【作者】 尚书；

【导师】 马晓军；

【作者基本信息】 兰州大学 ， 微生物学， 2012， 硕士

【摘要】 由于氮的可利用性往往会成为限制生态系统初级生产的因素,因此氮循环-直是整个生态学研究的热点。随着近年来分子生物学的发展,微生物生态学家研究发现氮循环与微生物有着非常密切的关系。氨氧化菌和反硝化菌所参与的硝化和反硝化作用是全球氮循环的重要步奏,因此受到了广泛关注。覆膜技术能有效地提高我国半干旱区的作物产量而被大面积推广。然而长期覆膜会改变农田生态系统表层土壤的含水量、温度以及土壤养分,特别是土壤中的N素；有可能直接影响生产的可持续性。本文采用构建克隆文库和实时荧光定量PCR的方法,研究了长期覆膜处理的黄土高原半干旱区农田生态系统中的氨氧化微生物和反硝化微生物的群落结构多样性、组成及丰度变化。实验设置3个处理：对照不覆膜处理(non-mulching, CK)、半覆膜处理(half-mulching, HM)和全膜处理(full-mulching, FM)。地膜于播种时覆盖,待来年播种时更换。得到以下结果：一、不同覆膜处理氨氧化微生物群落结构多样性1、构建amoA基因文库结果显示,不同覆膜处理的氨氧化细菌(AOB)的群落结构有较大差异。1)AOB群落由β-Proteobacteria的Nitrosomonas-like序列和Nitrosospira-Like序列组成。6个Cluster,分别为Cluster3a.1、Cluster3a.2、 Cluster3b、Cluster6、Cluster2-related和Cluster11/12;其中Cluster3a.2和Cluster3b为优势类群。2)不同覆膜处理AOB的群落组成有差异,Cluster6随着不同覆膜处理(FM, HM到CK)有增加的趋势,并且CK中所占的比例显著地高于其他两个处理。而Cluster3却随着覆膜处理(FM、HM到CK)有明显减少的趋势。3)不同处理样品中AOB的群落组成在OTUs水平上也有较大变化。AOB-CK1-101在FM中未能检测到,在HM中占较低比例,而在CK中占比例较高,达到12.31%；AOB-CK3-78在FM中有较高的丰度(5.51%)而在HM(0)和CK(0.77%)中几乎检测不到。2、不同覆膜处理下氨氧化古菌(AOA)的群落结构没有显著变化。1)442条古菌amoA基因序列均属于soil and sediment,分为Soil and Sediment1和Soil and Sediment2两个Cluster,其中Soil and Sediment1所占比例在30%-58%之间。2)在OTUs水平上AOA的群落结构没有显著性的变化。AOA-FM2-8, AOA-HM1-19和AOA-FM3-16为优势的OTU。3、不同覆膜处理的AOB和AOA amoA基因荧光定量结果PCR显示,AOB amoA基因的拷贝数高于AOA, AOA的拷贝数在1.16×107～3.12×107copies之间,AOB拷贝数在5.66×107-1.64×108copies之间。除10月份外,不同覆膜处理下(FM, HM到CK) AOB amoA基因拷贝数呈逐渐增加的趋势,且6月份,CK处理AOB拷贝数显著的高于FM处理。在不同季节AOA的拷贝数不同,处理间变化不明显。4、群落结构和环境因子相关性分析结果显示：NO3-和TIN与AOA和AOB群落结构的变化有相关性,NH4+与AOB的群落结构有相关性,但与AOA的群落结构影响不大。AOA和AOB amoA基因拷贝数与环境因子相关性分析,得到,AOB的丰度与土壤中的NO3-和TIN有显著的相关性。二、不同覆膜处理反硝化细菌的变化1、nirK基因文库结果显示不同覆膜处理下nirK基因的多样性较低,631个克隆子共得到10个OTUs;其中nirK-6为优势OTU,占97.7%。2、nirK基因荧光定量PCR的结果显示,在不同覆膜处理下,反硝化细菌nirK基因拷贝数变化不明显,但有明显的季节变化。nirK基因拷贝数与环境因子相关性分析结果显示,反硝化细菌nirK基因的丰度与土壤含水量有显著的相关性。综上所述,不同覆膜处理影响土壤样品中氨氧化微生物的群落结构和丰度；而对反硝化细菌nirK基因的群落结构没有明显地影响,但其丰度具有明显地季节性变化。更多还原

【Abstract】 Nitrogen (N) cycling is a major question in ecology, because the availability of N often limits the primary production of plants in many ecosystems. With the rapid development of culture-independent techniques, microbial ecologists have discovered the there are close relationship between N cycling and microbial communities. The nitrification and denitrification which are driven by ammonia oxidizers and denitrifying bacteria, play important roles in the global nitrogen cycle, and therefore receive widespread interest. Mulch technology, can effectively improve crop production in semi-arid Loess Plateau of China. However, long-term mulch treatment changes water content, temperature, and soil nutrients, especially N concentrations, which may-be direct impacts on the sustainability of production. We assessed abundance of functional genes affiliated with nitrification (amoA) and denitrification(nirK) using clone library and real time PCR. The effects of mulch on the community structure of ammonia oxidizers and denitrifying bacteria were investigated in a4-year mulch experiment site in the Loess Plateau of China. Three treatments were tested, non-mulching (CK), half-mulching (HM) and full-mulching (FM). The plastic film was mulched at sowing and reserved in field till the next sowing time. Some main results were obtained:The community structure of ammonia oxidizers in different mulch treatments1. amoA gene clone librarys showed that there are highly difference on the AOB community structure among different mulch treatments.1) The community structure of AOB consisted of Nitrosomonas-like sequences and Nitrosospira-Like sequences, attached to β-Proteobacteria. All AOB sequences fell within six clusters, Cluster3a.1、Cluster3a.2、Cluster3b、Cluster6、Cluster2-related and Cluster11/12。AOB communities were dominated by representatives of clusters3b and3a.2.2) The communities of AOB shifted in different mulch treatments, from a Nitrosomona-cluster6hardly any in FM soils, and increased in CK soil. Moreover, the percentage of cluster6in CK is significantly higher than mulch treatments (FM and HM).3) The Communities of AOB shifted in different mulch treatments at OTUs levels. AOB-CK1-101was absent in FM soil, and with lower proportion in HM soil. However, it accounted for12.31%in CK soil. AOB-CK-78had highly percengage in FM soil(5.51%). However, it was hardly detected in HM and CK soil smples. 2. There was no significantly change in AOA community structure.1)442AOB sequences fell within soil and sediment1and soil and sediment2, Soil and sediment1accounted for30%-58%in each clone library.2) There was no significant shift at OTUs level in AOA community structure. AOA-FM2-8, AOA-HM1-19and AOA-FM3-16was dominance in AOA community structure.3. Differences were also seen in the relative abundances of archaeal and bacterial amoA genes, bacterial amoA abundance (5.66×107～1.64×108copies/g) was significantly higher than Archaeal amoA abundance in all soils (1.16×107～3.12×107copies/g). Except October, the bacterial amoA gene abundances increased with FM, HM and CK. The abundance of bacterial amoA in June of CK was significant higher than FM. The abundance of archaeal amoA gene was no significant change in different treatments.4. Correlation analyses among community structure and environmental factor demonstrate that NO3-and TIN had a huge influence on the community structure of AOB and AOA, NH4+had an influence on the community struture of AOB instead of AOA. Correlation analyses among the abundance of AOA/AOB and environmental factor showed that there were significantly negative correlations between the abundance of AOB and the concentrations of NO3-and TINThe community structure of nirK gene in different mulch treatment1. nirK gene clone library results showed that the diversity of nirK gene was extremely lower, and only10OTUs was obtained from631clones. The dominate OTU was nirK-6, accountting for97.7%.2. Real time PCR results revealed abundance of denitrifying bacteria nirK gene did not change significantly, but had a significant seasonal variation. Correlation analyses among the abundance of nirK gene and environmental factor showed soil moisture should be the influencing factor.In general, the community structure of ammonia oxidizers changed in different plastic-film mulching methods; but no shift in denitrifying bacteria community structure.更多还原