【作者】 芦晓飞；

【导师】 谢丙炎；

【作者基本信息】 中国农业科学院 ， 生物安全， 2009， 博士

【摘要】 青藏高原是世界上海拔最高的高原,其平均海拔在4 000米以上,高寒、缺氧、降水少、日照长、辐射强,独特的自然地理环境使其富含特殊的微生物资源。但是由于全球气候的变化导致青藏高原气温不断升高,加之人类过度放牧的影响使得高寒草甸逐年退化,微生物生态系统被严重破坏。此外,由于能够利用培养技术获得的微生物种类不足自然环境中存在的1%,目前对该类型土壤的微生物多样性和资源调查存在很大的不足,阻碍了这些特殊微生物资源的开发应用。提取环境微生物宏基因组DNA及构建文库是研究未培养微生物的重要方法。因此,本研究利用宏基因组学技术首次对西藏米拉山高寒草甸土壤的微生物群落结构、功能微生物及微生物基因资源多样性进行了系统的研究,主要结果与结论如下:(1)针对高寒草甸土壤中腐植酸和有机质含量高、DNA提取非常困难的特点,首次选择和优化了适合该类型土壤样品不同分子量DNA提取及纯化的技术路线。通过对比不同DNA提取和纯化方法的研究表明:直接法提取的DNA片段小、杂质含量高,但是由于其提取效率高,结合操作简便的柱纯化方法,适合于微生物多样性研究;结合低速离心和Nycodenz密度梯度离心先分离出微生物细胞的间接法虽然DNA产率降低,但是纯度高、片段长,结合电泳纯化法更适合于构建大插入片段文库。(2)分别以pUC19质粒载体和pCC2FOS?载体构建了西藏米拉山高寒草甸土壤微生物宏基因组文库,为进一步开发利用其微生物资源奠定了基础。其中两个pUC19质粒文库(ZMp-1和ZMp-2)的平均插入片段长度分别为1 kb和4 kb;Fosmid文库(ZMf-vsmfba)包含30 624个克隆,平均插入片段为35 kb左右,库容量超过1 Gb;部分文库克隆末端测序结果表明,尽管构建两种文库的DNA提取和纯化方法不同,但是文库插入片段序列多样性丰富,都含有大量的未知微生物物种。(3)在直接提取宏基因组DNA的基础上,采用16S rRNA基因克隆文库技术研究表明:西藏米拉山高寒草甸土壤中细菌和古菌多样性具有一定特殊性;但是由于受到气候变化和人类活动的影响,退化草甸土壤的微生物群落结构发生了改变。古菌文库中与GenBank中序列相似性很低的未分类古菌类型占有很大比例,但是古菌类型非常单一,仅发现了嗜泉古菌界(Crenarchaeota)的未分类热变形菌纲门(Unclassified_Thermoprotei),其余为未分类古菌(Unclassified_Archaea),没有发现广域古菌界(Euryarchaeota)克隆;相比之下细菌类型丰富,基本涵盖了大部分土壤细菌基本类群。(4)由于草甸的退化使储藏在土壤中的大量碳氮加速释放将造成严重的环境问题,本研究进一步以氨单加氧酶(ammonia monooxygenase, amoA)基因为分子标记比较了不同退化程度草甸土壤样品中的氨氧化微生物(ammonia-oxidizing microorganisms, AOM)多样性发现:氨氧化古菌(ammonia-oxidizing archaea, AOA)在伴随草甸退化的硝化作用增强过程中的作用高于氨氧化细菌(ammonia-oxidizing bacteria, AOB),并且退化草甸土壤的硝化作用模式类似于温室保护地土壤。(5)根据已报道许多微生物能够分泌胞外蛋白酶降解线虫体壁及卵鞘的原理,新型蛋白酶的开发和利用在植物病原线虫的生物防治中具有重要作用。本研究在西藏高寒草甸土壤微生物的宏基因组DNA和文库的基础上,以蛋白酶为靶标对其微生物基因资源进行了调查,结果根据同源克隆方法得到了75条新丝氨酸蛋白酶基因片段;通过pUC19质粒文库克隆末端测序分析得到了7条新的类蛋白酶基因片段;利用Fosmid文库的大插入片段表达特性筛选到了2个新蛋白酶基因序列,表明西藏高寒草甸中蕴含着丰富的可利用新基因资源,具有极高开发利用价值。综合以上结果,本研究表明西藏高寒草甸土壤微生物群落的组成具有特殊性,其中有大量的未知微生物及其基因信息有待挖掘和开发利用。但是由受到环境和人为活动的影响,高寒草甸的退化导致微生物群落为适应环境压力而通过改变其活性、生物量和结构来进行调整。无论是从资源角度还是环境角度来看,对西藏高寒草甸的保护及恢复都至关重要。更多还原

【Abstract】 Qinghai-Tibetan Plateau is the highest and largest plateau on the Earth, which is frequently referred to as an extreme environment for special microbial resources. But most of them could not be utilized for their unculturable property. And togather with the global climate change and human impact, the microbial ecosystem of the Plateau is being destroyed. Extraction of total microbial DNA with cultured-in-dependent method and construction of metagenomic library is an alternative method to exploit uncultured microorganisms. To exploiting new microbial resources and investigating ecological changes on the Plateau, meadow soil from Tibetan Mila mountain was collected as alpine soil type. And this dissertation first systematically studied the microobial diversity of the alpine meadow soil in Tibetan Plateau with metagenomic method.A pre-experiment indicated that the alpine meadow soil has high organic matter and humic acid content, which resulted in a great difficulty in DNA extraction. We compared different extraction and purification methods, the result showed that the DNA obtained by directly extraction from the soil was in small DNA fragments with high contamination. For its easy operating and high efficiency, it is suitable for mircrobial diversity research after column purification step. Comparison with direct extraction, the DNA extracted from cells separated from combing low speed centrifugation and Nycodenz gradient centrifugation has larger molecular weigh and higher purity with comparative diversity.Metagenomic libraries were constructed by pUC19 and pCC2FOS? vector respectively. The average insert fragments of the two constructed pUC19 libraries (ZMp-1 and ZMp-2) were 1 kb and 4 kb individually, and the Fomisd library (ZMf-vsmfba) contained 30 624 clones of 35 kb average insert fragments, which made it has a total genomic storage capacity over 1 Gb. Bioassay demonstrated that both of the constructed libraries have large capability of insert DNA with unkown species. The method for constructing library provides a powerful tool for soil samples from extreme environment and the constructed library provides resources for finding and utilizing new functional genes from Tibetan altiplano.As 16S rRNA and its gene has proven to be useful and powerful markers for the presence of microorganisms in environmental samples, we determined the diversity of 16S rRNA gene of Archaea and Bacteria with library method. The result showed that the microorganisms, especially Archaea, have special characteristics. Taxonomic analysis recovered only Unclassified_Thermoprotei of Crenarchaeota and Unclassified_Archaea, no Euryarchaeota clone was found. Comparing to the Archaea, the bacterial group covered most of the basic soil bacteria types. But for the climate change and human effective, the microbial structure of degrading alpine meadow soil sample was changed.And with the meadows’retrogressing, the carbon/nitrogen are releasing and affecting environment severely. Much study has been focused on the process of biogeochemical cycle, whereas none was done with the ammonia-oxidizing microorganisms (AOM), whose diversity and factors influencing them are keys to understanding the movement of nitrogen. We estimated the amoA (ammonia monooxygenase subunits A) gene diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in two soil samples with different human and climate impact. The result suggested that AOA might play more important role in the degenerated grassland than AOB, and the opposite was true in undegraded sample. Besides, the amoA sequences from degenerated sample were closest to protected lands’sequences, which indicated that the model of nitrification in degenerated grassland of the alpine meadow soil in Tibetan Plateau is likely to that of protected lands in greenhouse.Furthermore, for exploring new protease, which was considered to be important nematode biocontrol reagent, we designed PCR amplification primers according to the serine proteases isolated from nematophagous fungi, which could decompose nematode body wall and egg cuticle. The amplified result with metagenomic DNA of alpine meadow soil showed that no achieved sequences were close to target genes, but the high diversity implicated rich available resources. Moreover, partial bidirectional-terminal sequencing of pUC19 plasimid library (ZMp-2) clones resulted into three protease-like genes with double-ends and four with single-end liking protease genes, and functional screening of the Fosmid library resulted into two new genes similar to protease. But the retrieved nine protease-like genes from two metagenomic libraries had very low similarity to reported sequences in GenBank (30~62%), we deduced they are brand-new protease genes.To sum up, there are much undiscovered special microbes and their gene information in the alpine meadow soil of the Tibetan Plateau. But with the environmental and anthropic impacts, the alpine meadows were degenerating and the microbial communities were adapting by changing their activity, biomass and structure. Therefore, the alpine meadows require our protection and recover for further utilizing and environmental protection purpose.更多还原