宋玉翔(1995-),男,安徽定远人,硕士研究生,主要从事土壤微生物研究。E-mail:
氨氧化古菌(Ammonia-oxidizing archaea,AOA)被认为是酸性土壤硝化过程的主要微生物类群,但AOA如何适应酸性胁迫并发挥作用一直是研究难点,而ATP酶(ATPase)是能量代谢的关键,其编码基因可能在AOA适应酸性胁迫过程中发生了趋同性演化。据此,本研究针对5个不同种植年限的马尾松人工林酸性土壤(15 a、24 a、45 a、55 a、63 a),通过深度宏基因组测序获得7 360亿碱基对,重构AOA氨单加氧酶
Ammonia-oxidizing archaea (AOA) is considered the main microbial group catalyzing the nitrification process in acidic soils. However, how AOA adapts to acid stress remains largely uncertain, and we hypothesize that the gene encoding ATPase for microbial energy metabolism may likely play a key role in the adaption of AOA to acidic stress in these soils.
In this study, 736 billion base pairs were obtained by ultra-deep metagenomics sequencing of acidic soils of Masson pine plantations with five different planting years (15 a, 24 a, 45 a, 55 a, 63 a). The phylogenetic congruency between the AOA
The habitat expansion of AOA to the acidic environment cannot be explained by the phylogenetic evolutionary trajectory of canonic
These results indicate that the AOA populations in acid plantation soils under different planting years were clearly differentiated, and the horizontal transfer of V-ATPase gene may be an important mechanism that enables AOA to survive acidic stress for the habitat expansion.
地球上酸性土壤约占无冰陆地面积的30%(pH < 5.5)[
氮素是所有生物生长的重要营养元素,而微生物将氨氧化为亚硝酸盐则是地球氮素循环的关键限速步骤[
ATP是所有生物生长的能量货币,而氨氧化古菌是严格化能无机自养菌,通过氧化氨态氮获得能量,同化无机碳自养生长。然而,值得注意的是,尽管氨单加氧酶
ATPase广泛存在于古菌、细菌和真核生物,主要包括古菌A-ATPase,细菌、线粒体和叶绿体中常见的F-ATPase,以及真核生物V-ATPase等[
据此,本研究利用深度宏基因组测序技术,针对浙江省建德市不同种植年限的马尾松人工林酸性土壤,研究不同林龄马尾松人工林土壤中AOA群落组成及影响AOA生态分化的环境因素,通过对
土壤样品采自浙江省建德市马尾松人工林(29.48°N,119.28°E),土壤类型为凝灰岩发育的红壤。该区为亚热带海洋型季风气候,海拔约200 m,年均气温17.4℃,年均降雨量1 600 mm。选择中龄林(15 a)、近熟林(24 a)、成熟林(45 a)、过熟林Ⅰ(55 a)、过熟林Ⅱ(63 a)的马尾松人工林土壤样品采样。具体设计如下:针对每一种年限,选择3个不同小区作为重复,空间间隔约2 000 m,每个小区由5个土柱样品混匀而成,采样深度为0~20cm,样品分成两份,一份保存于–20℃用于提取土壤总DNA,另一份经风干、磨细、过2 mm筛、混匀后在4℃保存,参照《土壤农化分析》[
土壤微生物组总DNA使用FastDNA Spin Kit for Soil(MP Biomedicals,USA)试剂盒提取,并通过DNeasy PowerClean Pro Cleanup Kit(QIAGEN,Germany)试剂盒纯化获得高质量DNA,具体步骤参考相关操作指南。通过微量紫外分光光度计(Nanodrop ND-1000)测定DNA的浓度和纯度后,保存于–20℃。
宏基因组测序主要通过Illumina Hi Seq 2000平台完成。首先利用自动聚焦声波基因组剪切仪Covaris M220,将土壤总DNA随机打断为约300 bp的片段,然后采用TruSeqTM DNA Sample Prep Kit试剂盒构建PE(Paired-End)文库,最后通过HiSeq 3000/4000 PE Cluster Kit试剂盒进行桥式PCR反应,质量检测合格后进行双末端(Paired-End,PE)测序。5种不同种植年限的森林土壤及其3个生物学重复均开展了深度测序,每个样品约50 G bp原始数据,共计获得约750 G bp高通量数据。
高质量数据分析具体如下:利用软件SeqPrep剪切序列3´端和5´端的adaptor序列,随后删除所有长度小于50 bp的序列reads;通过软件Sickle去除平均质量值低于20以及含N碱基的序列reads,保留高质量的reads,每个样品最终得到约49 G bp高质量数据。
以NCycDB数据库中AOA
根据已有的AOA物种多样性报道[
利用CD-HIT软件对
利用Microsoft Excel 2016对数据进行前处理,IBM SPSS Statistics 20进行差异显著性分析(LSD法,α=0.05)和Spearman分析;利用R软件vegan包开展Mantel分析、ANOISM分析;利用OriginPro 8.0制作箱线图;并利用软件Adobe Illustrator CS5优化调整。
人工种植导致马尾松林土壤性质发生显著变化。如
马尾松人工林土壤基本理化性质
Physico-chemical properties of
龄组 |
含水率 |
pH | 有机碳 |
全氮 |
水解性氮 |
有效磷 |
速效钾 |
注:表中数据为平均值±标准差( |
|||||||
中龄林①(15 a) | 27.6±1.4a | 4.70±0.21a | 17.0±1.8a | 1.44±0.12a | 187±4a | 6.60±1.84b | 69.7±6.7a |
近熟林②(24 a) | 27.0±3.0a | 4.31±0.11b | 14.6±0.7ab | 1.18±0.17ab | 140±23b | 7.52±1.79b | 57.7±4.0b |
成熟林③(45 a) | 15.3±0.9b | 4.09±0.17b | 15.4±3.4ab | 0.95±0.33b | 151±44ab | 8.71±1.03b | 21.7±1.5c |
过熟林Ⅰ④(55 a) | 18.56±1.6b | 4.04±0.15b | 15.0±1.4ab | 0.92±0.14b | 134±10b | 8.25±2.09b | 57.3±2.5b |
过熟林Ⅱ⑤(63 a) | 15.6±2.9b | 4.17±0.11b | 12.4±1.3b | 0.88±0.13b | 117±13b | 17.0±6.14a | 55.7±4.0b |
针对5种土壤3个重复的750 G bp高质量测序数据,通过4种方法重构
系统发育分析
马尾松人工林土壤AOA
Phylogenetic tree and convergence analysis of AOA
然而,ATPase subunit A基因分析则得到完全不同的结果,土壤中优势AOA具有较高的序列亲缘相似度,均可归类为V-ATPase并占所有序列96.3%。采用
这些结果表明,V-ATPase基因可能是古菌AOA适应酸性胁迫并发生趋同进化的分子基础。本研究中所有5种土壤均为酸性(pH < 5.0),而基于
进一步在更精细化水平,研究了不同种植年限下酸性森林土壤AOA
不同注释方法马尾松人工林土壤AOA
The results of different annotation methods for AOA
尽管AOA古菌ATPase subunit A基因与
ATPase subunit A基因分析结果也发现
总体而言,系统发育进化发现酸性土壤中占据绝对优势AOA的ATPase subunit A序列具有高度同源性,这些NT-α和NS-γ-2.3亚类与已知的嗜酸/耐酸AOA相似并可归类为V-ATPase。同时,这些AOA的
在微生物分类学不同水平,如纲(Class)、目(Order)、亚支(Subclade),将不同林龄马尾松人工林土壤AOA分类,计算其相对丰度的变异系数并与土壤理化性质进行相关分析。结果表明,AOA
不同分类水平下AOA
Coefficient of variation of abundance of AOA
Spearman分析不同AOA基因型与土壤理化性质发现,不同环境要素对AOA基因型的影响不同。pH、速效钾与NT-α亚支极显著正相关,但与NS-γ-2.3和NS-ζ亚支显著或极显著负相关(
不同马尾松种植年限下氨氧化古菌AOA类群与土壤理化性质的相关性
Relationship of AOA groups with soil physicochemical properties
AOA | 含水率 |
pH | 有机碳 |
全氮 |
水解性氮 |
有效磷 |
速效钾 |
注:*表示显著相关( |
|||||||
NT-α(Acidophilic) | 0.65** | 0.75** | 0.20 | 0.48 | 0.25 | –0.12 | 0.65** |
NS-γ-2.3(Acid-tolerant) | –0.36 | –0.60* | 0.00 | –0.39 | –0.01 | –0.06 | –0.71** |
NS-ζ(Acid-tolerant) | –0.49 | –0.89** | –0.51 | –0.78** | –0.46 | 0.37 | –0.57* |
NS-δ(Neutrophilic) | 0.41 | 0.28 | 0.14 | 0.23 | –0.05 | –0.05 | –0.02 |
Total |
–0.29 | –0.48 | 0.12 | –0.35 | 0.12 | –0.02 | –0.67** |
已有研究表明,酸性土壤中存在大量尚未可知的嗜酸/耐酸氨氧化古菌,基于经典的16S rRNA基因和氨单加氧酶
通过对马尾松人工林土壤总DNA深度宏基因组测序结果分析,表明V-ATPase基因协助AOA适应酸性胁迫机制可能具有普适性。目前AOA耐酸机制研究主要集中在NT类群,推测其可能通过一些典型机制适应酸性环境[
人工林种植可能促进了酸性AOA的种群分化。通过深度宏基因组测序发现,马尾松人工林酸性土壤中AOA主要是NT和NS-γ-2.3类群,并且随林龄增加,NT类群丰度先减少后增加,而NS-γ-2.3类群丰度先增加后减少,这可能是不同林龄土壤理化性质差异导致的。有研究表明pH是影响AOA群落的重要因子[
针对马尾松人工林酸性土壤总DNA深度宏基因组测序,研究发现通过不同注释策略对
此外,大片度基因拼接也可能产生误差,尤其是AOA的ATPase subunit A序列更长,拼接难度更大。尽管高通量测序导致微生物学、生态学、进化生物学等学科交叉发展态势迅猛[
基于16S rRNA基因和
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