【作者】 孟祥伟；

【导师】 谢丙炎；

【作者基本信息】 中国农业科学院 ， 生物安全， 2009， 硕士

【摘要】 古菌不仅存在于极端环境下,而且广泛分布于自然环境中,甚至作为优势菌群存在,在全球的生物地球化学过程中的作用不可忽视。细菌曾被认为是单独负责催化硝化过程的限速步骤,但是最近一些研究表明泉古菌同样具有氨氧化的能力,是土壤中居主导地位的氨氧化微生物。西藏米拉山的高寒草甸土壤,是青藏高原的典型土壤类型。在全球气候变暖的大背景下,青藏高原的气温正呈现较大幅度的变暖趋势,是全球气候变暖的敏感区,对全球气候变化的先兆性具有特殊的研究意义。为了分析西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况,本研究以西藏米拉山MP(位于米拉山口附近的公路旁,植被覆盖率小于50%)和GY(位于工布江达县,离村庄较远,植被覆盖率大于90%)两个地点土壤为样品,每个样品提取10份DNA,每份DNA进行3次PCR扩增古菌16S rRNA和amoA基因,分别以30次PCR扩增产物构建MP和GY两地古菌16S rRNA克隆文库(ARC-MP, ARC-GY)和amoA基因克隆文库(AOA-MP, AOA-GY)。并且为了检验本研究构建文库的方法是否具有重复性,采用同一土壤样品MP,使用相同的方法重复构建了古菌16S rRNA和amoA基因克隆文库(ARC-MPr和AOA-MPr)。使用DOTUR、∫-Libshuff、TreeClimber以及SONS等软件对西藏米拉山古菌16S rRNA基因克隆文库和古菌amoA基因克隆文库的多样性进行了分析。三个古菌16S rRNA基因克隆文库均包括嗜泉古菌界(Crenarchaeota)和未分类的古菌(unclassified_Archaea)两类,未发现广域古菌界(Euryarchaeota),并且获得的所有泉古菌均为热变形菌门(Thermoprotei)。三个古菌amoA基因克隆文库中氨氧化古菌主要为嗜泉古菌界。获得的古菌和氨氧化古菌与来自其它环境土壤中的序列具有很高的相似性,其中MP土壤中有一些古菌及氨氧化古菌序列与来自保护地土壤中的序列相似性很高,这一现象可能与MP土壤退化有关。使用DOTUR软件将古菌和氨氧化古菌序列按照相似性97%的标准分成若干个可操作分类单元(OTUs)。三个古菌16S rRNA基因克隆文库ARC-MP、ARC-MPr和ARC-GY分别包括54、51、和51个OTUs,表明MP与GY两地土壤古菌丰度基本相同。三个古菌amoA基因克隆文库AOA-MP、AOA-MPr和AOA-GY分别包括51、44和23个OTUs,表明MP土壤氨氧化古菌丰富度高于GY土壤,随着草甸的退化,氨氧化古菌类群逐渐扩大,作用逐渐加强。利用∫-Libshuff和TreeClimber两种软件对文库ARC-MP和ARC-GY进行比较分析,两种分析方法得到的P值均小于0.05(P<0.05),表明两文库在古菌群落组成上存在显著差异;对文库AOA-MP和AOA-GY进行比较分析,两种分析方法得到的P值均小于0.05(P<0.05),两文库存在显著差异。表明MP和GY两种不同类型土壤中古菌和氨氧化古菌的群落组成均存在显著差异;对文库ARC-MP和ARC-MPr进行比较分析,得到的P值均大于0.05(P>0.05),表明两文库间无显著差异;对文库AOA-MP和AOA-MPr进行比较分析,得到的P值大于0.05(P>0.05),表明两文库间无显著差异。以上两组采用同一土壤样品MP重复构建的文库间均无显著差异,说明采用本研究方法对同一土壤样品构建文库具有可重复性。使用SONS软件分析文库ARC-MP和ARC-GY的重叠率为36.05%,文库AOA-MP和AOA-GY间的重叠率为31.26%,说明MP和GY两地区的古菌和氨氧化古菌群落组成上有明显的不同,与利用∫-Libshuff和TreeClimber两种方法分析得到的AOA-MP和AOA-GY两文库在古菌群落组成上存在显著差异相呼应。SONS软件分析文库ARC-MP和ARC-MPr间的重叠率为98.29%,文库AOA-MP和AOA-MPr间的重叠率为84.01%,表明以上两组重复构建的文库间重叠率很高,与利用∫-Libshuff和TreeClimber两种方法分析得到的两文库在氨氧化古菌群落组成上无显著差异相吻合,再次验证了采用本文构建克隆文库的方法具有可重复性。总之,本研究使用DOTUR、∫-Libshuff、TreeClimber以及SONS等多种软件对西藏米拉山高寒草甸土壤古菌16S rRNA和amoA基因克隆文库的多样性进行分析,表明不同植被覆盖率的MP和GY两地古菌和氨氧化古菌群落组成存在显著差异,并且古菌资源比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用,同时本研究也为开发利用极端环境微生物奠定了基础。更多还原

【Abstract】 Archaea could not only survive under extreme conditions, but also exist in normal environments. With its huge abundance, Archaea is playing a highly important role in the biogeochemistry processes on the Earth. Nitrification in soil plays an important role in the global nitrogen cycle. Although bacteria were thought to be solely responsible for catalyzing the rate-limiting step of this process, several recent studies have suggested that Crenarchaeota are capable of performing ammonia oxidation. This area is a sensitive key region to the global warming. Alpine meadow is the key plant community type, which covered most area of Tibetan Plateau. Here we examine the diversity of archaea and ammonia-oxidizing archaea (AOA) within the soil of MP (at the entrance of Mila Mountain where the grass cover below 50%) and GY (at the Gongbo’gyamda County where the grass cover above 50%) of Mila Mountain. The four clone libraries were named ARC-MP, ARC-GY, AOA-MP and AOA-GY respectively. DNA was extracted from 10 replicate subsamples from each of the two soil samples. For each library, three replicate PCRs were conducted per soil DNA template (for a total of 30 replicate PCRs per library) using group-specific primers. To verify whether the method has the properties of good repeatability and high reliability or not, the archaea 16S rRNA and amoA gene libraries were generated for the soil of MP again.Phylogenetic analysis revealed that archaea in the three archaeal 16S rRNA clone libraries of Mila Mountain including the Crenarchaeota and unclassified_Archaea phyla, not found the Euryarchaeota. All the Crenarchaeota belong to the Thermoprotei. Mila Mountain belonged to the kingdom Crenarchaeota. The archaea and AOA have high similar with the sequences of protected soil.The sequences of archaea and AOA were divided into operational taxonomic units (OTUs) according to the 97% similarity threshold for OTU assignment was performed using the software program DOTUR. Archaea species composition from the three archaeal 16S rRNA gene clone libraries of ARC-MP, ARC-MPr and ARC-GY included 54, 51 and 51 OTUs respectively. AOA species composition from the three archaeal amoA gene clone libraries of ARC-MP, ARC-MPr and ARC-GY included 51, 44 and 23 OTUs respectively. The MP has roughly the same abundance of archaea as the GY, but the AOA abundance of MP are more than those in the GY.In order to compare the microbial populations present in the soil samples from two sites with statistic test, P values obtained by∫-Libshuff and TreeClimber. The P value between ARC-MP and ARC-GY clone libraries are 0.0000 (P <0.05), there have significant differences between the two libraries. The P value between AOA-MP and AOA-GY clone libraries are 0.0000 (P <0.05), there is significant differences between the two libraries. It means that the microbial populations of MP and GY are significant differences. It shows that the composition of the community of the AOA of MP and GY that related to the nitrogen cycle are significant change. The P value between ARC-MP and ARC-MPr clone libraries are above 0.05 (P >0.05), The P value between ARC-MP and ARC-MPr clone libraries are above 0.05 (P >0.05). It means that the microbial populations of the soil from same site have no significant difference. So conclude that the method used contruct libraries has the properties of good repeatability and high reliability.To compare the memberships and structures of two communities at a particular operational taxonomic units (OTUs) definition and implement nonparametric estimators for the fraction and richness of OTUs shared between clone libraries. Community overlap of inter and inner sample comparisons for OTUs obtained by the program SONS. For the archaeal 16S rRNA gene libraries of two samples (ARC-MP and ARC-GY), there are 36.5% overlapped community and for the archaeal amoA libraries of two samples (ARC-MP and ARC-GY), there are 31.26%overlapped community, which reconfirmed the microbial populations of MP and GY is significant differences. For the archaeal amoA libraries of two samples (ARC-MP and ARC-MPr), there are 98.29% overlapped community, and for the archaeal amoA libraries of two sites (ARC-MP and ARC-GY), there are 84.10%overlapped community. The overlapped communities between reconstruction libraries both for archaeal 16S rRNA and amoA genes are obviously large, which reconfirmed the method we used has the properties of good repeatability and high reliability.In a conclusion, to analysis the diversity of Archaeal 16S rRNA and amoA genes in Mila Mountain meadow soil of Tibet, the program DOTUR,∫-Libshuf, TreeClimber and SONS were used. Reconfirmed the microbial populations of MP and GY is significant differences. These findings show prolific archaeal diversity in the alp prairie soil of Mila Mountain, where they may be actively involved in nitrification.更多还原