氨及甲烷氧化微生物在淹水稻田不同土层中的分布规律

发布时间：2018-03-25 09:52

本文选题：氨氧化微生物　切入点：甲烷氧化菌　出处：《西南大学》2017年硕士论文

【摘要】：氨氧化微生物(AOA与AOB)是主导硝化反应限速步骤的微生物,甲烷氧化菌(MOB)主导土壤中CH4的氧化反应限速步骤的微生物,研究发现甲烷氧化菌和氨氧化微生物拥有同源的甲烷单加氧酶(pmoA)和氨单加氧酶(amoA),甲烷单加氧酶可作为氨单氧化酶参与反应。另外,两种酶的氧化底物CH4和NH4+的结构相似,两种底物与酶之间的反应可能存在竞争。但尚未有充分证据证实甲烷氧化菌(MOB)与两种氨氧化微生物(AOA与AOB)之间是否存在相互作用。淹水稻田在我国西南丘陵地区分布广泛,淹水土壤表层是一层水层,一般3~5cm,下面是薄薄的氧化层,厚度不到1cm,再下面是还原层,厚度一般为10~20cm,这种垂直深度分层导致土壤Eh值、pH值、温度、水分和养分等的差异。这些差异将对土壤中的微生物生态位产生一定影响。研究淹水稻田中垂直深度分层的氨氧化微生物和甲烷氧化菌的分布规律,有利于了解土壤中碳氮循环的微生物作用机制,为提高农田土壤氮肥利用率、减缓土壤温室气体排放提供科学依据。本研究采用普通PCR、实时荧光定量PCR(qPCR)、克隆测序等分子生物学技术,深入探究分析菌群关键功能基因的丰度及其多样性,厘清氨氧化微生物和甲烷氧化菌的的垂直空间生态位。本论文的主要研究内容和结论如下:本实验选取的三个采样区均属于中性紫色水稻土区,分别位于重庆市北碚区西南大学试验田、合川区大石镇高马村八社丘陵宽谷区、沙坪坝区青木关镇凤凰镇金塘三社低山地貌区。分别采集淹水层(0~3 cm)、氧化层(3~5 cm)、还原层(5~20 cm)土壤样品。通过室内培养、定量PCR技术及克隆测序,研究淹水稻田分异土层的淹水层、氧化层、还原层中氨氧化微生物和甲烷氧化菌的活性及群落结构。研究结果表明:(1)以硝化势表征土壤氨氧化微生物的活性,检查三种淹水稻田不同土壤层次的氨氧化微生物活性,结果发现,3种淹水稻田中硝化势大小表现为:沙坪坝淹水稻田北碚淹水稻田合川淹水稻田,不同淹水稻田均检测出氧化层(3~5 cm)的硝化势显著大于淹水层(0~3 cm)和还原层(5~20 cm),说明3~5cm土层的土壤环境更适宜于氨氧化作用的发生。并且不同淹水稻田及其不同土层中的硝化势与土层总氮浓度呈正相关;(2)以平均净氧化速率表征淹水稻田的甲烷氧化能力,结果发现,甲烷氧化能力沙坪坝淹水稻田合川淹水稻田北碚淹水稻田,其中沙坪坝与合川淹水稻田中不同土层的净甲烷氧化速率差异的趋势一致:还原层5~20 cm显著低于其他淹水层与氧化层,说明淹水稻田甲烷氧化主要由淹水层与氧化层完成。(3)通过定量PCR和基因测序,并对目的基因进行聚类分析,结果发现3种淹水稻田的AOB amoA丰度及多样性(AOB amoA绝大部分属于Nitrosomonas属)显著小于AOA及MOB;AOA amoA种群类别差异不大,但种群分布随着土层深度的增加,Group1.1b类逐渐减少而Group 1.1a类逐渐增加;MOB pmoA多样性远大于amoA基因,两种淹水稻田Type I型甲烷氧化菌分属于Methylococcacea type I methanotroph属(Type I a)与Methylocaldum属(Type I b),Type II型甲烷氧化菌则为Methylocystis属。随着土层深度的增加,Type I型甲烷氧化菌在淹水层及氧化层富集,Type II型甲烷氧化菌在还原层富集。综上所述,对氨及甲烷氧化微生物在3种淹水稻田的分布规律可归结为以下两点:(1)氨氧化与甲烷氧化作用在淹水稻田不同土层中发生具有规律性;(2)氨氧化与甲烷微生物在淹水稻田不同土层中分布有一定规律。
[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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S154.3

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