免耕稻田不同剖面层土壤中的二氧化碳固定自养微生物多样性、固定通量与影响因素

Autotrophic microbes are distributed widely in the nature，and have five pathways to fix CO2, so they have a huge potential for biological carbon fixation. This project selects no-tillage paddy soils in southern China as typical study area, and the CO2-fixation autotrophic microbial assamblage as the research object. The total DNAs of soil samples will be recovered by direct extract method. Then microbial gene libraries of cbbL、cbbM、aclB and coxL encoding key enzymes for CO2 fixation in different paddy soil profiles under non-tillage condition will be constructed. Clones of above libraries will be analyzed by PCR-RFLP method, and the functional genes of some typical clones will be sequenced and blast to study the phylogenetic characteristics of CO2-fixation microbes and elucidate the main groups of CO2-fixing autotrophic organisms in the paddy soil. Real-time quantitative PCR technology will be also used to determine the functional gene copies of CO2-fixation organisms in the paddy soil. Furthermore, stable isotope-coded affinity tag approach will be adopted to track the behavior of CO2 assimilation by soil microorganisms, so the the CO2-fixing rate can be calculated. Combined with the soil physical、chemical and biological properties in different profiles of no-tillage rice fields, major influential factors for CO2-fixation organisms will be revealed using multivariate analysis (PCA, CCA et al.). The result will promote the understanding on CO2 fixation by autotrophic microorganisms, and provide a new insight for reduction of CO2 emission in paddy soil environment.

自养微生物在自然界中广泛分布，其固定CO2有五条途径，有巨大的生物固碳潜力。本项目选择南方免耕稻田为典型研究区域，以CO2固定自养微生物类群为研究对象，通过提取不同剖面土壤微生物总DNA构建CO2固定的关键酶基因cbbL、cbbM、aclB和coxL文库，并对文库中的克隆进行酶切分型；通过功能基因序列测定与比对，研究其系统发育特征，阐明稻田土壤CO2固定自养微生物的主要类群；采用实时定量PCR技术，研究稻田土壤CO2固定微生物的数量与剖面分布；采用13C稳定同位素标记技术，示踪土壤中CO2同化过程，明确土壤自养微生物固定CO2通量；结合对免耕稻田不同土壤剖面物理、化学和生物学性质的系统测定，通过多元分析方法（PCA、CCA等），揭示影响CO2固定微生物的主要因素。预期结果将促进对稻田土壤CO2固定的深入理解，为稻田土壤CO2减排提供一条新的思路。

采集了皖南农田5种土壤的3个剖面层土壤样品，采用qPCR和PCR-RFLP方法研究了固碳微生物功能基因拷贝数和多样性。结果表明，固碳微生物cbbM基因拷贝数介于0.62×108 ~9.80×108 拷贝·g-1干土，cbbL green-like基因拷贝数从0.97×107~2.10×107 拷贝·g-1干土，cbbL red-like基因拷贝数5.84×109~10.50×109 拷贝·g-1干土，coxL基因在不同剖面土壤中的数量从0.42×108~5.79×108拷贝g-1；固碳自养微生物功能基因丰度指数随土壤剖面深度而降低，Thiobacillus、Sulfuritalea、Rhodopseudomonas、Magnetospirillum、Rubrivivax、Methulibium、Rhizobium、Alcaligenes、Bradyrhizobium、Burkhllderia、Bradyrhizobium为优势菌属。.采用稳定性同位素核酸探针(DNA-SIP)技术和微宇宙模拟的方法，对两种免耕水稻土中的固定CO2自养微生物多样性进行了研究。培养10周后，土壤13C-SOC含量范围为70.88～350.15mg·kg-1，土壤固碳速率为0.10～0.49μg13Cg-1d-1；Bradyrhizobium和Rubrivivax是为FG土壤的主要类群，而Rhodopseudomonas、Rhodospirillum、Methylibium和Variovorax是TF土壤的主要类群，化能自养型固碳微生物是免耕水稻土中的优势微生物类群；两种13CO2标记土样的cbbLR基因文库OTU类型、多样性指数均高于12CO2对照文库，表明固碳微生物对大气CO2有的一定负反馈调节作用。因此，免耕水稻土存在高度多样性的二氧化碳固定自养微生物，在农田土壤碳素循环方面具有重要作用。