【摘要】：昆蟲病原線蟲共生菌(Entomopathogenic bacteria)以其獨特的作用方式和能夠產(chǎn)生大量的抗菌、殺蟲活性物質(zhì),越來越得到國內(nèi)外專家學者的重視。目前,我國對昆蟲病原線蟲共生菌的研究主要集中在發(fā)酵液的活性和殺蟲蛋白的活性研究,而對于發(fā)酵液中含有的小分子化合物的抑菌活性的研究較少,因此本文的研究重點放在以下幾個方面:昆蟲病原線蟲共生菌SN22分子生物學鑒定、分離純化次生代謝產(chǎn)物中的小分子單體化合物、鑒定小分子化合物的化學結(jié)構(gòu)并測定小分子化合物的抑菌活性,結(jié)果如下:1.SN22菌株的鑒定:對SN22菌株進行生理生化特性測定和16S rRNA序列分析。PCR擴增SN22菌株的16S rRNA序列,選擇同源序列進行Clustal比對,采用Mega6.0軟件構(gòu)建系統(tǒng)發(fā)育樹,結(jié)果顯示SN22菌株與Xenorhabdus ehlersii DSM 16337T/AJ810294同源相似性為100%,將其編號為Xenorhabdus ehlersii SN22.2.SN22菌株次生代謝產(chǎn)物的分離純化:對埃勒斯致病桿菌SN22進行大批量發(fā)酵,采用活性追蹤法對其活性成分進行追蹤,發(fā)現(xiàn)BC組分抑菌活性較好。利用硅膠柱層析結(jié)合凝膠柱層析技術(shù)從埃勒斯致病桿菌SN22(Xenorhabdus ehlersii SN22)發(fā)酵液中分離得到5個單體化合物,并分別命名為化合物1,化合物2,化合物3,化合物4和化合物5。通過核磁共振波譜、質(zhì)譜及X-Ray單晶衍射技術(shù)分析,鑒定化合物1為3-(環(huán)己-2-烯)丙烯酸,文獻調(diào)研發(fā)現(xiàn)該化合物為新的天然產(chǎn)物,命名為Xenorhabic acid。化合物2、化合物3、化合物4和化合物5均為已知化合物,分別鑒定為:Xenorhabdins4、Xenorhabdins5、Xenorhabdins2 和 Xenorhabdins1。3.SN22菌株次生代謝產(chǎn)物生物活性測定:采用菌絲生長速率法測定了該化合物對植物病原真菌的抑菌活性;采用微量稀釋法測定了其對病原細菌的最小抑制濃度。結(jié)果表明:其對向日葵菌核病原菌Sclerotinia sclerotiorum和瓜果腐霉病原菌Pythium aphanidermatum菌絲生長具有明顯的抑制作用,其EC50值分別為27.99和29.55 μg/mL;對大腸埃希桿菌Escherichia coli 的最小抑制濃度(MIC)為62.5 μg/mL。采用微量稀釋法測定二硫吡咯酮類化合物(化合物2、化合物3、化合物4和化合物5)對病原細菌的活性。其結(jié)果表明:Xenorhabdins 4對枯草芽孢桿菌和金黃色葡萄球菌的最小抑制濃度(MIC)活性僅為4 μg/mL和2 μg/mL。Xenorhabdins 5對枯草芽孢桿菌的活性最好為32μg/mL。Xenorhabdins 2對枯草芽孢桿菌的活性表現(xiàn)最好,其最小抑制濃度為2 μg/mL,其次為金黃色葡萄球菌的最小抑制濃度為32 μg/mL;Xenorhabdins 1對金黃色葡萄球菌的活性最好,其最小抑制濃度為16 μg/mL。本文發(fā)現(xiàn)的二硫吡咯酮類化合物對柞蠶鏈球菌沒有表現(xiàn)為良好的生物活性。Xenorhabdins 4和Xenorhabdins 2濃度為256 μg/mL對柞蠶鏈球菌表現(xiàn)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.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S476

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