【作者】 张于光；

【导师】 肖启明； 李迪强；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2005， 博士

【摘要】 土壤微生物是自然物质循环不可缺少的成员,担负着分解动植物残体的重要作用,直接关系到土壤养分的有效性,对植物生长起到重要的作用,同时微生物在土壤肥力评价和生物净化等方面也有着重要的作用。土壤微生物对环境变化很敏感,能够较早地指示生态系统功能的变化,对于退化生态系统的恢复和治理以及提高不同植被的生产力均有重要的理论和实践意义。 三江源自然保护区是青藏高原的主体部分,是黄河、长江和澜沧江的发源地,平均海拔4400m,孕育了世界上海拔最高、面积最大的高寒草甸和高原湿地。该地区原始自然状态保存较好,是气候、环境变化信息最敏感最完整的载体和自然环境演变过程最忠实的科学记录。由于全球气候变化和人为破坏,“三江源”的生态环境不断恶化,加强三江源地区的生态保护刻不容缓。研究三江源地区的土壤微生物,揭示三江源土壤微生物的生态分布和区域特异性,将为评价三江源地区的生态环境,了解三江源地区土壤微生物在全球变化中的影响,为三江源自然保护区保护策略的制定提供科学依据。 本研究主要通过PCR扩增、基因克隆、RFLP和序列分析对三江源自然保护区不同植被和海拔高度土壤环境中的固氮酶基因nifH、反硝化酶基因nirK与nosZ和16S rDNA的分子多样性进行了分析,同时探讨了不同植被和海拔高度土壤中固氮微生物、反硝化细菌等功能微生物的多样性及其主要影响因子。其主要结果如下: 1、用于分子生态学研究的土壤微生物DNA提取方法研究 土壤微生物分子生态学研究的关键技术之一是从各种环境样品中高效的获得可进行分子生物学研究的基因组DNA。本研究利用SDS高盐法、变性剂加SDS高盐法和溶菌酶加SDS法三种方案对土壤微生物总DNA进行了提取,然后通过电泳加树脂柱回收和连续2次树脂柱回收两种方法进行了纯化,结果表明,变性剂加SDS高盐法的DNA提取效率明显高于其它两种方法,电泳加树脂柱法的纯化效果更好。PCR扩增表明,经过两种纯化方法后得到的DNA,都可以进行16SrDNA扩增和nirK、nosZ、nifH等功能基因的扩增。因此,认为变性剂加SDS高盐法是一种更为高效、可靠和适合于环境微生物分子生态学研究的DNA提取方法。 2、三江源地区土壤固氮微生物的分子多样性研究 为了了解青藏高原地区土壤固氮微生物的群落结构及其差异,本研究首次利用PCR-RFLP和测序分析对位于青藏高原腹地的三江源自然保护区土壤微生物固氮基因(nifH)的多样性和系统发育进行了探讨。研究选取了来自于不同海拔高度、植被类型的6个样品,根据样地的生物地理化学性状的主成分分析(PCA)表明,6个样品可以聚类分为3个不同的组,其中样地YS-2、YS-3、ZD-1和ZD-2较明显的聚类在更多还原

【Abstract】 Soil microorganism is the necessary part in global material cycle of carbon, nitrogen, and so on, thus it affects the material balance, the composition of the atmosphere and geochemistry procedure in the global scale. Soil microorganisms play a key role in degrading the animal and plant body, which directly influence the available nutrient of soil and vegetation growth. Soil microorganisms are sensitivity to the environmental change and easily indicated the change of the ecosystem functions, which had important significance to renew and protect the ecosystem. Understanding the structure and composition of soil microbial communities and their responses and apaptions to global change is critical in maintaining or restoring desirable ecosystem functions. Due to the irrational exploitation and utilization for natural resources, more and more factors impact on the diversity of soil microorganisms, such as the increasing of CO₂ concentration, vegetation degeneration and environmental pollution.Sanjiangyuan national nature reserve located in the southwestern part of Qinghai Province and is the center part of Tibet Plateau which is the highest and youngest plateau in the world and was named "The Third Ploar of Earth" for its unique location. Sanjiangyuan area is the biggest alpine prairie and wet area in the world, and its average altitude is about 4400 meter. With the upgrade of Himalaya’s motion, the complex geology and physiognomy was formed in Sanjiangyuan area. Because the particular environment and location, Sanjiangyuan area come into being the unique biological region and ecological system. Alpine prairie, alpine grassland and mountain forestry are the main plant community types in this area. Therefore, Sanjiangyuan natural reserve has the important significance value in scientific research. However, the global change and man-made destruction in Sangjianyuan area lead to the environmental deterioration. Research on the distribution and regional character of soil microorganisms in this area could help to evaluate environment, understand the influence in global change and offer scientific data for making the protected policy.In this study, a PCR-based cloning and sequencing approach was used to investigate the molecular diversity and community structure of nitrogen-fixing microbes and denitrifying bacteria and their key impact factors in the different plant community types and altitude sites. Moreover, the influence of plant community type changing to soil microbial diversity in Sangjiangyuan natural reserve was discussed by using the traditional cultural technology and analysing the diversity of 16S rDNA. The key results from these studies are summarized as bellow.1. Extraction method of Soil Microbial DNA for Molecular Ecology StudyIn environmental microbiology, molecular ecology study has been widely concerned in the world, while one of the key technologies of study uncultured microorganism in molecular level is obtain the high quality genome from environmental microbiology. In this study, soil microbial DNA was extracted using SDS lysis method, denaturing solution plus SDS lysis method and lysozyme plus SDS lysis, respectively. The crude DNA extraction was purified by gel electrophoresis plus minicolumn method and double minicolumns method, respectively. The result showed that the denaturing solution plus SDS lysis method could extract DNA more efficiently, and the gel electrophoresis plus minicolumn method could help to obtain purer DNA that was available in amplifying its 16S rDNA and functional genes fragment （such as nifH, nosZ, nirK） by polymerase chain reaction （PCR）. Therefore, denaturing solution plus SDS lysis could be an efficient and reliable method to extract DNA in the molecular ecology studies.2. Molecular Diversity of Nitrogen-Fixing Genes （nifH） in Sanjiangyuan Natural ReserveTo understand the composition and structure of nitrogen-fixing bacteria communities in the Tibet Plateau, the molecular diversity and phylogenetic analysis of nifH gene in Sanjiangyuan natural reserve were examined by using the PCR-RFLP based cloning approach.The samples were come from different sites which have different altitudes and plant community types in Sanjiangyuan natural reserve. The biogeochemical parameters were analyzed by principal-components analysis （PCA） to assess biogeochemical variability among sites and try to identify the biogeochemical parameters driving this variability. Six samples were divided into 3 clusters by PCA, sites YS-2、YS-3、 ZD-1 and ZD-2 clustered into a cluster, and sites YS-1 and NQ-1 had significant difference with the other 4 samples, respectively. For the nifH gene segment, diverse PCR products were characterized by cloning, restriction fragment length polymorphism （RFLP） analysis and sequencing. A total of 446 clones and 162 operational taxonomic units （OTUs） which were digested by the restriction enzymes MspI and RsaI were obtained from all samples. There are 1-2 significant dominant clusters of clones and shared 3 OTUs in all samples. Pairwise site comparisons showed that 6.2% to 31.3% and 4.3 to 18.4% of the total number of clones and OTUs were shared, respectively. This result showed they shared more clones and OTUs between samples which had closely biogeochemical properties. The PCA of the OTUs and biogeochemical parameters showed that C, N content and C/N ratio are the key factors to impact on the community structure of nitrogen-fixing bacteria community. Withthe increasing of altitude, the clones and OTUs decreased, and have not significant difference among the same plant community type sites.A wide range of sequence divergence was observed in the 59 nifH clones that were sequenced from all samples. Sequence comparison showed that the nifH clones were 64% to 98% similar. The phylogenetic tree was constructed by using the Clustal W and Mega softwares. 59 sequences could be subdivided into 4 clusters, and some of them were closely related with a> p\ y^ h-Proteobacteria, but The majority of the clones were not closely related to any known nitrogen-fixing bacteria, Therefore, most of them are unique and may represent novel sequences of nitrogen-fixing bacteria.3. The Molecular Diversity of Denitrifying Bacteria in Alpine Prairie of Sanjiang-yuan Natural ReserveThis was the first description the molecular diversity of denitrifying bacteria by analysis the molecular diversity of nirK and nosZ gene using PCR-RFLP and sequencing analysis in Sanjiangyuan natural reserve in Tibet plateau. 4 samples, over 4600m of altitude and alpine prairie of plant community type, had different biogeochemical property by principal component analysis （PCA）. sites ZD-1> ZD-2 and YS-1 clustered into a cluster, and site ZD-3 had significant difference with the other 3 samples. Based on the PCR and RFLP analysis, 253 nirK clones and 283 nosZ clones were received in four samples, and received 78 different OTUs of nirK and 120 different OTUs of nosZ by the restriction enzymes Mspl and Rsal digested, respectively. For nirK gene, there are different significant dominant clusters of clones in all samples, while for nosZ gene, there are not significant dominant cluster except site YS-1. RFLP-PCA showed that the RFLP patterns had significant similarity with biogeochemical property for nirK gene, but nosZ gene is not. The analysis of environmental factors showed that altitude and C/N ratio maybe the key factors to influence the denitrifying community structure.36 nirK clones and 17 nosZ clones were sequenced, and their levels of nucleotide identity were from 69% to 98% and 57% to 97% by DNAMAN software, respectively. None of the sequences were completely identical to any deposited in the data banks, and therefore each of them belong to a noncharacterized bacterium. Finally, the phylogenetic tree was constructed by using the Clustal W and Mega softwares. All the sequences could be subdivided into 4 groups, and most of them had similarity with different bacteria species （such as Blastobacter denitrifwans , Nitrosomonas sp. ■> Achromobacter xylosoxidans^ Sinorhizobium melilot^ Azospirillum irakensei） which showed many bacteria had denitrify gene and are the potential denitrify bacteria in soil. However, denitrifying bacteria in all samples had not significant different distribution in phylogenetic tree.更多还原