碳穩(wěn)定性同位素示蹤免耕土壤微生物多樣性研究
發(fā)布時間：2019-01-28 20:21
【摘要】：溫室氣體的排放導(dǎo)致全球變暖和海平面上升,引起了人們的廣泛關(guān)注。土壤是溫室氣體的主要排放源之一,大氣中約有5%-20%的CO_2來自土壤,自養(yǎng)微生物有極強的環(huán)境適應(yīng)性,在二氧化碳固定方面有很大潛力,但免耕耕作對水稻土CO_2固定自養(yǎng)微生物數(shù)量和種類的影響仍不清楚。本實驗選擇南方免耕稻田為典型研究區(qū)域,以CO_2固定自養(yǎng)微生物類群為研究對象,采用13C穩(wěn)定同位素標記技術(shù),示蹤土壤中CO_2同化過程,明確土壤自養(yǎng)微生物固定CO_2通量;采用穩(wěn)定性同位素核酸探針(DNA-SIP)技術(shù),結(jié)合PCR-RFLP方法和高通量測序技術(shù),研究了南方免耕水稻土中CO_2固定自養(yǎng)微生物的多樣性。結(jié)果表明,經(jīng)過10周的連續(xù)培養(yǎng),實驗土壤13C-SOC含量范圍為70.88～350.15mg.kg-1,其中 FG-PL 為 350.15 mg.kg-1,FG-PP 為 133.05 mg kg-1 FG-PH 為70.88mg·kg-1 TF-PL 為 328.76mg kg-1 TF-PP 為 171.62 mg kg-1 TF-PH 為 92.01 mg·kg-1�？梢姴煌拭嫱寥牢⑸锕潭–O_2通量差異顯著,表現(xiàn)為PLPPPH。兩種土壤13C-C02固定速率為0.10 0.49μg13Cg-1d-1～其中 FG-PL 為0.49μg13Cg-1d-1,FG-PP為0.19μg13Cg-1d-1,FG-PH為0.10μg13Cg-1d-1,TF-PL為0.46μg13Cg-1d-1,TF-PP 為 0.23μg13Cg-1d-1,TF-PH 為0.13μg13Cg-1d-1。與以往的研究結(jié)果比較發(fā)現(xiàn),添加較高濃度的C02能夠提高免耕水稻土中二氧化碳固定自養(yǎng)微生物的多樣性,表明在CO_2升高環(huán)境條件下土壤具有一定的調(diào)節(jié)功能,有減緩CO_2溫室氣體效應(yīng)的潛力。熒光實時定量PCR定量結(jié)果顯示,鐵滲潛育水耕人為土 FG和普通鐵滲水耕人為土TF經(jīng)13CO_2標記處理80d后,密度梯度離心分離的15層DNA樣品中的cbbLR基因第8層最高,分別為1.36×1O5拷貝g-1干土和2.21×1O5拷貝.g-1干土。高通量測序結(jié)果表明,Proteobacteria(42.4%)為土 壤微生物的優(yōu)勢門,Betaproteobacteria(25.20%)和A1phaproteobacteria(10.00%)為土 壤微生物的優(yōu)勢綱,Burkho1deria1es(20.60%)Rhizobia1es(5.10%)、Rhodospiri11a1es(3.00%)為土 壤微生物的優(yōu)勢目,Burkho1deriaceae(20.1%)和 Rhodospiri11aceae(2.40%)為土壤微生物的優(yōu)勢科。以分離的7、8層DNA為模板,構(gòu)建了包括兩種土壤13CO_2標記和12CO_2對照的cbbLR基因文庫,8個基因文庫中672個克隆經(jīng)酶切后分為30個OTUs類型,分布在5個目、9個科和12個已知屬。土壤FG以Bradyrhizobium和Rubrivivax為主要類群,占全部克隆數(shù)的60.40%和 13.86%,土壤TF中的微生物種屬分布比較均勻,Rhodopseudomonas、Rhodospirillum、Methylibium和Variovorax分別占全部克隆數(shù)的20.90%、11.94%、16.42%和10.45%。兩種土壤13CO_2標記的cbbLR基因文庫OTU類型、多樣性指數(shù)均高于12CO_2對照文庫,其群落結(jié)構(gòu)也有明顯的變化。因此,免耕水稻土存在高度多樣性的二氧化碳固定自養(yǎng)微生物,在農(nóng)田土壤碳素循環(huán)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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S154.3
本文編號：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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S154.3
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