【作者】 陈双喜；

【导师】 邵宗泽；

【作者基本信息】 国家海洋局第三海洋研究所 ， 海洋生物学， 2008， 硕士

【摘要】 自然界中的微生物在砷的生物地球化学循环中起着重要作用。海洋砷的主要来源是岩浆活动,深海热液口活动是砷的一个重要来源。微生物通过累积、甲基化、氧化、还原等作用参与砷价态改变和元素循环,但是目前关于海洋环境的砷抗性菌知之甚少,尤其是深海环境。为了解深海环境中的微生物主要的砷抗性细菌,本论文对印度洋、大西洋中脊深海沉积物砷抗性菌的多样性进行研究。本文用含有2 mmol/L NaAsO2的PTA培养基富集来自印度洋和大西洋七个不同站点的深海沉积物样品,得到七个砷抗性菌群。我们对这些菌群的砷抗性菌进行了筛选,同时构建16SrDNA文库与DGGE相结合的方法对其中三个菌群(站点IR-TVG1、IRTVG2、IR-TVG3)的结构进行了解析;分析了不同培养时间和不同砷浓度下IR-TVG2、IR-TVG3站点的抗性菌群结构的变化。从七个砷抗性菌群中共分离到102株单菌,分属于28个属50个种,主要为变形菌纲α和γ亚群与Actinobacteria(放线菌门,高G+C革兰氏阳性菌)类群。微杆菌、食烷菌、海杆菌、Idiomarina、盐单胞菌和假交替单胞菌占优势,其中1株菌可能属于新属新种,15株菌可能属于新种。通过构建16SrDNA文库与DGGE结合的方法,确定站点IR-TVG1抗性菌群中的优势抗性菌为假交替单胞菌;站点IR-TVG2抗性菌群中的优势抗性菌为食烷菌与微杆菌;站点IR-TVG3抗性菌群中的优势抗性菌为微杆菌。从IR-TVG2、IR-TVG3分离到的微杆菌虽然16S rRNA序列完全一样,但它们的重复PCR指纹图谱和砷抗性能力不一样。为了了解取样时间和砷浓度对菌群结构的影响,我们选取IR-TVG2、IR-TVG3站点的抗性菌群做进一步的DGGE分析。结果显示,不同取样时间和不同砷浓度下两站点的抗性菌群结构变化明显。在2mM富集培养条件下,IR-TVG2在培养的初期只有一优势条带,但28h后出现另一条优势条带;但在20mM富集培养条件下没有随时间发生变化。IR-TVG3在两种浓度的富集培养条件下,菌群结构一直没有变化。在0-60mM的砷浓度下,IR-TVG2样品的优势条带随着砷浓度的提高而变少,而IR-TVG3样品,在低浓度砷富集培养条件下仅一条优势条带,但随着砷浓度的提高而出现了第2条优势条带。对筛选到的102株单菌进行了砷抗性范围测定,可在10mM NaAsO2存在下正常生长的菌株仅占总菌数的16.6%,这些高抗性的单菌主要筛选自IR-TVG2和IR-TVG3。这可能是这两站点混合菌群比其他菌群抗性高的原因。这些具有高抗性的菌株主要属于Microbacterium属,占52.6%;但实验结果表明,菌株抗性与种属没有必然联系,同属甚至同种的细菌有时表现出差异极大的抗性能力。此外不同的培养基培养条件,相同的菌株也会表现出差异的抗性能力。利用兼并引物扩增砷流出泵基因(arsB与ACR3),共获得17个目的大小的基因片段,测序比对结果显示,这些基因与已报道的砷流出泵基因相似度在75%左右。实验结果表明,单菌是否具有砷流出泵与其种属和抗性能力没有必然联系。在获得的优势菌中,均未扩的砷流出泵基因,说明它们存在其他的砷抗性机制。对从热液口虾的头部富集分离到的单菌D8-2(Brachybacterium paraconglomeratum,100%)进行初步的研究,结果显示该菌具有良好的砷抗性能力,但不具有砷氧化能力,D8-2对砷具有很好的胞内累积作用,累积量达到2313.7μg As g-1细胞干重,为目前的细菌砷累积量之最高。对来自站点IR-TVG1的单菌CK-I1-6(Pseudomonas alcalophila,99.9%)进行初步的研究,扩增得到一750bp大小的基因片段,该基因与Pseudomonas sp. A07的砷流出泵ACR3基因相似性76%。该菌有良好砷抗性,但不具有砷氧化能力,其抗性机制可能与砷流出泵相关。更多还原

【Abstract】 More evidences suggest that microbes participate in the geochemical circulation of arsenic, and play an important role in the biocirculation. However,little is know about the diversity of arsenite-resistant bacteria in marine environment, especially deep sea. Aims of this study are to isolate such bacteria and detect the diversity of these bacterial species from deep sea sediments.Seven deep sea sediment samples were collected from the Indian and Atlantic oceans, with a depth range from 1420m to 4019m. Seven arsenite-resistant bacterial communities were obtained by enrichment in PTA medium with 2 mmol/L NaAsO2 . Arsenite-resistant bacteria were isolated from the communities. Structure of bacterial communities of three samples (IR-TVG1, IR-TVG2, IR-TVG3) was analyzed by 16S rDNA library analyses and DGGE of V3 region of 16S rDNA. In arsenite-resistant bacterial community of IR-TVG2 and IR-TVG3, the dynamic changes in response to different incubation times and increasing arsenite concentration were examined further.Total of 102 strains were isolated from the communities, they were tentatively identified to be 50 species of 28 genera. Most of them belong toα-Proteobacteria,γ-Proteobacteria and Actinobacteria. The predominant genera were Microbacterium, Alcanivorax, Marinobacter, Idiomarina, Halomona and Pseudoalteromonas. One isolate possibly belonged to a novel species of novel genus, while fifty isolates possibly belonged to novel species according to the 16S rDNA sequence.DGGE and 16S rDNA library analyses results showed that the predominant bacteria in IR-TVG1 community was Pseudoalteromonas, corresponding isolate was AS-I1-3; The predominant bacteria in IR-TVG2 community were Alcanivorax and Microbacterium, corresponding isolates were CK-I2-8 and AS-I2-1; The predominant bacterium in IR-TVG3 community was Microbacterium, corresponding to isolate AS-I3-5. The two isolates, AS-I2-1 and AS-I3-5, had the same 16S rDNA sequence, but varied in BOX-PCR and arsenite tolerance. In addition, community structure of IR-TVG2 and IR-TVG3 changed dynamically in response to incubation periods and arsenite concentration, and varied from each other.Among all the 102 isolates, only 16.6% can grow in present of 10 mmol/L NaAsO2, which were mainly isolated from IR-TVG2 and IR-TVG3, and the most key bacteria are Microbacterium, occupying 52.6%.With degenerate primers, genes encoding arsenite efflux pump (ars B and ACR3) were PCR detected, showing the expected size of 750bp. Of the 102 isolates, 17 showed successful amplifications with either one or two of the specific primer sets. These fragments showed ~75% similarities to the reported arsenite transporters.Strain D8-2(Brachybacterium paraconglomeratum,100%), isolated from a deep sea shrimp, and strain CK-I1-6(Pseudomonas alcalophila,99.9%),isolated from sediment of IR-TVG1, were further studied. Both two showed high resistance but both could not oxidize arsenite to arsenate. D8-2 showed high ability of arsenite absorption, after 48h incubation arsenic accumulation in cells amounted to 2313.7μgAsg-1 (dry weight). A gene of 76% similarity to the arsenite transporter of ACR3 of Pseudomonas sp. A07 was amplified from CK-I1-6.更多还原