本文選題：氨氧化微生物　切入點：甲烷氧化菌　出處：《西南大學》2017年碩士論文

【摘要】：氨氧化微生物(AOA與AOB)是主導硝化反應限速步驟的微生物,甲烷氧化菌(MOB)主導土壤中CH4的氧化反應限速步驟的微生物,研究發(fā)現(xiàn)甲烷氧化菌和氨氧化微生物擁有同源的甲烷單加氧酶(pmoA)和氨單加氧酶(amoA),甲烷單加氧酶可作為氨單氧化酶參與反應。另外,兩種酶的氧化底物CH4和NH4+的結構相似,兩種底物與酶之間的反應可能存在競爭。但尚未有充分證據(jù)證實甲烷氧化菌(MOB)與兩種氨氧化微生物(AOA與AOB)之間是否存在相互作用。淹水稻田在我國西南丘陵地區(qū)分布廣泛,淹水土壤表層是一層水層,一般3~5cm,下面是薄薄的氧化層,厚度不到1cm,再下面是還原層,厚度一般為10~20cm,這種垂直深度分層導致土壤Eh值、pH值、溫度、水分和養(yǎng)分等的差異。這些差異將對土壤中的微生物生態(tài)位產生一定影響。研究淹水稻田中垂直深度分層的氨氧化微生物和甲烷氧化菌的分布規(guī)律,有利于了解土壤中碳氮循環(huán)的微生物作用機制,為提高農田土壤氮肥利用率、減緩土壤溫室氣體排放提供科學依據(jù)。本研究采用普通PCR、實時熒光定量PCR(qPCR)、克隆測序等分子生物學技術,深入探究分析菌群關鍵功能基因的豐度及其多樣性,厘清氨氧化微生物和甲烷氧化菌的的垂直空間生態(tài)位。本論文的主要研究內容和結論如下:本實驗選取的三個采樣區(qū)均屬于中性紫色水稻土區(qū),分別位于重慶市北碚區(qū)西南大學試驗田、合川區(qū)大石鎮(zhèn)高馬村八社丘陵寬谷區(qū)、沙坪壩區(qū)青木關鎮(zhèn)鳳凰鎮(zhèn)金塘三社低山地貌區(qū)。分別采集淹水層(0~3 cm)、氧化層(3~5 cm)、還原層(5~20 cm)土壤樣品。通過室內培養(yǎng)、定量PCR技術及克隆測序,研究淹水稻田分異土層的淹水層、氧化層、還原層中氨氧化微生物和甲烷氧化菌的活性及群落結構。研究結果表明:(1)以硝化勢表征土壤氨氧化微生物的活性,檢查三種淹水稻田不同土壤層次的氨氧化微生物活性,結果發(fā)現(xiàn),3種淹水稻田中硝化勢大小表現(xiàn)為:沙坪壩淹水稻田北碚淹水稻田合川淹水稻田,不同淹水稻田均檢測出氧化層(3~5 cm)的硝化勢顯著大于淹水層(0~3 cm)和還原層(5~20 cm),說明3~5cm土層的土壤環(huán)境更適宜于氨氧化作用的發(fā)生。并且不同淹水稻田及其不同土層中的硝化勢與土層總氮濃度呈正相關;(2)以平均凈氧化速率表征淹水稻田的甲烷氧化能力,結果發(fā)現(xiàn),甲烷氧化能力沙坪壩淹水稻田合川淹水稻田北碚淹水稻田,其中沙坪壩與合川淹水稻田中不同土層的凈甲烷氧化速率差異的趨勢一致:還原層5~20 cm顯著低于其他淹水層與氧化層,說明淹水稻田甲烷氧化主要由淹水層與氧化層完成。(3)通過定量PCR和基因測序,并對目的基因進行聚類分析,結果發(fā)現(xiàn)3種淹水稻田的AOB amoA豐度及多樣性(AOB amoA絕大部分屬于Nitrosomonas屬)顯著小于AOA及MOB;AOA amoA種群類別差異不大,但種群分布隨著土層深度的增加,Group1.1b類逐漸減少而Group 1.1a類逐漸增加;MOB pmoA多樣性遠大于amoA基因,兩種淹水稻田Type I型甲烷氧化菌分屬于Methylococcacea type I methanotroph屬(Type I a)與Methylocaldum屬(Type I b),Type II型甲烷氧化菌則為Methylocystis屬。隨著土層深度的增加,Type I型甲烷氧化菌在淹水層及氧化層富集,Type II型甲烷氧化菌在還原層富集。綜上所述,對氨及甲烷氧化微生物在3種淹水稻田的分布規(guī)律可歸結為以下兩點:(1)氨氧化與甲烷氧化作用在淹水稻田不同土層中發(fā)生具有規(guī)律性;(2)氨氧化與甲烷微生物在淹水稻田不同土層中分布有一定規(guī)律。
[Abstract]:Ammonia oxidizing microorganisms (AOA and AOB) is the rate limiting step of nitrification of dominant microorganisms, methane oxidizing bacteria (MOB) oxidation reaction rate limiting step leading CH4 in soil microorganisms, the study found that methane oxidizing bacteria and ammonia oxidizing microorganisms have homologous methane monooxygenase (pmoA) and ammonia monooxygenase (amoA) methane monooxygenase, as ammonia monooxygenase reaction. In addition, the structure of two kinds of enzyme substrate oxidation CH4 and NH4+ were similar between the two substrate and enzyme reaction may compete. But there is no sufficient evidence to confirm that the methane oxidizing bacteria (MOB) and two kinds of ammonia oxidizing microorganisms (AOA and AOB) the interaction between paddy field. Widely distributed in southwest hilly area of our country, flooding the soil surface is a layer of water, generally 3~5cm, below is a thin oxide layer, thickness is less than 1cm, then the following is the reduction of layer thickness is 10~20cm, the vertical In depth stratification of soil Eh value, pH value, temperature, moisture and nutrient differences. These differences will have a certain impact on soil microbial ecological niche. The distribution law of flooded paddy fields in vertical depth stratification of ammonia oxidizing microorganisms and methane oxidizing bacteria, is conducive to understanding the mechanism of action of microbial carbon and nitrogen cycling in soil in order to improve the utilization of farmland, soil nitrogen, reduce greenhouse gas emissions and provide a scientific basis. This study uses ordinary PCR and real-time fluorescence quantitative PCR (qPCR), cloning, sequencing and other molecular biology techniques, explore in depth analysis of flora abundance and diversity of key functional genes, clarify the ammonia oxidizing microorganisms and methane oxidizing bacteria vertical ecological space the bit. The main research contents and conclusions are as follows: three the sampling area in the experiments are neutral purple soil area, located in Beibei District of Chongqing City Southwestern University experimental field, high Macun eight hilly gully area Dashi Town, Hechuan District, Shapingba Qingmuguan Jintang town Fenghuang town three agency low mountain landform area. Flooding layer were collected (0~3 cm), oxide layer (3~5 cm) (5~20 cm), the reduced layer soil samples. Through indoor culture, quantitative PCR technology cloning and sequencing of waterlogging layer, paddy soil on differentiation of oxidation layer, activity and community structure in ammonia oxidizing microorganisms and methane oxidizing bacteria reduction. Research results show that: (1) to characterize the nitrification potential of soil ammonia oxidizing microbial activity, check three kinds of paddy in different soil layers of ammonia oxidation microbial activity the results showed that, 3 kinds of paddy field in Shapingba as follows: the nitrification potential size of paddy field in Beibei paddy field in Hechuan paddy field, different paddy field were detected in the oxide layer (3~5 cm) the nitrification potential significantly greater than flooding layer (0~3 cm) and reduction layer (5~ 20 cm), indicating that the soil environment in 3~5cm soil layer is more suitable for the ammonia oxidation occurs. Different flooding and total nitrogen nitrification potential and soil and paddy soil layer in different concentrations were positively related; (2) the average net rate of oxidation characterization flooding methane oxidation capacity of paddy field, the results show that the methane oxidation capacity of paddy field in Shapingba Hechuan Beibei flooded paddy paddy field, including Shapingba and Hechuan flooded net methane oxidation rates of different soil paddy fields in the same trend: the reduction of 5~20 cm layer was significantly lower than other flooding layer and oxide layer, that is mainly composed of methane oxidation in paddy flooding layer and oxide layer. (3) by quantitative PCR and gene sequencing, and for the purpose of gene cluster analysis results showed that 3 kinds of paddy field AOB amoA abundance and diversity (AOB amoA most belong to the genus Nitrosomonas) was significantly lower than that of AOA and MOB; AOA amoA Group class difference is not big, but the population distribution with the increase of soil depth, the Group1.1b class and Group class 1.1a decreased gradually increased; the diversity of MOB pmoA is much larger than the amoA gene, two kinds of paddy field Type type I methanotrophs belonging to Methylococcacea type I methanotroph (Type I a) and Methylocaldum (Type I b) Type, II type methane oxidizing bacteria is Methylocystis. With the increase of soil depth, Type type I methanotrophs in the flooded layer and the oxide layer enrichment, Type type II methanotrophs in the reduced layer enrichment. In summary, the ammonia and methane oxidizing microorganisms in 3 kinds of distribution of paddy field can be attributed to the following two points: (1) ammonia oxidation and methane oxidation in paddy soil with different regularity; (2) the distribution of ammonia oxidizing microorganisms and methane have certain rules in paddy field in different soil layers.

【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S154.3

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