【作者】 常显波；

【导师】 张晓华；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2011， 博士

【摘要】 本研究首先比较了不同方法的分离效果,以此为基础,通过纯培养和16S rDNA系统发育分析对黄海冷水团、北极海区及青岛盐池的沉积物样品进行放线菌多样性研究,并对分离菌株进行抗菌活性及胞外酶活性筛选,取得以下结果:1.海洋放线菌不同分离方法的比较研究:以青岛海区潮间带沉积物为研究对象,分析了不同样品预处理方式、稀释液种类、海水浓度、培养基种类等因素对放线菌分离效果的影响。结果表明:以55℃预处理样品6 min、纯海水配制培养基、1/4林格氏溶液稀释样品、在培养基M1、M6、M7和M8上可以分离到更多的放线菌。2.黄海冷水团沉积物中海洋放线菌的多样性研究:利用4种分离效果较好的培养基从黄海冷水团沉积物中共分离到172株放线菌;选取51株代表菌株进行16S rDNA测序分析,发现这些放线菌分别属于链霉菌属、拟诺卡氏菌属、小单孢菌属、异壁放线菌属和一个新属;在172株放线菌中,有58株对病原菌有抑菌活性,有94株可同时产生三种胞外酶活性。3.北极海洋沉积物中放线菌的分离培养与活性筛选:利用6种培养基从北极海洋沉积物中共分离出109株放线菌,选取34株代表菌株进行16S rDNA测序分析,结果表明它们分别属于链霉菌属、假诺卡氏菌属和拟诺卡氏菌属;在109株放线菌中,有39株对病原菌有抑菌活性。4.青岛泊子盐场放线菌分离培养:利用3种培养基从青岛即墨市田横镇泊子盐场盐池底泥样品中共分离纯化出15株放线菌。对分离菌株进行16S rDNA测序分析,发现这15株放线菌分别属于链霉菌属、拟诺卡氏菌属和一个新属。在15株放线菌中,分别有12株、3株、2株和1株放线菌产淀粉酶、产酯酶、蛋白酶和纤维素酶。5.耐碱刺孢放线菌新属新种的分类鉴定:菌株CXB654~T基内菌丝有分支,不断裂,气生菌丝分化成长孢子链,孢子呈椭圆形,孢子表面有刺突,生长需要NaCl,核糖和葡萄糖是细胞壁特征糖。16S rDNA序列比对结果显示,菌株CXB654~T与亲缘关系最近的标准菌株的的同源性分别为96.6%、96.5%和96.1%,并在N-J和M-L系统进化树上形成独立分支。综合该菌在生理生化特征、16S rDNA同源性和化学指标等方面的实验结果,可以确定CXB654~T菌株是拟诺卡氏菌科中的一个新属,命名为耐碱刺孢放线菌属。6.青岛盐生放线菌新属新种的分类鉴定:菌株CXB832~T基内菌丝由黄白色到深黄色,有分支,不断裂,丰富的白色气生菌丝分化成长孢子链,孢子呈杆状,孢子表面光滑,最适盐度范围为9～12% (w/v) NaCl,葡萄糖和木糖是细胞壁特征糖。16S rDNA序列比对结果显示,菌株CXB832~T与亲缘关系最近的标准菌株的同源性分别为95.4%和94.9%,并在N-J和M-L系统进化树上形成独立分支。综合该菌在生理生化特征、16S rDNA同源性和化学指标等方面的实验结果,可以确定菌株CXB832~T是拟诺卡氏菌科中的一个新属,命名为青岛盐生放线菌属。更多还原

【Abstract】 The diversity of marine actinomycetes isolated from different environments was studied based on comparative study of different isolation methods by pure cultures and 16S rDNA sequence-based phylogenetic analysis, and the strains were screened for antibacterial activity and enzymatic activity. The main results are as follow:1. Study of different isolation methods of marine actinomycetes: The impacts of different sample preparation methods, type of diluents, seawater concentration and culture medium on the isolation of actinomycetes from the inter-tidal mud at Qingdao were investigated. The results showed that samples pretreated with 55°C for 6 minutes, grow on the culture media made of pure seawater and diluted with 1/4 Ringer’s solution can improve the isolation of actinomycetes. The medium M1、M6、M7 and M8 are more effective when compared to others.2. Study of the diversity of marine actinomycetes from sediments of the Yellow Sea Cold Water Mass: In this study, 172 actinomycetes were isolated by using four culture media which are more effective on separation of actinomycetes. Bioinformatics analysis of the 16S rDNA sequences from the 51 representative isolates can be assigned to five actinobacteria genera (Streptomyces Nocardiopsis, Actinoalloteichus, Micromonospora and a novel genus). Out of 172 isolates, 58 isolates exhibited antimicrobial activity against human pathogens, and 94 isolates can produce three kinds of enzymes simultaneously.3. Isolation and screening of actinomycetes from Arctic marine sediments: In this study, 109 actinomycetes were isolated using six different culture media. Bioinformatics analysis of the 16S rDNA sequences from the 34 representative isolates can be assigned to three genera (Streptomyces, Pseudonocardia and HNocardiopsis). Out of 109 isolates, 39 isolates exhibited antimicrobial activity against human pathogens.4. Isolation and culture of actinomycetes from a salt pond, Qingdao: In this study, 15 actinomycete were isolated using three different media. Bioinformatics analysis of the 16S rDNA sequences from the isolates can be assigned to three genera (Streptomyces, Nocardiopsis and a novel genus). Out of 15 isolates, 12, 3, 2 and 1 isolates produces amylase, lipase, caseinase and cellulose accordingly.5. Taxonomic analysis of Spinactinospora alkalitolerans gen. nov. sp. nov.: The strain CXB6542~T formed branched substrate mycelium without fragmentation. Abundant aerial mycelium differentiated into long or short chains of spores, and the spores were elliptic and cylindrical with spinous surface. Strain CXB6542~T required NaCl for growth, and contained ribose and glucose as the major whole-cell components. Comparative analysis of 16S rDNA sequence showed that the novel strain was most closely related to genera within the family Nocardiopsaceae, but formed a separate lineage. The strain CXB6542~T had only 96.6%, 96.5% and 96.1% similarity to the standard strains with the closest relationship respectively. On the basis of phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain CXB6542~T was considered to represent a new genus and species in the family Nocardiopsaceae, and the name Spinactinospora alkalitolerans gen. nov. sp. nov. is proposed.6. Taxonomic analysis of Salinactinospora qingdaonensis gen. nov. sp. nov.: The strain CXB832~T formed pale-yellow to deep yellow and branched substrate mycelium without fragmentation. Abundant white aerial mycelium differentiated into long chains of spores, and the spores were rod-shaped with smooth surfaces. Optimal growth occurred at 9～12% (w/v) NaCl. Strain CXB832~T contained glucose and xylose as the major whole-cell components. BLAST results for the 16S rDNA sequence of strain CXB832~T showed that its closest relatives were members of genera Nocardiopsis and Haloactinospora in the family Nocardiopsaceae, with the highest gene sequence similarity of 95.4% and 94.9% with the standard strains respectively. The 16S rDNA based neighbour-joining and maximum-likelihood trees showed the phylogenetic relationship between strain CXB832~T and the closely related genera, and that strain CXB832~T formed a separate lineage. On the basis of phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain CXB832~T was considered to represent a new genus and species in the family Nocardiopsaceae, and the name Salinactinospora qingdaonensis gen. nov. sp. nov. is proposed.更多还原