【作者】 王红；

【导师】 张晓华；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2010， 硕士

【摘要】 黄海冷水团位于黄海中部洼地的深层和底部,是一种独特的水文现象。黄海冷水团每年5月左右开始形成于黄海中部,7、8月份达到强盛期,11,12月份完全消失；其主要特征是显著低温(4.6～9.3℃),温差大,盐差小,温盐性质较为稳定。在黄海冷水团这一特殊的海洋生态环境中会形成特殊的海洋微生物群落。海洋微生物是海洋生态系统中的重要组成部分,在海洋生物地球化学循环中起重要作用,因此,研究海洋微生物群落组成有助于更深入掌握海洋微生物分布特征及其在整个海洋生态系统中的作用。本论文对黄海冷水团可培养细菌多样性及群落组成进行了研究,这对于了解冷水团的形成对微食物环乃至整个近海生态系统产生的影响有重要意义。2008年7月21日至8月2日随中国科学院海洋研究所科学3号考察船出海采集海水样品,分别在3200-3和3850-5两个非冷水团(冷水团外部)站点,以及3500-5,3600-6,3600-8,3400-6和3800-2五个冷水团站点,取0米,10米,30米深度的海水,以原样,1/10,1/100三个稀释度进行涂布2216E平板。从涂布的2216E平板以菌落计数法计算出海水中异养细菌浓度为0.56×103～2.2×103 cfu/ml,从测得的AODC (Acridine Orange Direct Count), DVC (Direct Viable Count)数据计算得出海水中的总菌数和活菌数分别为1.3×105～4.8×105个/ml和6.0×104～1.6×105个/ml。从2216E平板上共分离到475株细菌并保种,选取163株细菌进行16S rDNA扩增,并用限制性内切酶HhaⅠ对PCR产物进行ARDRA (Amplified Ribosomal DNA Restriction Analysis)多态性分析,选取114株不同带型的细菌进行16SrDNA测序。结果显示,冷水团区域细菌归为4个细菌类群：变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)。其中变形菌门包括α-,β-,γ-变形菌纲。冷水团外部分离到的细菌也归为：变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes),但没有分离到β-变形菌纲。γ-变形菌纲在冷水团外部和内部的不同深度都是优势菌群,不同的是冷水团区域α-变形菌纲比例较高(26.8%),而非冷水团区域α-变形菌纲比例相对较低(15.6%),而且两个区域γ-变形菌纲包括的细菌属也不同。这表明黄海冷水团和非冷水团区域细菌多样性都很丰富,但其群落组成和优势菌群有所不同。在对冷水团区域细菌多样性研究中,基于16S rDNA序列分析,发现3200-3(123°0.153′E,31°59.968′N)站点10米深分离到的WH134T菌株为潜在的海洋细菌新种,进一步通过形态学鉴定方法(如革兰氏染色、鞭毛染色、电镜观察等)、生理生化特征(如温度、盐度、pH、淀粉酶、药敏等试验)、GC含量测定、脂肪酸含量测定以及API 20E、API 20NE、API 50CH、API ZYM和BIOLOG快速鉴定系统等对该菌株进行了分类鉴定。经鉴定,WH134T为革兰氏染色阴性,弧形,极生单鞭毛,运动,无泳动性和发光性,兼性厌氧,G+C含量为42.5 mol%,在弧菌属的40～50%范围内；16S rDNA序列比对结果显示,其与V. rumoiensis DSM 19141T亲缘关系最近,相似度为96.6%；通过选取7个看家基因进行多位点序列分析(MLSA),其gyrB、topA、recA、ftsZ、mreB、gapA和rpoA基因序列与V. rumoiensis DSM 19141T的相似度分别为75.7,74.6,83.6,78.9,82.9,86.0和89.4%；因此从分子生物学和生理生化角度可以确定其进化地位为一株弧菌新种,定名为冷水团弧菌(Vibrio marisflavi)。WH134T在三个菌种保藏中心的保藏号为CGMCC 1.8994, LMG 25284T和DSM 23086T。此外,以同样的方法对分离自浒苔灾害发生海域的菌株ZXM137T的分类地位进行了分析,该菌株革兰氏染色为阴性、菌体呈杆状、无鞭毛、用2216E液体培养基培养超过24h菌体自动聚集、严格需氧、主要的脂肪酸是C18:1ω7c、G+C含量为60.1mol%、较耐盐、能在11%(w/v) NaCl含量下生长；16S rDNA序列比对结果显示,该菌株与Jannaschia pohangensis同源性最高,相似度为95.3%。通过生理生化和分子生物学特征比较,可以确定该菌为α-变形菌纲红细菌目红细菌科新属新种,定名为嗜盐怀恕菌(Huaishuia halophilus)。ZXM137T在两个不同国家的菌种保藏中心的保藏号为CGMCC 1.8891T和LMG 24854T。更多还原

【Abstract】 The Yellow Sea Cold Water Mass (YSCWM) is an important phenomenon in the shelf sea of China. It is prominent in summer and autumn, and renewing in winter every year. The study of YSCWM in the past focused on physical oceanography but rarely on microbiology. Microorganisms play a major role in the biogeochemical cycling. However, phylogenetic diversity of cultivable bacteria in this area has not been reported yet.Phylogenetic diversity of cultivable bacteria from the inter of YSCWM area was studied in July 2008, and was compared with that from the exterior of YSCWM area. Sea water sample was collected in five stations 3500-5,3600-6, 3600-8,3400-6 and 3800-2 from the inter of YSCWM, and two stations 3200-3 and 3850-5 from the exterior of YSCWM. From the colony count method with 2216E plates, the abundance of the heterotrophic bacteria of sea water was 0.56×103～2.2×103cfu/ml; according to the AODC and DVC results, the total bacterial number and the total number of viable cells of the marine water samples were 1.3×105～4.8×105/ml and 6.0×104～1.6×105/ml, respectively.475 bacterial strains were isolated from 2216E plates, and the PCR-ARDRA technique was performed on these strains for cluster analysis, with fragments of 16S rDNA digested by restriction enzyme HhaⅠ. Samples from the inter of YSCWM stations showed as many RFLP patterns as those from exterior of YSCWM. The 16S rDNA of all major RFLP patterns were sequenced. Phylogenetic analysis of 114 bacterial strains showed that bacterial phylotypes from the inter of YSCWM were made up of four phyla bacteria, i.e. Proteobacteria, Actinobacteria, Firmicutes and Flavobacteria, including 24 genera and from the exterior were made up of the same four taxon, including 15 genera.α-,β- andγ- Proteobacteria subdivisions of the Proteobacteria were discovered from the inter of YSCWM. Howeverβ-Proteobacteria was not discovered from the exterior of YSCWM.γ-Proteobacteria represented the vast majority of bacterial isolates at every depth. By comparing,α-Proteobacteria represented the higher proportion with percentage of 26.8% inter of YSCWM than the proportion of exterior of YSCWM with percentage of 15.6%. It indicated that samples from the inter of YSCWM and the exterior of YSCWM showed the same high bacterial diversity, but bacterial community composition, as well as the predominant bacterial groups from the inter of YSCWM was different from the exterior of YSCWM.This study also aimed to identify two novel marine bacteria discovered in YSCWM and in Qingdao offshore during the massive green algae bloom. After sequencing the 16S rDNA, the sequences was blasted in Genbank database. Two strains WH134T and ZXM137 T were found to have low 16S rDNA sequences similarity to V. rumoiensis DSM 19141T and Jannaschia pohangensis with 96.6% and 95.3%. Further identification had been made by using classical morphologic identification methods including Gram-staining, flagella-staining, TEM, tranditional physiological and biochemical methods (such as temperature, pH and salty for growth, drug sensitivity, amylase activity, etc.), GC content, polar lipid composition, and the rapid identification systems, i.e. API 20E, API 20NE, API 50CH, API ZYM and BIOLOG. WH134Twas Gram-negative, cells of strain WH134T were slightly curved rods that were motile by means of a polar flagellum, facultatively anaerobic bacterial strain, negative for luminescence and swarming. The G+C content was 42.5 mol%, within the range of Vibrio 40～50%. Phylogenetic analysis based on gyrB, topA, recA, ftsZ, mreB, gap A and rpoA gene sequences revealed that strain WH134T belongs to the genus Vibrio, and showed similarities of 75.7,74.6,83.6,78.9,82.9,86.0 and 89.4% with the most closely relative Vibrio rumoiensis DSM 19141T. On the basis of phenotypic, phylogenetic and genetic evidence, strain WH134T should be classified as a novel Vibrio species, for which the name Vibrio marisflavi sp. nov. is proposed, with the type strain WH134T(=CGMCC 1.8994T=LMG 25284T=DSM 23086T).ZXM137T was isolated from seawater collected from a coastal region of Qingdao, P.R. China, when the massive green algae (Enteromorpha prolifera) bloomed. According to the same methods for identification, strain ZXM137T is Gram-negative, non-motile bacterium, strictly aerobic. The cells aggregated together when incubated at 28℃for more than 24h in marine 2216E broth. Growth occurred with 11%(w/v) NaCl. The major cellular fatty acid of strain ZXM137T was C18:1ω7c The G+C content of DNA was 60.1 mol%. It contained Q-10 as the predominant ubiquinone and the major polar lipids were phosphatidylglycerol, unidentified aminolipid and lipid. A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ZXM137T formed a distinct evolutionary lineage within the family Rhodobacteraceae of the phylum a-Proteobacteria. On the basis of phenotypic, chemotaxonomic and phylogenetic evidences, strain ZXM137T was considered to represent a novel genus and species, for which the name Huaishuia halophilus gen. nov. sp. nov. was proposed. The type strain was ZXM137T(=CGMCC 1.8891T= LMG 24854 T).更多还原