碳稳定性同位素示踪免耕土壤微生物多样性研究
发布时间：2019-01-28 20:21
【摘要】：温室气体的排放导致全球变暖和海平面上升,引起了人们的广泛关注。土壤是温室气体的主要排放源之一,大气中约有5%-20%的CO_2来自土壤,自养微生物有极强的环境适应性,在二氧化碳固定方面有很大潜力,但免耕耕作对水稻土CO_2固定自养微生物数量和种类的影响仍不清楚。本实验选择南方免耕稻田为典型研究区域,以CO_2固定自养微生物类群为研究对象,采用13C稳定同位素标记技术,示踪土壤中CO_2同化过程,明确土壤自养微生物固定CO_2通量;采用稳定性同位素核酸探针(DNA-SIP)技术,结合PCR-RFLP方法和高通量测序技术,研究了南方免耕水稻土中CO_2固定自养微生物的多样性。结果表明,经过10周的连续培养,实验土壤13C-SOC含量范围为70.88～350.15mg.kg-1,其中 FG-PL 为 350.15 mg.kg-1,FG-PP 为 133.05 mg kg-1 FG-PH 为70.88mg·kg-1 TF-PL 为 328.76mg kg-1 TF-PP 为 171.62 mg kg-1 TF-PH 为 92.01 mg·kg-1。可见不同剖面土壤微生物固定CO_2通量差异显著,表现为PLPPPH。两种土壤13C-C02固定速率为0.10 0.49μg13Cg-1d-1～其中 FG-PL 为0.49μg13Cg-1d-1,FG-PP为0.19μg13Cg-1d-1,FG-PH为0.10μg13Cg-1d-1,TF-PL为0.46μg13Cg-1d-1,TF-PP 为 0.23μg13Cg-1d-1,TF-PH 为0.13μg13Cg-1d-1。与以往的研究结果比较发现,添加较高浓度的C02能够提高免耕水稻土中二氧化碳固定自养微生物的多样性,表明在CO_2升高环境条件下土壤具有一定的调节功能,有减缓CO_2温室气体效应的潜力。荧光实时定量PCR定量结果显示,铁渗潜育水耕人为土 FG和普通铁渗水耕人为土TF经13CO_2标记处理80d后,密度梯度离心分离的15层DNA样品中的cbbLR基因第8层最高,分别为1.36×1O5拷贝g-1干土和2.21×1O5拷贝.g-1干土。高通量测序结果表明,Proteobacteria(42.4%)为土 壤微生物的优势门,Betaproteobacteria(25.20%)和A1phaproteobacteria(10.00%)为土 壤微生物的优势纲,Burkho1deria1es(20.60%)Rhizobia1es(5.10%)、Rhodospiri11a1es(3.00%)为土 壤微生物的优势目,Burkho1deriaceae(20.1%)和 Rhodospiri11aceae(2.40%)为土壤微生物的优势科。以分离的7、8层DNA为模板,构建了包括两种土壤13CO_2标记和12CO_2对照的cbbLR基因文库,8个基因文库中672个克隆经酶切后分为30个OTUs类型,分布在5个目、9个科和12个已知属。土壤FG以Bradyrhizobium和Rubrivivax为主要类群,占全部克隆数的60.40%和 13.86%,土壤TF中的微生物种属分布比较均匀,Rhodopseudomonas、Rhodospirillum、Methylibium和Variovorax分别占全部克隆数的20.90%、11.94%、16.42%和10.45%。两种土壤13CO_2标记的cbbLR基因文库OTU类型、多样性指数均高于12CO_2对照文库,其群落结构也有明显的变化。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。
[Abstract]:Greenhouse gas emissions have caused global warming and sea-level rise, causing widespread concern. Soil is one of the main sources of greenhouse gas emissions. About 5- 20% of CO_2 in the atmosphere comes from soil. Autotrophic microorganisms have strong environmental adaptability and have great potential in carbon dioxide fixation. However, the effect of no-tillage on the number and species of CO_2 fixed autotrophic microorganisms in paddy soil is still unclear. In this experiment, the southern no-tillage paddy field was selected as the typical research area, and the CO_2 fixed autotrophic microorganism group was used as the research object. The 13C stable isotope labeling technique was used to trace the CO_2 assimilation process in the soil. The fixed CO_2 flux of soil autotrophic microorganisms was determined. The diversity of autotrophic microbes fixed by CO_2 in no-tillage paddy soils in southern China was studied by using stable isotope nucleic acid probe (DNA-SIP) technique combined with PCR-RFLP method and high-throughput sequencing technique. The results showed that, after 10 weeks of continuous culture, the range of 13C-SOC content in soil was 70.88 ~ 350.15 mg 路kg ~ (-1), and FG-PL was 350.15 mg.kg-1,. FG-PP is 133.05 mg kg-1 FG-PH, 70.88mg kg-1 TF-PL is 328.76mg kg-1 TF-PP, 171.62 mg kg-1 TF-PH is 92.01 mg kg-1.. It can be seen that the fixed CO_2 fluxes of soil microorganisms in different profiles are significantly different, showing that PLPPPH.. The 13C-C02 fixation rate of the two soils was 0.100.49 渭 g 13Cg-1d-1d-1 ~ where FG-PL was 0.49 渭 g 13Cg-1d-1FG-PP was 0.19 渭 g 13Cg-1d-1FG-PH was 0.10 渭 g 13Cg-1d-1d -1 TF-PL was 0.46 渭 g 13Cg-1d-1d-1, The TF-PP was 0.23 渭 g 13Cg-1d-1d ~ (-1) TF-PH was 0.13 渭 g ~ (13) Cg-1d-1d ~ (-1). Compared with the previous studies, it was found that the diversity of CO2 autotrophic microorganisms in no-tillage paddy soil could be improved by adding higher concentration of CO2, which indicated that the soil had a certain regulating function under the condition of elevated CO_2. It has the potential to slow down the greenhouse gas effect of CO_2. The quantitative results of fluorescence real time quantitative PCR showed that the cbbLR gene was the highest in the 15 DNA samples separated by gradient centrifugation after 80 days of treatment with 13CO_2 marker for FG and TF in ferric osmotic soil and common iron permeable soil, and the highest level of cbbLR gene was found in the 15 layers of DNA samples separated by density gradient centrifugation. It was 1.36 脳 1O5 copy g-1 dry soil and 2.21 脳 1O5 copy g-1 dry soil, respectively. The results of high throughput sequencing showed that, Proteobacteria (42.4%) was the dominant, Betaproteobacteria (of soil microbes (25.20%) and A1phaproteobacteria (10.00%) was the dominant class of soil microbes, Burkho1deria1es (20.60%) Rhizobia1es (5.10%). Rhodospiri11a1es (3.00%) is the dominant order of soil microbes, Burkho1deriaceae (20.1%) and Rhodospiri11aceae (2.40%) are the dominant families of soil microbes. A cbbLR gene library consisting of two kinds of soil 13CO_2 markers and 12CO_2 control was constructed using the isolated 7G / 8 DNA as template. 672 clones of the 8 gene libraries were divided into 30 OTUs types and distributed in 5 orders after restriction endonuclease digestion. 9 families and 12 known genera. Soil FG was mainly composed of Bradyrhizobium and Rubrivivax, accounting for 60.40% and 13.86% of the total clones. The distribution of microbial species in soil TF was more uniform, and Rhodopseudomonas,Rhodospirillum,Methylibium and Variovorax accounted for 20.90% and 11.94% of the total clones, respectively. 16.42% and 10.45%. The diversity index of cbbLR gene library of two soil 13CO_2 markers was higher than that of 12CO_2 control library, and the community structure was also significantly changed. Therefore, there is a high diversity of carbon dioxide autotrophic microorganisms in no-tillage paddy soil, which plays an important role in carbon cycling of farmland soil.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S154.3
本文编号：2417286
[Abstract]:Greenhouse gas emissions have caused global warming and sea-level rise, causing widespread concern. Soil is one of the main sources of greenhouse gas emissions. About 5- 20% of CO_2 in the atmosphere comes from soil. Autotrophic microorganisms have strong environmental adaptability and have great potential in carbon dioxide fixation. However, the effect of no-tillage on the number and species of CO_2 fixed autotrophic microorganisms in paddy soil is still unclear. In this experiment, the southern no-tillage paddy field was selected as the typical research area, and the CO_2 fixed autotrophic microorganism group was used as the research object. The 13C stable isotope labeling technique was used to trace the CO_2 assimilation process in the soil. The fixed CO_2 flux of soil autotrophic microorganisms was determined. The diversity of autotrophic microbes fixed by CO_2 in no-tillage paddy soils in southern China was studied by using stable isotope nucleic acid probe (DNA-SIP) technique combined with PCR-RFLP method and high-throughput sequencing technique. The results showed that, after 10 weeks of continuous culture, the range of 13C-SOC content in soil was 70.88 ~ 350.15 mg 路kg ~ (-1), and FG-PL was 350.15 mg.kg-1,. FG-PP is 133.05 mg kg-1 FG-PH, 70.88mg kg-1 TF-PL is 328.76mg kg-1 TF-PP, 171.62 mg kg-1 TF-PH is 92.01 mg kg-1.. It can be seen that the fixed CO_2 fluxes of soil microorganisms in different profiles are significantly different, showing that PLPPPH.. The 13C-C02 fixation rate of the two soils was 0.100.49 渭 g 13Cg-1d-1d-1 ~ where FG-PL was 0.49 渭 g 13Cg-1d-1FG-PP was 0.19 渭 g 13Cg-1d-1FG-PH was 0.10 渭 g 13Cg-1d-1d -1 TF-PL was 0.46 渭 g 13Cg-1d-1d-1, The TF-PP was 0.23 渭 g 13Cg-1d-1d ~ (-1) TF-PH was 0.13 渭 g ~ (13) Cg-1d-1d ~ (-1). Compared with the previous studies, it was found that the diversity of CO2 autotrophic microorganisms in no-tillage paddy soil could be improved by adding higher concentration of CO2, which indicated that the soil had a certain regulating function under the condition of elevated CO_2. It has the potential to slow down the greenhouse gas effect of CO_2. The quantitative results of fluorescence real time quantitative PCR showed that the cbbLR gene was the highest in the 15 DNA samples separated by gradient centrifugation after 80 days of treatment with 13CO_2 marker for FG and TF in ferric osmotic soil and common iron permeable soil, and the highest level of cbbLR gene was found in the 15 layers of DNA samples separated by density gradient centrifugation. It was 1.36 脳 1O5 copy g-1 dry soil and 2.21 脳 1O5 copy g-1 dry soil, respectively. The results of high throughput sequencing showed that, Proteobacteria (42.4%) was the dominant, Betaproteobacteria (of soil microbes (25.20%) and A1phaproteobacteria (10.00%) was the dominant class of soil microbes, Burkho1deria1es (20.60%) Rhizobia1es (5.10%). Rhodospiri11a1es (3.00%) is the dominant order of soil microbes, Burkho1deriaceae (20.1%) and Rhodospiri11aceae (2.40%) are the dominant families of soil microbes. A cbbLR gene library consisting of two kinds of soil 13CO_2 markers and 12CO_2 control was constructed using the isolated 7G / 8 DNA as template. 672 clones of the 8 gene libraries were divided into 30 OTUs types and distributed in 5 orders after restriction endonuclease digestion. 9 families and 12 known genera. Soil FG was mainly composed of Bradyrhizobium and Rubrivivax, accounting for 60.40% and 13.86% of the total clones. The distribution of microbial species in soil TF was more uniform, and Rhodopseudomonas,Rhodospirillum,Methylibium and Variovorax accounted for 20.90% and 11.94% of the total clones, respectively. 16.42% and 10.45%. The diversity index of cbbLR gene library of two soil 13CO_2 markers was higher than that of 12CO_2 control library, and the community structure was also significantly changed. Therefore, there is a high diversity of carbon dioxide autotrophic microorganisms in no-tillage paddy soil, which plays an important role in carbon cycling of farmland soil.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S154.3
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