利用高通量测序技术研究典型草原根际土壤细菌群落结构及其对氮沉降的响应

There is growing concern about the effects of atmospheric nitrogen (N) deposition, which is one of the most serious environmental issues worldwide, on various ecosystems. Grassland is an ideal model for evaluating the effects of N deposition on terrestrial ecosystems, science it is the biggest terrestrial biome with high sensitiveness to N and frequently subject to N limitation. However, most previous related studies have intrinsic questions to be solved. Firstly, they used to only concentrate on the effects of total quantity of yearly N deposition but neglected that of N deposition regime (the distribution of N deposition over a year). In those studies, the experimental plots simply received a dose of N with only 1 or 2 additions per year to mimic N deposition, while any given natural ecosystems receives N deposition form atmosphere continuously, subsequently, the results from such kind of experiments are questionable. Secondly, a comprehensive understanding of how elevated N affects soil microbial community remains incomplete, especially the specific roles of plant species under N deposition in structuring rhizosphere and bulk soil microbial communities is poorly described. Therefore, we propose to utilize MiSeq sequencing of 16S rDNA gene, a high-throughput sequencing technique, with bioinformatics analysis, to characterize the structure and diversity of soil bacterial communities in rhizosphere and bulk soils in this study. And it will be carried out with an undergoing experiment of the Typical Grassland Nitrogen Deposition Experiment, which has been manipulated since September 2008. The Experiment is located in a Leymus chinensis steppe in Xilin River Basin, Inner Mongolia. For rhizosphere soil collection, we will choose 6 dominant plant species in each plot out of 60 plots treated with different amounts and regimes of N deposition. Our objectives are: 1) to characterize bacterial richness, diversity, and relative abundances of taxa between bulk soil and the plant rhizospheres using high throughput sequencing of bacterial 16S rDNA genes, as well as variations in soil properties and corresponding traits of the dominant plant species, between plots treated with different amounts and regimes of N deposition. 2) to link plant-soil-bacteria and quantify the contributions of plant traits, soil properties and N deposition to shape bacterial communities in rhizosphere and bulk soils. 3) to address the mechanisms responsible for the observed responses of microbial communities in rhizosphere and bulk soil to N deposition and the key driving factors .

大气氮沉降增加是目前全球普遍发生的严重环境问题之一，它对各类生态系统的影响已受到广泛关注。由于氮是草原这一最大陆地生态系统最主要的限制因子，故草原生态系统对氮沉降十分敏感， 使草原成为研究氮沉降效应的理想的模式生态系统。已有的研究首先是大多仅关注总氮沉降量的效应而忽略了沉降格局（氮沉降在一年内的分布），其结果难以准确反映氮沉降在自然界中连续发生而引起的生态效应的现实，其次是根际与非根际土壤微生物群落的信息十分缺乏。本研究拟以内蒙古典型草原为研究对象，应用16S rDNA扩增高通量测序和生物信息学技术，通过研究不同氮沉降量和沉降格局下， 6种优势植物根际与非根际土壤细菌群落结构与多样性的特征与异同，以及相应土壤与植物群落的变化，建立植物-土壤-细菌之间的关系，定量分析植物、土壤和氮沉降对土壤细菌群落结构的贡献， 揭示根际与非根际土壤细菌群落结构与多样性变化对氮沉降的响应机制与关键驱动因素。

由于草原是地球上面积大且常常受氮素限制的陆地生态系统，因此是研究氮沉降效应的理想模式生态系统，但目前对土壤微生物群落与氮沉降关系的理解并不完全，特别对植物在根际与非根际土壤微生物群落结构形成中的作用知之甚少。根际是土壤与植物根系紧密结合的区域，大量的微生物栖居于此，调控着植物的生长、健康和生物地球化学循环过程。分析根际微生物群落组成及其影响因素，对于我们理解植物与微生物的种间关系、生物地球化学循环过程、植物生长和健康以及农业生产具有重要的意义。草原是对氮沉降十分敏感的生态系统，本研究以内蒙古典型草原为研究对象，应用16S rRNA基因扩增高通量测序和生物信息学技术分析，分析了优势禾本科植物羊草（Leymus chinensis）、大针茅（Stipa grandis）、糙隐子草（Cleistogenes squarrosa）、羽茅（Achnatherum sibiricum）、冰草（Agropyron cristatum）、克氏针茅（Stipa krylovii）和冷蒿（Artemisia frigida）等根际土壤原核生物群落结构特征及其对不同氮沉降处理的响应。研究发现根际原核生物优势门的组成与非根际存在一定差异，拟杆菌门、变形菌门、后壁菌门、绿菌门的相对多度在五种植物根际都有不同程度的增加，而泉古菌门、酸杆菌门、放线菌门、浮霉菌门、单芽孢菌门、FBP和硝化螺旋菌门的相对多度在五种植物根际有不同程度的降低。植物根际原核生物群落的alpha多样性（Faith’s PD、Richeness、Shannon和Simpson）一定程度上低于非根际，但大部分差异不显著。对原核生物群落进行基于Bray-Curtis距离矩阵的ward聚类分析，基于Unifrac距离矩阵的主坐标分析以及anosim分析均表明，根际与非根际土壤原核生物群落结构存在显著差异，具有根际效应；不同植物之间根际原核生物群落没有明显差别，不具有物种特异性效应。群落系统发育结构显示，根际与非根际原核生物群落的净种间亲缘关系指数（NTI）均显著大于0，表明根际与非根际原核生物群落的构建过程均受到较强的确定性过程影响。比较不同氮沉降处理的数据，发现氮沉降主要是通过改变根际与非根际优势菌门的比例而影响土壤微生物群落，而根际微生物的变化反过来影响着植物多样性。