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| 西沙鹿角珊瑚共附生细菌多样性及其与珊瑚白化关系的研究 | |
| 其他题名 | Studying of the bacterial composition associated with Acropora solitaryensis and the relation to the coral bleaching |
| 刘助红 | |
| 出版年 | 2012 |
| 学位类型 | 硕士 |
| 导师 | 陈偿 ; 胡超群 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 西沙群岛是我国南海诸岛四大群岛之一，位于我国南海的核心海域，是我国南海代表性的珊瑚礁群岛和渔民赖以生存的渔业捕捞海域。西沙群岛珊瑚礁生态系统作为我国代表性的珊瑚礁生态系统，珊瑚礁系统最为完整，其造礁石珊瑚种类约占我国石珊瑚种类的四分之三，地位十分重要。近年来，由于各种原因，西沙群岛珊瑚出现大面积白化和死亡现象，珊瑚礁生态系统也正在朝着荒漠化方向发展。针对这种情况，本研究用培养和免培养技术分析了西沙鹿角珊瑚白化过程共附生微生物种群动态，发现了鹿角珊瑚白化与微生物种群变化的紧密联系，确定了感染鹿角珊瑚并导致白化的传染性病原菌，具体研究结果如下：\n1）优化了珊瑚共附生微生物16SrDNA 变性凝胶梯度电泳分析（denaturing gradient gel electrophoresis，DGGE）技术体系，发现用CTAB/SDS的方法提取珊瑚总细菌DNA的效果最佳，选择16SrDNA V3区域引物扩增的片段结合35%到50%的变性剂梯度能获得较高分辨率的DGGE图谱。\n2）用DGGE方法比较了西沙不同生理状态的鹿角珊瑚共附生细菌的多样性。DGGE分析发现细菌种群多样性随着珊瑚白化程度的变化而改变，相对正常珊瑚，在部分白化的珊瑚的白化部位，细菌种群多样性显著增多，但在完全白化的样品中，条带数量急剧下降，且明显少于正常珊瑚所能检测到的条带，完全白化的鹿角珊瑚中只有最优势的菌群被检测到。将主要条带切胶并测序鉴定，共获得17个序列；最主要的条带都是根瘤菌目的细菌，分别为Ochrobactrum sp.，Ensifer sp.和Methylobacterium sp.；随着珊瑚白化程度的加强，弧菌和其他潜在病原的序列大量增加，其中Vibrio coralliilyticus，Aquabacterium sp., Shewanella algae等机会致病菌在白化珊瑚样品中出现或密度大大增加。\n 3）分别构建了健康珊瑚和白化珊瑚的16SrDNA克隆文库, 从每个文库中挑取约500个克隆进行AFLP分析，双酶切结果显示，正常鹿角珊瑚的细菌种群多样性十分丰富，共获得76个亚型，测序鉴定了71个亚型。而白化珊瑚样品的种群大大降低，共鉴定到48个亚型，成功测定了其中41个亚型。序列比对结果显示健康珊瑚中的细菌包括α-proteobacteria，β-proteobacteria，γ-proteobacteria，Firmicutes，Fusobacteria，Epsilonproteobacteria等6个门（纲），在白化的鹿角珊瑚中，只鉴定出4个门（纲）的细菌，Fusobacteria和Epsilonproteobacteria没有检出，而且每个门（纲）所包含的细菌多样性也大大降低，与健康珊瑚相比，具有固氮作用的Methylobacterium、Microvirga等种类急剧减少，而以弧菌为代表的菌群成为了优势菌群。\n4）分别用4种培养基从健康和白化鹿角珊瑚中分离了大量菌株，并对其种类进行16SrDNA分子鉴定。结果发现，两种样品中分离的细菌多样性存在很大的差异。在健康的鹿角珊瑚中，分离到34株不同的细菌，分属于8个不同的属，其中弧菌占据了很大的比例（68%），其它细菌包括Rhodobacter，Staphylococcus，Alteromonadaceae，Nautell，Thalassobius，Photobacterium等；从白化珊瑚中只分离到41株细菌，但只有5个不同的属，包括Vibrio，Pseudoalteromonas，Bacillus，Exiguobacterium等，其中弧菌占80%。然而两种珊瑚样品中弧菌种类也有差异，例如Vibrio fortis种只在白化珊瑚中分离到，Vibrio azureus却只在健康的鹿角珊瑚中分离到。\n5）从白化珊瑚中分离的疑似病原细菌进行了人工感染和菌株鉴定，结果显示其中一株细菌对鹿角珊瑚有很强致病性，108CFU/ml浸泡六小时后，可在一周左右导致鹿角珊瑚发生白化现象，大概9天后，珊瑚彻底白化而死亡；将被感染的珊瑚与健康珊瑚共培养一周左右也导致了白化和死亡现象，显示该细菌能够引起珊瑚白化并具有传染性。16SrDNA测序和BioLog生化鉴定结果显示该病原细菌为弧菌属细菌，其16SrDNA序列与裂珊瑚菌（Vibrio corallilyticus）存在较高相似性（98%），而Biolog生化分析则显示与哈氏弧菌（V. harveyi）有较高同源性（97%），显示该细菌可能是一种新的珊瑚病原菌。\n根据这些结果我们可以得到如下结论：第一，共附生微生物在珊瑚共生体系中起着重要作用，固氮细菌作为优势种群可能是珊瑚氮源的重要来源，是维持珊瑚礁的健康状态的重要因素；第二，珊瑚白化过程中，微生物种群也是动态变化的。随着白化加重，微生物种群多样性开始加强，后逐步减弱，完全白化珊瑚中绝大部分菌群消失，与此同时，弧菌等潜在病原菌种类和密度大大加强，显示珊瑚礁白化与病原菌存在密切联系；第三，感染实验证实珊瑚白化可以由病原弧菌引起，而且具有传染性。据此我们推测认为，多样性的共附生微生物是珊瑚共附生体系健康发育和成长不可或缺的部分，当珊瑚礁处于内外因子胁迫环境时，珊瑚虫和虫黄藻共生体系平衡被打破，并导致共附生微生物种群发生变化，机会致病性微生物大量繁殖并最终导致整个系统的崩溃，从而产生白化和死亡。 |
| 英文摘要 | Xisha islands, one of the biggest islands in South China Sea, are very important for our country and they are the representive coral reefs and fishing area for the fishers. As the representive coral reef system, it is the best in China, and the stony coral there accounts up to three out four in our country. Now, the coral reef system is also degrading year by year. Given that, this study analyzed the bacterial population associated with Acropora solitaryensis in the course of coral bleaching, and found that they were linked closely, finally a pathogen that could infect Acropora solitaryensis and lead it to bleaching was comfired. The detail results as follows:\n1) Optimization of DGGE (denaturing gradient gel electrophoresis) system, we found that the CTAB / SDS method for the extraction of total bacterial DNA were best, selection of 16S rDNA V3 regional primers binding 35% to 50% of the denaturant gradient obtained better resolution of DGGE pattern.\n2) Compared the diversity of bacteria associated with the Acropora solitaryensis by the molecular ecology methods, including DGGE and clone library method. From the DGGE pattern, we found that the diversity of bacteria associated with Acropora solitaryensis was changed with the extent of coral bleaching. Compared with the healthy coral, the bacteria was much more in the diseased tissue, but in the whole bleaching coral, the number of bands declined sharply and only the prominent band were detected. Seventeen sequences was cut and sequenced, the main strips were rhizobia to bacteria, which were Ochrobactrum sp., Ensifer sp. and Methylobacterium sp.; with the coral bleaching degree strengthened, Vibrio and other potential pathogens sequence increases in great quantities, including Vibrio coralliilyticus, Aquabacterium sp., Shewanella algae and other opportunistic pathogens.\n3) Constructing 16SrDNA clone libraries for the healthy and diseased coral separately. For each library, approximately 500 clones were selected for AFLP analysis, the result showed that diversity of bacterial populations in staghorn coral were very rich, a total reception of 76 subtypes and identification of the 71 subtypes, while albino coral were gained 48 subtypes and 41 subtypes were successfully identified. The result of clone library method showed that the bacteria associated with Acropora solitaryensis belonged to many phyla. including α-proteobacteria，β-proteobacteria，γ-proteobacteria，Firmicutes，Fusobacteria，Epsilonproteobacteria，unclassified bacreria etc. In contrast, the diversity of bacteria was not so rich and the two phyla, Fusobacteria and Epsilonproteobacteria, were not identified. Even in each phylum, the species were less than the healthy coral. Compared with healthy coral, such bacteria with nitrogen fixation as Methylobacterium, Microvirga drastically reduced, while Vibrio accounted for high proportion.\n4) Many heterotrophic bacteria were isolated form the coral with the plates 2216E and M1. Meantime described the morphic characterization and identified the species. The result showed that the bacteria cultured from the coral were different between the healthy and the diseased. Form the healthy coral, we isolated 34 strains belonged to eight phyla, and the Vibrio accounted much(68%), but Rhodobacter capsulatus，Staphylococcus epidermidis，Alteromonadaceae，Nautella italica，Thalassobius mediterraneus，Photobacterium ganghwense etc were also isolated. However, only five phyla were found, and they were Vibrios which were also the main genus(80%)，Pseudoalteromonas，Bacillus cereus，Exiguobacterium. Though the Vibrio was both found much whatever in the healthy or the diseased, the species of Vibrio were some different, for example Vibrio fortis were found in the diseased and Vibrio azureus in the healthy. In addition, if 98% is the definite of the new species, many were isolated from the healthy and the bleached corals. \n5) After the biochemistry identification and the artification infection of coral with the probable pathogen isolated form the bleached Acropora solitaryensis, the results showed that the bacteria had some similarity with Vibrio coralliilyticus and Vibrio harveyi, and it leaded the coral to bleaching and the sea water to unclear after about a week. The Acropora solitaryensis died when nine days passed. Then cultured the healthy coral with the albino one in the same tank, the healthy also died, which suggested that the coral bleaching caused by the pathogen was infectious.\nTherefore, according to the data, the following conclusions obtained: First, symbiotic microorganisms in coral symbiotic system played an important role, nitrogen-fixing bacteria, as the dominant populations, may be an important source of nitrogen source for coral, were important factors for the maintenance of coral reef’s healthy status. Second, in the coral bleaching process, the change of microbial populations was dynamic, with the aggravation of coral bleaching, diversity of bacteria began to strengthen, then gradually weakened. In the fully bleached coral, the vast majority of flora disappeared, at the same time, the number of potential pathogenic such as Vibrio species greatly enhanced, which suggested the coral bleaching had the close relation to pathogenic bacteria. Finally, infection experiments confirmed that coral bleaching was caused by pathogenic Vibrio, and had contagious. We speculated that diversity of symbiotic and epiphytic microorganisms was the indispensable part for coral’s healthy growth and development. When the coral reefs were in the stress environment of internal and external factors, the balance between the coral and Symbiodinium was broken, and the symbiotic microbial population changed, then the opportunity pathogenic bacteria may grow quickly and eventually leaded to the collapse of the whole system, resulting in albino and death of coral. |
| 中文关键词 | 鹿角珊瑚 ; 细菌多样性 ; DGGE ; 珊瑚白化 |
| 英文关键词 | Acropora solitaryensis，diversity of bacteria， DGGE ，coral bleaching |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 海洋生物学 |
| 来源机构 | 中国科学院南海海洋研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287111 |
| 推荐引用方式 GB/T 7714 | 刘助红. 西沙鹿角珊瑚共附生细菌多样性及其与珊瑚白化关系的研究[D]. 中国科学院大学,2012. |
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