【作者】 王淑芳；

【导师】 耿美玉； 肖湘；

【作者基本信息】 中国海洋大学 ， 药物化学， 2008， 博士

【摘要】 热液口生态系统自从1977年发现以来,被誉为“生命绿洲”。热液口生态系统的发现使人们对“生物圈”的认识有了进一步的了解。热液喷发物含有大量的化学物质和能量,由此孵育了耐高温、高压和分解有毒气体并且不依靠太阳能生活的大量生物。这个生态系统的环境特征被认为与地球生命形成早期的环境特征相似,因此研究热液生命系统中的嗜压、嗜热等极端微生物无疑具有重大的意义。古菌的氨氧化作用是在2005年以来发展的新课题。一直以来,科学家认为β-和γ-类型的细菌在氨氧化作用中占主导地位,古菌并不具备氨氧化的功能。但在2005年科学家通过基因组的研究发现古菌16S rRNA与类似细菌amoA的基因并排排列。同年分离得到了以氨为唯一能量来源的中温泉古菌,使得古菌能够进行氨氧化这一理论得到证实。随之而来的问题是,在其他生态系统中,是否也存在古菌的氨氧化作用?其多样性如何?不同生态系统古菌与细菌的氨氧化作用如何?哪些类型的古菌具有氧化氨的功能?本论文以2005年Atlantic/Alvin科学考察船中美联合取样得到的硫化物和同年中国“大洋一号”科学考察船取得的热液区沉积物样品为实验材料,对3个硫化物和15个沉积物样品进行氨氧化古菌的多样性及定量分析,并对2个硫化物和墨西哥湾沉积柱的甲烷菌进行多样性调查。对硫化物氨氧化古菌的多样性分析表明热液口硫化物的氨氧化古菌多样性并不丰富,但是序列比较特异。温度较低的样品的多样性比较丰富,温度较高的样品的多样性较低。所有3个样品的克隆子分成两个类型,定义为Chimney Group I和Chimney Group II。两个类型间序列具有明显的区别,平均相似性为80%。其中Chimney Group I与日本海的沉积物有较高的同源性（94%）,而Chimney Group II与NCBI基因库的克隆子最高同源性也仅为89%。说明这个类型的克隆子序列比较新,有可能是存在于热液口硫化物中的特异氨氧化古菌。对硫化物的一个样品进行古菌16S rRNA的多样性分析表明氨氧化古菌并不属于MG1。16S rRNA来自嗜热泉古菌Desulfurooccales、Thermoproteales和Thermococcales。定量分析也表明氨氧化古菌amoA基因与古菌16S rRNA基因的拷贝数比例为6:94,这样的一个比例应该是比较容易的检测到,所以有可能这个样品中的氨氧化古菌与MG1并无关系。对热液口沉积物的古菌amoA基因及古菌16S rRNA的多样性分析表明,古菌amoA序列与深海来源的克隆子序列有比较高的同源性,表明热液口沉积物与深海沉积物的氨氧化古菌有相似起源。同样对16S rRNA多样性的分析表明,热液口沉积物的克隆子全部属于MG1,因此热液口沉积物的氨氧化古菌可能来自MG1这个广泛存在的类型。在热液口沉积物与西太平洋暖池区沉积物的比较中,两者有比较大区别,两者的OTUs类型完全独立,不互相包含。此外暖池区沉积物中部分克隆子来自Marine Bethic Group。说明热液口沉积物与其它沉积物的氨氧化古菌还是有比较大区别。通过Real-Time PCR定量分析所有沉积物样品（16个）中的古菌16S rRNA、AOA-amoA和AOB-amoA。结果表明古菌amoA基因在沉积物中的含量丰富,且与细菌amoA的含量相似或更高,表明在热液口沉积物中古菌氨氧化比细菌氨氧化的作用更重要。在热液口沉积物中的氨氧化古菌16S r RNA基因数量从4.598×10⁴拷贝/克（干重）～7.027×10⁷拷贝/克（干重）,古菌amoA基因数量从4.597×10⁴拷贝/克（干重）～6.701×10⁶拷贝/克（干重）,细菌amoA基因的数量从1.182×10⁴拷贝/克（干重）～2.302×10⁶拷贝/克（干重）,氨氧化古菌与氨氧化细菌的数量比值在0.75～170之间。相关性分析表明古菌16S rRAN与古菌amoA基因存在相关性（r=0.652,n=16,P﹤0.01）,古菌amoA拷贝数与有机物含量（包括有机氮、有机碳、有机质）、无机氮、总氮、及NH4+、NO3-、NO2-和硝化潜势、pH值有显著（P﹤0.01）或相关（P﹤0.05）关系,细菌amoA拷贝数与有机质含量、无机氮、总氮、及NH₄⁺、NO₃^-、NO₂^-和硝化潜势、pH值有显著相关（P﹤0.01）或相关（P﹤0.05）关系。古菌amoA基因数量与细菌amoA基因数量间也存在显著相关（P﹤0.01,12个位点）或相关（P﹤0.05,4个位点）关系。此外本论文利用甲烷菌辅酶M的编码基因mcrA对热液口硫化物及墨西哥湾沉积物的甲烷菌群进行多样性调查,结果发现在这两个甲烷含量丰富的地区甲烷菌类型完全不同。在热液喷发成因的热液口地区,甲烷产生菌占主导地位;在热成因墨西哥湾地区,甲烷氧化菌占主导地位。本论文回答了前文提出的氨氧化古菌在洋脊热液区生态系统中的生态多样性及生态作用问题,无疑对古菌氨氧化作用的研究更进了一步,也必将推动古菌氨氧化作用的更深入的研究。同时本文以热液口样品为研究对象,对揭示热液口生态系统起到了一定的作用。更多还原

【Abstract】 Hydrothermal vent was discovered in 1979, and its discovery improved our understanding about lives. Because the hydrothermal vent plume including kinds of chemicals and enormous energy, and a lot of life-form were brougnt up. Many of them are pizotrophic and hyperthermophilic and can decompound noxious gases. This mechianism is different with our understanding about lives on the world. Many scientists think the environments of hydrothermal vent are similar with that when the lives appeared first on the earth. The study towards the deep-sea microbes will be helpful to the understanding of the origin of life, the character of biodiversity and to the exploitation of microbial resources in deep sea. The metabolic and ecological study of life on hydrothermal vent environments will give some insights on evolution and adaptation mechanism of extremophiles.Ammonia-oxidizing archaea was dicovered in 2005. For a long time,β-andγ-proteobacteria were thought as the most important microbial, and studied as a amodel. But in 2005, scientists screened a fosmid clone contained a 16S rRNA gene of group 1.1b Crenarchaeota alongside genes encoding potential homologues of Bacterial AMO A and B subunits. The ultimate confirmation of AOA was achieved by cultivation of a mesophilic Crenarchaeota from a marine aquarium in Seattle, USA. This Crenarchaeota strain grows chemolithoautotrophically and uses ammonia as the sole energy source. It further raises the questions,for example, are there any AOA in other microbial system? What’s their diverisity and ubiquity? Which play more important role in ammonia oxidation, AOA or AOB? What’s kind of arhaea has the capability oxidizing annomina?Here, we studied 3 sulfide chimneies obtained by Atlantic/Alvin in the fall of 2005 from Juan de Fuca Ridge and 15 sediments from Middle Atlantic Ridge, East Pacific Ridge and India Ridge obtained by“Dayang number 1”in the same year. We reported here the diversity, ubiquity and quatitative of ammonia-oxidizing archaea in the 18 samples. 2 sulfide rocks and 1 sediment core were analyzed through mcrA gene survy.The results of diversity of AOA-amoA survey on sulfide from hydrothermal vent indicated the diversity and richness was not high, but some differential AOA-amoA sequences were derived. Low temperature sample had high richness and diversity, and high temperature has low richness and diversity. All the sequences from sulfide defined to 2 groups: Chinmney Group I and Chimney Group II. The 2 groups had observed differences and the average similarity between the 2 groups was 80%. Chimney Group I had the closest relationship with the sediment A-1 cluster from Janpan Sea （94%）. The Chimney Group II had low similarity with sequences deposited in NCBI （≦89%） which indicating this group was a new cluster, and maybe the differential ammonia oxidizing arhaea habited in hydrothermal vent system.In order to derive the possible origin of the new defined cluster, an archaeal 16S rRNA library was constructed. The analysis indicated only hyperthermophilic Crenarchaeota including Desulfococcas, Thermoproteas, were detected, but no Marine Group I existed. Assuming that one AOA cell contains only one copy of amoA genes, the AOA occupies at least 6% of the archaeal community. This large proportion of AOA should be easily detected by 16S rRNA library analysis. So,we think the AOAs don’t related to MG1.Another results of diversity of AOA-amoA survey on sediments from hydrothermal vent field indicated the diversity and richness was high and the sequences obtained had closest relationship with deep-sea sequences, which indicated the similar origin of hydrothermal vent sediments and deep-sea sediments. The survey on archaeal 16S rRNA indicated all the sequences derived from hydrothermal vent sediments fall in MG1 group, which suggested ammonia-oxidizing archaea may belong to MG1. Comparison the sediments from hydrothermal vent and the sediment from West Pacific, the great difference was sequences didn’t fall in the same OTUs and some sequences from West Pacific fall in Marine Bethic Group, which suggested there are diffrences between hydrothermal vent sediments and other kind of sediments.Fluorescence PCR used to quantitative the quantity of archaeal 16S rRNA, AOA-amoA and AOB-amoA gene. The results indicted a high value of AOA-amoA genes existed in the hydrothermal vent sediments and AOB-amoA genes were same or many times lower than AOA-amoA genes, which suggested AOA play important role in the environments of hydrothermal vent sediments. The quantity of archaeal 16S rRNA was between 4.598×10⁴copies/dry g～7.027×10⁷copies/dry g, AOA-amoA gene quantity was between 4.597×10⁴ copies/dry g～6.701×10⁶ copies/dry g , and AOB-amoA genes quantity was between 1.182×10⁴ copies/dry g～2.302×10⁶ copies/dry g. The ratios of AOA-amoA to AOB-amoA was between 0.75～170. Correlation analysis indicated there were significant correlation bween arhcaeal 16S rRNA and AOA-amoA gene （r=0.652,n=16,P﹤0.01）. Significant （P﹤0.01） or positive correlations（P﹤0.05）were found between archaeal amoA gene copies number and organic matters （organic carbon and organic nitrogen）, mineral nitrogen, total nitrogen, concentrations of NH₄⁺、NO₃^-、NO₂^-, PNR, pH and similar correlations were found between bacterial amoA gene copies number and chemical or PNR except no positive correlations were found between organic carbon and nitrogen. Significant correlation between archaeal amoA gene copies number and bacterial amoA gene copies number was found from 12 sites, and positive correlation found from the other 4 sites.Inadditional, we investigated the microbial related to methano by constructing mcrA gene clone libraries of sulfide and Mexico sediments. The results indicated methanogenic archaea were predominant in sulfide, which surrounded by high concentration of H2 and CO2, and methano-oxidizing archaea （MOA） were predominant in Mexico Gulf sediment.Our study gave some explanations of the questions about ammonia-oxidizing arhaea, which will improve the study on this metabolize, and hydrothermal vent chimneys and sediments were used in the thesis, which is helpful for the discovery of this microbial system.更多还原