【摘要】： 蛭弧菌作為一種依靠裂解宿主菌使自身得以繁殖的寄生微生物,它可以裂解大多數(shù)科、屬的革蘭氏陰性細(xì)菌,尤其是對(duì)病原弧菌具有良好的裂解作用。本實(shí)驗(yàn)采用DNB雙層平板法以Escherichia coli DH5α作為宿主菌從湖南省東江水域采集到淡水水樣中篩選得到6株菌株(分別命名為HNAL,HNAS,HNBL,HNBS,HNCL和HNCS)。通過(guò)觀測(cè)6株菌在DNB雙層平板上形成的噬菌斑的形狀,同時(shí)利用透射電子顯微鏡觀察它們的形態(tài),并對(duì)照《伯杰氏細(xì)菌鑒定手冊(cè)》(第九版),最后進(jìn)行16S rRNA基因和16S-23S rRNA ITS擴(kuò)增鑒定、測(cè)序和系統(tǒng)進(jìn)化學(xué)分析,可以鑒定這6株菌為蛭弧菌,同時(shí)經(jīng)系統(tǒng)進(jìn)化學(xué)分析認(rèn)為菌株HNAL,HNAS,HNBL和HNBS屬于蛭弧菌的一個(gè)新種(Bdellovibrio sp.nov)。我們系統(tǒng)研究了6株蛭弧菌對(duì)能導(dǎo)致水產(chǎn)養(yǎng)殖病害的30株致病或潛在致病菌的裂解譜。此外,我們首次以湖南淡水蛭弧菌(HNAS)為研究對(duì)象,先在電鏡下初步觀察蛭弧菌與大腸桿菌的作用方式,之后利用超薄切片技術(shù)結(jié)合透射電子顯微鏡,初步研究淡水蛭弧菌作用大腸桿菌的裂解機(jī)制。 本研究結(jié)果表明,6株淡水蛭弧菌對(duì)30株致病或潛在致病菌具有較好的裂解能力,HNAL,HNAS,HNBL,HNBS,HNCL和HNCS的裂解率分別為70%、73.3%、66.7%、66.7%、50%和60%,如果考慮不同蛭弧菌株間的協(xié)同作用,它們的裂解率則達(dá)93.3%;通過(guò)負(fù)染法對(duì)菌體染色,在電鏡下觀察到蛭弧菌HNAS以機(jī)械鉆孔的形式侵染大腸桿菌;同時(shí)通過(guò)電鏡觀察比較,確定蛭弧菌與大腸桿菌最適濃度比為1:1;在電鏡下觀察蛭弧菌作用大腸桿菌過(guò)程的超微結(jié)構(gòu),根據(jù)所觀測(cè)到電鏡圖可進(jìn)一步確認(rèn)該株淡水蛭弧菌是以機(jī)械鉆孔的方式對(duì)宿主菌進(jìn)行裂解的,同時(shí)觀察到了蛭弧菌附著、侵染、蛭質(zhì)體和釋放的階段。
[Abstract]:Bdellovibrio bdellovibrio, as a parasitic microorganism which can reproduce itself by lytic host bacteria, can cleave Gram-negative bacteria of most families and genera, especially for pathogenic Vibrio. In this experiment, six strains (named HNAL,HNAS,HNBL,HNBS,HNCL and HNCS)., respectively) were isolated from fresh water samples collected from Dongjiang waters of Hunan province by DNB double-layer flat plate method with Escherichia coli DH5 偽 as host bacteria. By observing the shape of the plaque formed on the double-layer plate of DNB, the morphology of the plaque was observed by transmission electron microscope (TEM), and compared with the Burger's Manual of bacterial Identification (ninth edition). Finally, 16s rRNA gene and 16S-23S rRNA ITS amplification, sequencing and phylogenetic analysis showed that the six strains were identified as Bdellovibrio spp. At the same time, the phylogenetic analysis showed that the strain HNAL,HNAS, was identified as Bdellovibrio bdellovibrio. HNBL and HNBS are new species of Bdellovibrio (Bdellovibrio sp.nov). We have systematically studied the cleavage spectra of 6 strains of Bdellovibrio to 30 pathogenic or potential pathogenic bacteria which can cause aquaculture diseases. In addition, for the first time, we studied the interaction of vermiculovibrio vermiculosus and Escherichia coli under electron microscope, and then we used ultrathin section technique combined with transmission electron microscope (TEM) to study the interaction of Vibrio pellicularis (HNAS) with E. coli. The cleavage mechanism of E. coli by Vibrio leech was studied. The results showed that 6 strains of Vibrio leech had good cleavage ability to 30 pathogenic or potential pathogenic bacteria. The cleavage rates of HNAL,HNAS,HNBL,HNBS,HNCL and HNCS were 70%, 73.3%, 66.7%, 66.7%, respectively, and the lytic rates were 70.0%, 73.3%, 66.7% and 66.7%, respectively. 50% and 60%, if the synergetic effects among different strains of vermiculose arc were considered, the cleavage rate of the strains was 93.3%. Bdellovibrio HNAS was observed to infect E. coli in the form of mechanical drilling by negative staining method, and the optimum concentration ratio of Bdellovibrio to E. coli was determined by electron microscope, and the optimal concentration ratio of Bdellovibrio to E. coli was determined by electron microscope. The electron microscope was used to observe the ultrastructure of E. coli induced by Bdellovibrio vermiculosus. It was further confirmed that the strain of Vibrio vibrio was lysed by mechanical drilling, and the attachment of Vibrio vermiculoides was observed at the same time. Infection, vermiculite and release stages.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378

【引證文獻(xiàn)】

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2 何偉杰;蛭弧菌HKM的生物學(xué)特性分析及其相關(guān)應(yīng)用研究[D];華南理工大學(xué);2011年

3 鮑星;蛭弧菌控制鮮切果蔬致病菌數(shù)量的研究[D];華南理工大學(xué);2012年

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本文編號(hào)：2437077