL and 2 渭 g/mL.Xenorhabdins 5 against Bacillus subtilis was the best for 32 渭 g/mL.Xenorhabdins 2 against Bacillus subtilis. The activity of Bacillus grasses is the best. Its minimum inhibitory concentration was 2 渭 g / mL, followed by Staphylococcus aureus at 32 渭 g / mL. Xenorhabdins 1 had the best activity against Staphylococcus aureus with a minimum inhibitory concentration of 16 渭 g / mL. It was found that the dithiopyrrolidone compounds had no good biological activity against S. tussah, but the concentrations of Xenorhabdins 4 and Xenorhabdins 2 were 256 渭 g/mL and showed weak activity against Streptococcus tussah.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476
[Abstract]:(Entomopathogenic bacteria), a symbiotic bacterium of nematodes, has been paid more and more attention by experts and scholars at home and abroad for its unique way of action and its ability to produce a large number of antibacterial and insecticidal substances. At present, the studies on the symbiotic bacteria of insect nematodes mainly focus on the activity of fermentation broth and insecticidal protein, but less on the bacteriostatic activity of the small molecular compounds contained in the fermentation broth. Therefore, this paper focuses on the following aspects: SN22 molecular biological identification, isolation and purification of small molecular monomers from secondary metabolites of nematode symbiotic bacteria. The chemical structure and bacteriostatic activity of small molecular compounds were identified. The results were as follows: identification of 1.SN22 strain: physiological and biochemical characteristics of SN22 strain and analysis of 16s rRNA sequence. 16s rRNA sequence of SN22 strain was amplified by PCR. Homologous sequences were selected for Clustal alignment, and phylogenetic tree was constructed by Mega6.0 software. The results showed that the similarity between SN22 strain and Xenorhabdus ehlersii DSM 16337T/AJ810294 was 100%. The isolated and purified secondary metabolites of Xenorhabdus ehlersii SN22.2.SN22 strain were separated and purified. The SN22 of pathogenic bacillus Ehlers was fermented in large quantities and its active components were traced by activity tracing method. It was found that the bacteriostatic activity of BC component was better. Five monomers were isolated from the fermentation broth of pathogenic bacterium SN22 (Xenorhabdus ehlersii SN22 by silica gel column chromatography and gel column chromatography. They were named compound 1, compound 2, compound 3, compound 4 and compound 5 respectively. The compound 1 was identified as 3- (cyclohexene-2-ene) acrylic acid by NMR, mass spectrometry and X-Ray single crystal diffraction technique. It was found that the compound was a new natural product named Xenorhabic acid.. Compound 2, compound 3, compound 4 and compound 5 are known compounds, identified as Xenorhabdins4,Xenorhabdins5, respectively. Biological activity of secondary metabolites of Xenorhabdins2 and Xenorhabdins1.3.SN22 strains: the bacteriostatic activity of the compound against plant pathogenic fungi was determined by hyphal growth rate method. The minimal inhibitory concentration to pathogenic bacteria was determined by microdilution method. The results showed that it could inhibit the growth of sunflower sclerotia pathogen Sclerotinia sclerotiorum and Pythium aphanidermatum mycelium, and its EC50 values were 27.99 渭 g / mL and 29.55 渭 g / mL, respectively. The minimum inhibitory concentration (MIC) of Escherichia coli was 62.5 渭 g / mL. The activity of dithiopyrrolidone compounds (compound 2, compound 3, compound 4 and compound 5) against pathogenic bacteria was determined by microdilution method. The results showed that the minimum inhibitory concentration of (MIC) activity of Xenorhabdins 4 on Bacillus subtilis and Staphylococcus aureus was only 4 渭 g/mL and 2 渭 g/mL.Xenorhabdins 5 against Bacillus subtilis was the best for 32 渭 g/mL.Xenorhabdins 2 against Bacillus subtilis. The activity of Bacillus grasses is the best. Its minimum inhibitory concentration was 2 渭 g / mL, followed by Staphylococcus aureus at 32 渭 g / mL. Xenorhabdins 1 had the best activity against Staphylococcus aureus with a minimum inhibitory concentration of 16 渭 g / mL. It was found that the dithiopyrrolidone compounds had no good biological activity against S. tussah, but the concentrations of Xenorhabdins 4 and Xenorhabdins 2 were 256 渭 g/mL and showed weak activity against Streptococcus tussah.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476
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