Knowledge Resource Center for Ecological Environment in Arid Area
| 生物结皮中微生物群落结构与功能 | |
| 其他题名 | Microbial community structure and function in biological soil crusts |
| 张青一 | |
| 出版年 | 2017 |
| 学位类型 | 博士 |
| 导师 | 胡春香 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 生物土壤结皮广泛分布在荒漠和半荒漠地区稀疏的灌丛间隙，对表土的稳定、增肥、及保水起着重要功能。本论文以腾格里沙漠南缘的沙坡头试验站和库布齐沙漠东缘的达拉特旗试验站为研究地点，将直接显微观察、传统培养、生理生化分析、普通分子生物学方法、及宏基因组学手段等相结合，从不同演替阶段典型生物结皮中微生物的物种组成、群落结构、代谢活性、及功能潜力等方面进行了较系统地研究，主要结果如下：\n多种方法(直接观察、传统培养、DGGE、克隆文库、高通量测序等)同步研究了藻结皮、蓝藻地衣结皮、绿藻地衣结皮和藓结皮中的蓝藻多样性和群落结构，发现，藻结皮和藓结皮都是以Microcoleus为绝对优势种，两种地衣结皮中Nostoc和Scytonema的丰度显著高于其他结皮；随着结皮演替蓝藻多样性先增加后降低，使地衣结皮中蓝藻多样性最高，藓结皮中最低。但多种方法得到的群落结构存在巨大差异，甚至方法间差异大于样品间差异。\n鉴于DGGE方法较为精细的分辨率和高性价比，对4阶段9种自然结皮的蓝藻分析发现，除绿藻地衣结皮外，其他结皮在不同阶段都有变化，而且念珠藻地衣结皮和伪枝藻地衣结皮与藻结皮较近，拟色球藻地衣与绿藻地衣结皮较近。人工结皮的分析还发现，结皮覆盖类型影响不大，坡度和时间都有影响。人工接种蓝藻促结皮恢复技术可直接在流沙上接种，但坡度都最好小于30°，同时保证结皮的发育时间。在细菌通用引物下，DGGE技术对各演替阶段结皮的分析发现，随着结皮演替细菌多样性有降低趋势，因藓结皮中检测到的主要是叶绿体基因；藻结皮和地衣较为接近，但绿藻地衣结皮中Stichococcus bacillaris非常丰富。在真菌特异引物下，DGGE技术发现，蓝藻地衣结皮中真菌的多样性最高，各结皮中的真菌差异主要在Taphrina johansonii和Paracostantinella sp.的丰度上，而且它们都是各结皮中的优势真菌。\n高通量测序发现，Proteobacteria，Actinobacteria，Cyanobacteria，Bacteroidetes和Acidobacteria等是各演替阶段结皮中的优势原核微生物；最主要的真核微生物为Ascomycota。但结皮在演替过程中多样性和群落结构都有着明显的不同，而不同优势种形成的地衣结皮之间的微生物群落结构相对而言更为接近。在影响结皮群落结构的环境因素中，水分和土质对原核微生物群落的影响最大，土质和电导率对真核微生物群落影响最大。\n宏基因组测序通过蛋白编码序列的NR库比对发现，Cyanobacteria, Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes等是结皮中的优势微生物门类；真菌Ascomycota丰度在地衣结皮中最高，Euryarchaeota等古生菌的丰度随着结皮演替增加。NR库比对结果同时显示，随着结皮演替，其中微生物丰富度和多样性都显著增加。固氮活性和固氮微生物的研究发现，藻结皮的固氮酶活性显著低于它阶段，但nifH基因的拷贝数随结皮演替显著增加。nifH基因克隆文库分析发现，随结皮演替，异形胞固氮蓝藻逐渐减少，固氮异养菌的丰度逐渐增加。\n土壤酶活性显示，随着结皮演替，β葡萄糖苷酶、多酚氧化酶、过氧化氢酶、几丁质酶、蛋白酶、硝酸还原酶活性有增加趋势，但蔗糖酶呈下降趋势；其余酶都在地衣和藓结皮高于藻结皮。CLPP分析再次证明，藓结皮在多种碳源利用方面最有优势，绿藻地衣结皮利用碳源的功能多样性最低，藻结皮在利用环式糊精方面有优势，而且绿藻地衣在纤维素、藓结皮在聚合碳和胺类利用等方面的能力与胞外酶活性反映的规律完全一致。基于宏基因组的分析表明，在COG层面，氨基酸转运和代谢、能量产生和转化、无机离子转运相关基因丰度在地衣结皮显著最高，而细胞壁合膜生物合成相关基因显著最低。SEED库表明，休眠和孢子、离子获取和代谢基因丰度随结皮演替显著增加，而光合作用、氮代谢和钾代谢等途径的基因丰度显著减少。KEGG分析发现，氨基酸代谢，糖代谢以及复制和修复方面基因丰度随演替有增加，能量代谢方面呈减少趋势。在固碳途径中，随演替卡尔文本森循环途径的基因丰度呈明显降低，而3-羟基丙酸/4-羟基丁酸途径途径呈显著增加。胞外聚合物降解中，果胶质、半纤维素、几丁质降解相关基因丰度呈增加趋势，而纤维素降解基因在绿藻地衣结皮与藓结皮中相似。参与碳固定的基因与物种关系上，演替早期主要是蓝藻，中期(地衣阶段)主要是放线菌和变形菌，晚期(藓结皮)是酸杆菌和变形菌；参与胞外聚合物降解的基因与物种关系，演替早期主要是变形菌，中期主要是放线菌，晚期主要是酸杆菌。统计学相关关系则表明，与碳固定基因相关性最高且最多的是放线菌、α变形菌和蓝藻，而与胞外聚合物降解基因最具相关性是放线菌、α变形菌、酸杆菌和蓝藻。 |
| 英文摘要 | Biological soil crusts (BSCs) are widely distributed in the sparse vascular vegetation desert and semi desert areas. BSCs have important functions on surface soil stability, soil fertility improvement, and water retention. This work carried out a series of direct microscopic observation, traditional culture, physiological and biochemical analysis, the common methods of molecular biology, and metagenomic approaches to research the microbial community composition and functional potential in the different succession stages. The main results are provided as follow:\nCombined with a variety of methods (traditional culture, DGGE, clone library sequencing, high-throughput sequencing etc.), we researched the cyanobacteria diversity and community structure in the different succession stages of BSCs [algae (A), cyanolichen (C), green algae-lichen (G), and moss crusts (M)]. We found that in algal crusts and moss crusts Microcoleus was absolute dominant species, the abundance of Nostoc and Scytonema in lichen crusts was significantly higher than that of other crusts, and with the development succession, in moss crusts stage the cyanobacteria gradually decline and in the algae crusts stages the diversity was the lowest. But the cyanobacteria community structures from each methods were obvious different, which method itself had more impact on the community composition than the stages of BSCs. \nBecause of the high precision and cost performance of DGGE approach, we studied the natural BSCs with DGGE and found that, in addition to the green algae lichen crusts, the cyanobacteria community composition in other BSCs also had differences, such as, Nostoc and Scytonema-dominant lichen crusts were close to algae crusts, and Chroococcidiopsis-dominant lichen crusts were even close to green algae lichen crusts. Artificial BSCs DGGE studies found that development time obvious impacted on the cyanobacteria diversity and community composition, slope had obvious effect on cyanobacteria community composition, and vegetation coverage had relatively minimal impact. So the artificial BSCs inoculation technology could directly apply to naked sand, but the slope maybe low than 30°, and the development time should be insured. \nThrough DGGE we researched the change of bacterial and fungal diversity in different succession stage of BSCs. We found that as the BSCs succession, bacterial diversity had a tendency to reduce, and we recovered a large number of chloroplast sequences in moss crusts. Looking from the bacterial community composition, algal crusts were close to lichen crusts, and in green algae-lichen crusts the abundance of Stichococcus bacillaris was high. By the fungi specific primers of DGGE analysis, we found that cyanolichen crusts had the highest fungal diversity. The abundance differences of Taphrina johansonii and Paracostantinella sp., which belongs to Ascomycetes fungus caused the main reason for the difference between different succession BSCs.\nThe 454 high-throughput sequencing found that Proteobacteria, Actinobacteria, Cyanobacteria, Bacteroidetes and Acidobacteria were the advantage of procaryote microbiology in each succession stage of BSCs, and the main eukaryotic microorganism was Ascomycota. But the microbial diversity and community structure were significantly different among BSCs, and the microbial community structure between two kinds of lichen crusts was relatively more close to each other. We also found that water and soil texture had the greatest effect in the prokaryotic microbial community structure, and the environmental factors which had the greatest influence on eukaryotic microbial community composition were soil texture and Ec. \nMetagenome sequencing results obtained the protein-code sequences, and the taxonomic analysis with NR library showed that Cyanobacteria, Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes were advantage microbes; for eukaryotic microorganisms, fungi Ascomycota abundance in lichen crust was the highest, Euryarchaeota increased along with succession. NR library comparing results also showed that as the BSCs succession, microbial richness and diversity were significantly increased both.\nThe activity of dinitrogenase and nitrogen-fixing microorganism community in different development succession stages of BSCs studies have found that the dinitrogenase activity of algal crusts was significantly lower than the rests, the rest of the BSCs had no significant difference, but the nifH genes showed the copy number of which increased significantly as the BSCs development. The nifH gene clone library analysis found that with the succession of BSCs, heterocyst nitrogen-fixing cyanobacteria community gradually reduced, the abundance of nitrogen fixation heterotrophic bacteria increased. \nSoil enzyme activity study showed that, as the crust succession, the activity of beta glycosidase, polyphenol oxidase, catalase, chitinase, protease and nitrate reductase had increased trend; the activity of invertase decreased; the rest of the main enzymes had not such variation trend, but also was lowest in algal crusts. Biolog ECO plate CLPP analysis showed that the moss crusts had the highest AWCD value, and in green algae-lichen crusts the microbial functional diversity was lowest. Alage crusts had the highest cyclodextrin degradation activity, and green algae crusts had the advantage in the cellulose degradation. The carbon polymer and amine degradation in moss crusts were advantage. Which were in accord with the rule of extracellular enzymes activities change among BSCs.\nFor the ecological function change in different succession stages of BSCs using metagenome sequencing approach, we has carried on the COG, SEED and KEGG annotations. COG functional genes annotated results showed that the relative abundance of amino acid transport and metabolism, energy production and conversion, inorganic ion transport and metabolism gene were significantly highest in middle stage, while Cell wall/membrane/envelope biogenesis gene was significantly lowest in two lichen crust. SEED library annotation results show that the dormancy and spores, iron acquisition and metabolism, membrane transport genes abundance significantly increased during the succession of BSCs, and photosynthesis, nitrogen metabolism and potassium metabolism genes abundance significantly reduced. Through KEGG metabolic pathways annotation analysis, we found that the change rule of the main several KEGG level 2 is that, the metabolism of amino acids, glucose metabolism and replication and repair genes had increasing trend, membrane transport and energy metabolism showed a trend of decrease. \nFor the analysis of the carbon fixation genes based on KEGG results, we found that during the succession, the Calvin-Benson-Bassham cycle (CBB) genes were declined distinctly, while the hydroxypropionate-hydroxybutyrate cycle (3HP/4HB) genes increased. As to the degeration of extracellular polymer, pectin, hemicellulose and chitin degeration genes aboundance increased with succession, and the aboundance of cellulose relative gene were similar in green algae lichen crusts and moss crusts. As to the relationship of carbon fixation genes and corresponding species, we found that at the early successional stage, the corresponding microbes mainly were Cyanobacteria, at the lichen stages mainly were Actinobacteria and Proteobacteria, at the late stage mainly were Acidobacteria and Proteobacteria. The research of the relationship between extracellular polymer degeration genes and corresponding species showed that, which mainly were Proteobacteria in algae crusts, Actinobacteria in lichen crusts, Acidobacteria in moss crusts. From the view of statistical correlation of genes and species, Actinobacteria, Alphaproteobacteria and Cyanobacteria had the closeset relationship with carbon fixation genes, and Actinobacteria, Alphaproteobacteria, Acidobacteria and Cyanobacteria had the closest relationship with extracellular polymer degeration genes. |
| 中文关键词 | 生物结皮 ; 群落结构 ; 功能潜力 ; 高通量测序 ; 宏基因组 |
| 英文关键词 | Biological soil crusts Community structure Functional potential High throughput sequencing Metagenome |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 水生生物学 |
| 来源机构 | 中国科学院水生生物研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287916 |
| 推荐引用方式 GB/T 7714 | 张青一. 生物结皮中微生物群落结构与功能[D]. 中国科学院大学,2017. |
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