【作者】 董小培；

【导师】 赵志光； 刘光琇；

【作者基本信息】 兰州大学 ， 植物学， 2010， 硕士

【摘要】 极端环境条件下微生物群落演替与气候演变的关系研究是生命科学和地球科学交叉的一个国际前沿领域。冻土微生物常年处于极端严酷的环境条件下,对环境变化特别敏感,因而,研究冻土中的微生物分布、遗传变化与自然选择对研究过去气候环境形成于变化、评价生物和人类活动对地球环境的影响有重要意义。本研究分别以乌鲁木齐河源区不同海拔表层土壤和不同层位冻土样品为介质,利用荧光显微计数技术、寡营养恢复培养技术、16SrDNA基因克隆技术、分子标记-变形梯度凝胶电泳(DGGE)、限制性片度长度多态性(T-RFLP)技术等,通过对冻土样品中微生物的数量和物种多样性的研究,分析了不同海拔、不同深度中微生物的群体结构和种类变化,并结合相关的土样理化性质资料,探讨冻土微生物数量、多样性和物种垂直结构的时空变化特征,揭示微生物的来源及其物种的演替规律以及与土壤理化参数的关联度。主要结果如下：1、在20个代表不同年代的冻土芯样品中,可培养细菌数量与年代、土壤含水量、总C和总N含量都成显著的正相关,与pH值呈显著负相关,表明深坑样冻土中可培养细菌数与环境有紧密联系。而且,可培养细菌数及其在细胞总数中所占比值随年代的增加而下降。海拔样品中可培养细菌数与含水量、总C、总N和pH值相关性不如深坑样。通过分析研究海拔、植被与可培养细菌数之间的相互关系,我们发现海拔高度并不是影响可培养细菌数的决定因素,但不同的植被类型对细菌生长有一定的决定作用：单子叶草本植物>乔木类植物>双子叶草本植物。2、恢复培养结果显示,从深坑样品中检测到的31株细菌分别属于Firnicutes、Actinobacteria、α-Protebacteria、β-Protebacteria和y-Protebacteria5个系统发育组的20个属;从海拔样品中检测到的23株细菌分别属于Firmicutes、Actinobacteria、α-Protebacteria、β-Protebacteria、γ-Protebacteria和Bacteroidetes 6个系统发育组的16个属。深坑样中Actinobacteria与α-,β-,γ-Protebacteria呈显著负相关,Firmicutes与β-Protebacteria呈显著正相关；海拔样中α-Protebacteria与β-Protebacteria呈显著负相关,与Firmicutes成显著正相关。而且,在深坑样中,γ-Protebacteria与pH呈显著正相关,与可培养细菌总数呈显著负相关；海拔样中Actinobacteria与海拔呈显著正相关。从而推断出,细菌的物种多样性不仅受环境因素影响,而且是种间相互作用的结果。产色素菌的比例较高,深坑样中为98.60%,深层样品中产色素菌含量较表层样品小；海拔样品中为47.06%,随海拔的增加呈减小趋势。进一步分析发现,分离出来的许多菌株都是功能菌,在净化乌鲁木齐河源区的环境及生态建设方面具有很大的应用价值。3、DGGE对细菌的分析显示,从深坑样中检测到的23条细菌16S rDNA片段分别属于Actinobacteria、α-Protebacteria、β-Protebacteria、δ-proteobacteria、Nitrospira、Acidobacteria和Gemmatimonadete 7个系统发育组；从海拔样中检测到的30条细菌16SrDNA片段分别属于Actinobacteria、a-Protebacteria、P-Protebacteria、8-proteobacteria、Nitrospira、Acidobacteria、Gemmatimonadete、Firmicutes、Thermodesulfobacteria、Chloroflexi和Bacteroidetes 11个系统发育组。对DGGE图谱中细菌物种多样性(shannon指数)和丰富度的分析表明,细菌物种多样性在不同深度及不同海拔样品中差异较小。然而需要指出的是,DGGE方法可以检测出来更多类群的细菌,但有些能恢复培养出来的而并未检测到,因而需要将分子方法与传统的培养技术相结合,才能更充分、更全面地研究冻土微生物。4、DGGE对古菌的分析显示,从深坑样中检测的21条古菌16SrDNA片段分别属于Crenarchaeota(泉古菌门)的Caldisphaera和Pyrolobus2属和Euryarchaeota：广古菌门)的Methanolinea、Thermogymnomonas、Methanosphaerula和Methanocella4属。海拔样检测到的17条古菌16SrDNA片段分别隶属于Crenarchaeota的Caldisphaera1个属和Euryarchaeota的Methanolinea、Thermogymnomonas和Methanosphaerula3属。对其进行物种多样性分析显示,深坑样的shannon指数基本在0.8-1.0之间,多样性随深度的增加呈上升趋势。海拔样的shannon指数在0.5-1.1之间,多样性随海拔的增加呈上升趋势。以上研究表明,不同深度及不同海拔梯度样品中微生物的数量、组成和分布有很大差异,而且都在一定程度上与气候环境的指标和时空变化相关,为阐明、推演乌鲁木齐河源区气候环境的演变规律,为当地生态环境建设的调控提供科学参考。更多还原

【Abstract】 On the condition of extreme environment, the relationship between microbial community succession and climate evolution is an international frontier. Permafrost microorganisms are particularly sensitive to environmental changes, thus, it is significant for the study of the distribution of permafrost microorganisms, genetic change and natural selection to evaluate past climate changes as well as the influence of biological and human activities on the Earth’s environment.In order to understand spatial and temporal variations of soil microorganism in the headwaters of the Urumqi river, soil from different altitudes and different depths has been sampled. Here, fluorescence microscopy counting techniques, few nutrients of cultivation techniques,16S rDNA gene cloning, molecular markers-deformation gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (T-RFLP), were used to reveal the microbial community structure and diversity. Soil physical, chemical and other environmental variables are mensured as well. The results obtained here were summarized as follows:1、In the samples which representing different age, the cultured bacterial amount have a significant, positive correlation with age, soil moisture content, total C, total N concentrations, and have a significant, negative correlation with pH value. It suggested that cultured bacterial amount and the environment are closely linked. Moreover, as age increasd, the cultured bacterial amount in the ratio of total cell number are decreasing. In the samples which at different altitudes, the correlation between the cultured bacterial amount and soil moisture content, total C, total N concentrations, pH value are not as well as the samples at different depths. We found that the altitude does not the determining factor affect the number of bacteria, but different vegetation types have some decisive on the growth of bacteria:monocot herb> arbor plants> dicotyledonous herbs.2、Cultivation-dependent approaches showed that the 31 strains at different depths belong to following group:Firmicutes、Actinobacteria、α-Protebacteria、β-Protebacteria and y-Protebacteria. Actinobacteria have a significant, negative correlation withα-,β-,γ-Protebacteria; Firmicutes have a significant, positive correlation with P-Protebacteria; y-Protebacteria have a significant, positive correlation with pH, and have a significant, negative correlation with the cultured bacterial amount. The 23 strains at different altitudes belong to following group: Firmicutes、Actinobacteria、α-Protebacteria、β-Protebacteria、γ-Protebacteria and Bacteroidetes.α-Protebacteria have a significant, negative correlation withβ-Protebacteria, and have a significant, positive correlation with Firmicutes; Actinobacteria have a significant, positive correlation with depths. It suggested that the types and diversity of bacteria not only affected by environmental, but also the result of the interaction between them.98.60% pigmented colonie was observed in the samples at different depths, the deep sample is smaller than the surface sample; 47.06% pigmented colonie was observed in the samples at different altitudes, as altitude increasd, the ratio is reduced. Further analysis found that many of the strains isolated were functional bacteria, they have great value at purifing the environmental of the headwaters of the Urumqi river and constructing ecological.3、DGGE analysis of bacteria showed that the 23 representative bacterial bands at different depths belong to following group:Actinobacteria、α-Protebacteria、β-Protebacteria、δ-proteobacteria、Nitrospira、Acidobacteria and Gemmatimonadete; the 30 representative bacterial bands at different altitudes belong to following group: Actinobacteria、α-Protebacteria、β-Protebacteria、δ-proteobacteria、Nitrospira、Acidobacteria、Gemmatimonadete、Firmicutes、Thermodesulfobacteria、Chloroflexi and Bacteroidetes. Analysis of the bacterial community structure diversity (shannon index) and richness showed that the difference of bacterial diversity at different depths and at different altitudes is small. However, it should point out that, DGGE method can be detected more groups of bacteria, but some can resume training were not detected out, and thus need to combined molecular methods with the traditional culture technique to more fully and more comprehensively study soil microorganisms.4、DGGE analysis of archaea showed that the 21 representative arehaeal bands at different depths belong to following kind:Caldisphaera、Pyrolobus、Methanolinea、Thermogymnomonas、Methanosphaerula and Methanocella; the 17 representative arehaeal bands at different altitudes belong to following kind:Caldisphaera、Methanolinea、Thermogymnomonas and Methanosphaerula. Analysis of the arehaeal community structure diversity (shannon index) showed that shannon index is between 0.8 to 1.0 in the samples at different depths, the diversity increase with depth increasing; shannon index is between 0.5 to 1.1 in the samples at different altitudes, the diversity increase with altitude increasing.Above all, these studies have shown that the number of microbes, composition and distribution in the samples at different depths and at different altitudes are very different, and to some extent, were interrelated with the indicators of environment and climate and spatial and temporal changes. They can provide scientific reference to deduce the evolution law of climate and environment in the headwaters of the Urumqi river, and to control the construction of local ecological environment.更多还原