典型水稻土中固碳基因及功能微生物研究

CORC > 生态环境研究中心 > 中国科学院生态环境研究中心 > 土壤环境科学实验室

题名	典型水稻土中固碳基因及功能微生物研究
作者	肖可青
学位类别	硕士
答辩日期	2014-05
授予单位	中国科学院研究生院
授予地点	北京
导师	朱永官
关键词	水稻土微生物固碳 marker基因环境因子根际效应 Paddy soil microbial carbon dioxide fixation marker genes environmental factors rhizosphere effect
其他题名	Genetic characterization of microbial CO2 fixation in typical paddy soil
学位专业	环境科学
中文摘要	陆地生态系统是地球上仅次于海洋生态系统的第二大碳（C）库。农田土壤是陆地生态系统的重要组成部分，由于人类活动的长期干扰（如耕作、施肥、排水和灌溉等）使得该C库成为最活跃的一部分，而这一特征在水稻土中尤为明显。微生物在C元素循环过程中扮演中极为重要的作用，它们有极高的丰度和代谢多样性，几乎参与了C循环的所有环节。由微生物调控的土壤C循环过程可以固定或释放温室效应气体CO2，进而影响全球气候。研究微生物固定CO2的分子生态机理对于缓解全球气候变暖具有重要科学意义，同时也可以为农田土壤肥力科学化管理提供技术支撑，为正确评估农田土壤固碳潜力及低碳农业规划提供科学依据。至今已发现的微生物固定CO2途径有5条，即Calvin循环、还原性三羧酸循环（rTCA）、还原性乙酰辅酶A途径、3-羟基丙酸盐/苹果酰-辅酶A循环和4-羟基丁酸盐循环，其中Calvin循环途径是公认的主要的微生物固碳途径。在这些生化途径中，催化关键反应步骤的酶常被认为是关键酶，其对应的编码基因常用作marker基因，在微生物生态学研究中广泛应用于功能微生物的定量和系统进化分析。然而，综合文献我们发现，目前的研究多集中于水生生态系统和旱地，对于水稻土中的微生物固碳过程关注较少，而且对于除Calvin循环以外的其他固碳途径研究也比较缺乏。基于此，本研究利用酶活测定、实时定量PCR（qPCR）、末端限制性长度多态性分析（T-RFLP）、克隆和测序等多种生物学技术，收集我国南方五种典型水稻土，对其中通过Calvin循环进行自养固碳的微生物进行了原位研究；其次选取其中两种不同类型的水稻土进行室内模拟实验，采用根袋区分和收集根际与非根际土壤，进行氮肥处理，并在不同的水稻生长期采样，重点考察根际效应对微生物固碳过程的影响。结合土壤理化性质测定和统计分析，探究影响微生物固碳过程的关键环境因子及其贡献。论文主要的研究内容与成果如下： 1. 原位土壤研究对我国南方五种典型水稻土中通过Calvin循环进行自养固碳的微生物进行原位研究。利用酶活性测定、qPCR、克隆文库以及T-RFLP方法结合土壤理化性质分析研究发现：Calvin循环途径的3种marker基因中，cbbLG多样性最低，且序列多和硫杆菌属的一些细菌相似度较高；与此对应，cbbLR和cbbM基因具有很高的多样性，其序列大多独立成簇，不能归类到已知的细菌类群中。qPCR 结果显示3种基因丰度范围在在106-109拷贝数/克干土，并且cbbLR的丰度均高于cbbM和cbbLG，且其丰度与土壤固碳酶活性显著正相关，我们推测cbbLR在3种基因中可能发挥着更大的作用。与土壤理化性质进行统计分析显示粘粒含量、碳氮比、阳离子交换量（CEC）、pH和土壤有机碳（SOC）含量对微生物群落组成和marker基因丰度变异显著相关。 2. 微宇宙实验选取其中两种不同类型的水稻土进行室内模拟实验，采用根袋区分和收集根际与非根际土壤，进行氮肥处理，并在不同的水稻生长期采样，重点考察根际效应对微生物固碳过程的影响。利用qPCR揭示了3种自养固碳途径的marker基因在水稻不同生长阶段在根际与非根际土壤中的变化与多样性。基于偏冗余分析（pRDA）的变量分离研究显示生长期、土壤类型和根际效应与基因丰度变异均显著相关（P < 0.05），分别贡献了37%，19%和12%的解释量，而氮处理（尿素添加）则没有显著贡献（P ＞0.05）。结果表明不同环境因素对土壤固炭基因丰度变异影响顺序为：生长周期＞土壤类型＞根际效应。可以说，水稻土中固定CO2的自养微生物受多种环境因素及其相互作用共同影响。以上研究表明，水稻土具有很大的微生物固碳潜力。Calvin循环的3个marker基因中，cbbLR基因由于其高丰度可能比另外两个基因发挥更重要的作用，而环境因子如CEC、pH、粘粒含量、C/N和SOC等对基因多样性有显著影响。自养固碳途径的marker基因在水稻不同生长阶段在根际与非根际土壤中的变化与多样性。而生长周期和土壤类型对这些基因丰度的变异影响更大，而根际效应贡献相对较少。因此水稻土微生物固碳受多种环境因素及其相互作用共同影响。本研究部分揭示了水稻土中微生物固碳分子生态机理，对于理解微生物在全球气C循环过程中的作用具有一定科学意义，也可以为农田土壤肥力科学化管理和构建低碳农业提供科学依据。
英文摘要	Terrestrial ecosystem is the second largest carbon (C) pool on the earth. Agricultural land is an important part of terrestrial ecosystem, and therein C pool is the most active part under frequent human disturbance (tillage, fertilization, drainage, and irrigation), especially for paddy soils. Microorganisms play an important role in the C turnover, as they are involved in almost all processes of the carbon cycle due to their high abundance and diverse metabolic pathways. The turnover of C during soil biomass formation and decomposition mediated by microorganisms leads to the release or sequestration of greenhouse gas CO2, affecting global warming potentially. Therefore, research on this process is of great significance for mitigating global warming, and also for scientific fertilization in agriculture and establishing low-carbon agriculture. Over the past 60 years, five CO2 fixation pathways have been found successively: the Calvin cycle, the reductive tricarboxylic acid (rTCA) cycle, the reductive acetyl-CoA pathway, the 3-hydroxypropionate/malyl-CoA cycle and the 4- hydroxybutyrate cycle. Among them, the Calvin cycle is known to be the major pathway for microbial CO2 fixation. The enzymes catalyzing mechanistically difficult steps in a given pathway are usually conserved and act as key enzymes; the corresponding coding genes, often named as marker genes, are ubiquitously used in microbial ecological studies. However, to data, previous studies have emphasized either upland soils or aquatic environment, and also paid little attention to other pathways apart from the Calvin cycle. Therefore, in this study, clone library, T-RFLP, qPCR, and enzyme assay were used to investigate three marker genes involved in the Calvin cycle in five typical paddy soils in China. At the same time, microcosm experiment was conducted to explore the “rhizosphere effect” on autotrophic microorganisms. In combination with soil physiochemical analysis and statistical method, we aimed to find out the main environmental factor and their relative contribution. The major contents and findings of this thesis are as follows: 1. In situ experiments The diversity of three ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbLG, cbbLR and cbbM) was investigated by clone library, T-RFLP, qPCR, and enzyme assay in five paddy soils in China. The cbbLG sequences revealed a relatively low level of diversity and were mostly related to the sequences of species from Thiobacillus. In contrast, highly diverse cbbLR and cbbM sequences were dispersed on the phylogenetic trees, and most of them were distantly related to known sequences, even forming separate clusters. Abundances of three cbbL genes ranged from 106 to 109 copies g-1 soil, and cbbLR outnumbered cbbM and cbbLG in all soil samples, indicating that cbbLR may play a more important role than other two cbbL genes. Soil properties significantly influenced cbbL diversity in five paddy soils, of which clay content, C/N ratio, CEC, pH, and SOC correlated well with variations in microbial composition and abundance. 2. Microcosm experiments Microcosm experiments were conducted using two different types of paddy soil, planted with rice and added with urea. At tillering and ripening stages, bulk and rhizosphere soils were sampled separately for DNA extraction and qPCR analyses. Six marker genes (cbbLG, cbbLR, cbbM, aclB, oorA, accA) of three autotrophic pathways (the Calvin cycle, the reductive tricarboxylic acid cycle, and the 4-hydroxybutyrate cycle) were detected, suggesting their pervasiveness in paddy soil. Redundancy analysis and variation partitioning based on partial redundancy analysis revealed higher contributions of growth stage (explaining 37%) and soil type (explaining 19%) on the variation of these genes, lower but significant impact of rhizosphere effect (explaining 12%), and no significant effect of urea addition (P > 0.05). In summary, our results highlighted the potential of CO2 sequestration for paddy soils. The cbbLR gene may play a more important role in CO2 fixation than the other two cbbL genes in the paddy soil, while local edaphic factors, such as CEC, pH, clay content, C/N ratio, and SOC, significantly influenced gene diversity. Marker genes for three autotrophic pathways in paddy soils varied with time. Statistical analysis revealed higher contributions of growth stage and soil type on the variation of gene abundances and also lower but significant impact of rhizosphere. It can be concluded that the CO2 fixation autotrophic microorganisms in the paddy soil were affected by complex interactions environmental factors. Our study revealed processes of microbial carbon dioxide fixation in paddy soil, which is important to our understanding of the role of microorganism in global carbon cycle, and also provides scientific basis for scientific fertilization in agriculture and establishing low-carbon agriculture.
公开日期	2015-06-11
内容类型	学位论文
源URL	[http://ir.rcees.ac.cn/handle/311016/13444]
专题	生态环境研究中心_土壤环境科学实验室
推荐引用方式 GB/T 7714	肖可青. 典型水稻土中固碳基因及功能微生物研究[D]. 北京. 中国科学院研究生院. 2014.