三江湿地与稻田细菌及T4型细菌病毒多样性比较研究
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摘要
为了阐述开发对三江湿地中细菌群落结构以及多样性的影响，同时探索湿地与稻田生态系统中T4型细菌病毒群落特征，本文通过两部分实验分别采用分子生物学技术对三江湿地和稻田生态系统中的细菌以及T4型细菌病毒群落进行了研究。
对三江湿地（毛果、小叶樟）以及开发后的稻田土壤DNA构建16S rRNA基因文库，每个文库中得到100个阳性克隆，将同源性≥97%的序列划分为一个OUT。稻田、毛果湿地、小叶樟湿地文库分别产生了70、75、75个OTUs。构建文库的覆盖率分别为49%，44%和42%，涵盖了样地大约一半的细菌种类。香农指数及物种丰富度指数Schao1表明，湿地土壤中细菌多样性高于稻田。在三个文库300个克隆中发现了变形菌门、厚壁菌门、放线菌门、酸杆菌门、绿湾菌门、疣微菌门、浮霉菌门、拟杆菌门和Armatimonadetes类细菌9个菌门。其中，变形菌门、放线菌门、厚壁菌门、疣微菌门、酸杆菌门、绿湾菌门为三个文库中共同拥有的菌门。湿地开发为稻田后，土壤中变形菌的数量和多样性明显增加。稻田土壤中变形菌占细菌群体的37%，毛果湿地和小叶樟湿地中变形菌分别占17%和24%。此外，稻田土壤中存在α-、β-、γ-、δ-四种变形菌纲，湿地中只存在α-、δ-两种变形菌纲。主成分及群落相似性分析结果表明，三个文库中的细菌群落总体上差异较大，毛果湿地与小叶樟湿地细菌群落组成比较接近，稻田与湿地中细菌群落相似性较低。三江湿地中酸杆菌数量偏低不同于其它湿地生态系统，说明三江湿地中存在着独特的细菌组成。厚壁菌门在稻田以及毛果湿地中数量波动不大，却远高于小叶樟湿地。小叶樟湿地中的疣微菌明显高于稻田和毛果湿地。此外，在稻田和毛果湿地中发现了浮霉菌，小叶樟湿地中未发现；稻田和小叶樟湿地中发现了拟杆菌，毛果湿地中未发现。因此，我们认为植被以及湿地类型（稻田属于人工湿地）会影响土壤中细菌群落结构及多样性。
通过对三江湿地（毛果、小叶樟）以及稻田生态系统中的T4型细菌病毒群落的g23基因序列构建系统进化树并结合UniFrac分析在湿地以及稻田水体中发现了g23基因序列的新组。同时发现，湿地生态系统中的T4型细菌病毒多样性不同于水生生态系统和旱地黑土，而与稻田生态系统中的T4型细菌病毒的多样性相似。UniFrac分析表明，湿地土壤中的T4型细菌病毒明显不同于水体中的T4型细菌病毒。随着时间和季节的变化，T4型细菌病毒的群落结构也有所变化，且水体中T4型细菌病毒群落结构的变化大于土壤中。通过与其它生态系统中的T4型细菌病毒进行比较发现，湿地生态系统中T4型细菌病毒的环境异质性大于湖泊、旱地黑土以及稻田土壤。
To illustrate the effects of the exploitation on bacterial community structure anddiversity in Sanjiang wetlands, and explore the diversity characteristic of T4-type phages inwetlands and paddy ecosystem as well, Molecular biology techniques were used to investigatethe community of bacteria and T4-type phages.
Bacterial16S rRNA gene clone libraries were constructed, and300clones were obtainedfrom three libraries (paddy, Carex lasiocarpa and Deyeuxia platyphylla wetlands) in total.Unique phylotypes were defned as OTUs with≤97%16S rRNA gene sequences similarity,and we obtained70,75,75OTUs from paddy, Carex lasiocarpa and Deyeuxia platyphyllawetlands, respectively. The coverages of clone libraries were49%,44%and42%, whichexplain approximately half of the total bacterial species in the study sites. Shannon index andspecies richness index Schao1show that bacterial diversity in wetlands is higher than that inpaddy. Proteobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Acidobacteria,Planctomycetes, Bacteroidetes, Chloroflexi and Armatimonadetes were identified among the300clones. And Proteobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Acidobacteria,Chloroflexi were the shared bacterial Phylums among the three libraries. Significant increasein the number and diversity of proteobacteria in the soil has been found after the exploitationfrom wetlands to paddy. Composition analysis result shows that the proportion ofProteobacteria is37%,17%and24%in paddy, Carex lasiocarpa and Deyeuxia platyphyllawetlands, respectively. There are4Proteobacteria classes (α-, β-, γ-, δ-)in paddy, while only2classes(α-, δ-) in wetlands. Results of principal component and community similarity analysisindicate that bacterial communities from the three libraries is divergence in general, bacterialcommunities in Carex lasiocarpa wetland is similar to that in Deyeuxia platyphylla wetland,similarity of bacterial communities between paddy and wetlands is relatively low. The lowerproportion of Acidobacteria obtained in this study inferred that the bacterial communities inwetlands in Sanjiang plain were unique and differed with those found in other wetlandsaround the world. We concluded that vegetation and wetland type will affect the diversity ofbacterial communities. The number of Firmicutes didn't fluctuate obviously between paddyand Carex lasiocarpa wetland, however, much higher than that in Deyeuxia platyphyllawetland. And Verrucomicrobia in Deyeuxia platyphylla wetland is much higher than that inpaddy and Carex lasiocarpa wetland. In our study, Planctomycetes was found in paddy andCarex lasiocarpa wetland, which didn't exist in Deyeuxia platyphylla Wetland. We also foundBacteroidetes in paddy and Deyeuxia platyphylla wetland, which didn't exist in Carex lasiocarpa wetland.
Based on the analysis of Phylogenetic tree and UniFrac, We found novol group of g23sequences in waters in the wetlands and the paddy field. The distribution of g23sequences inthe wetlands was distinctly differed with those from aquatic environments and upland blacksoils, but similar with those from the paddy fields. Unifrac analysis shows that the T4-typebacterial viruses between soil and water in wetland ecosystem changed significantly. Greatertemporal change of T4-type phage communities in waters than in soils was discovered in thisstudy. Heterogeneous of T4-type phage communities in the wetlands is much more than thosein lake freshwater, upland black soils and paddy field soils.
对三江湿地（毛果、小叶樟）以及开发后的稻田土壤DNA构建16S rRNA基因文库，每个文库中得到100个阳性克隆，将同源性≥97%的序列划分为一个OUT。稻田、毛果湿地、小叶樟湿地文库分别产生了70、75、75个OTUs。构建文库的覆盖率分别为49%，44%和42%，涵盖了样地大约一半的细菌种类。香农指数及物种丰富度指数Schao1表明，湿地土壤中细菌多样性高于稻田。在三个文库300个克隆中发现了变形菌门、厚壁菌门、放线菌门、酸杆菌门、绿湾菌门、疣微菌门、浮霉菌门、拟杆菌门和Armatimonadetes类细菌9个菌门。其中，变形菌门、放线菌门、厚壁菌门、疣微菌门、酸杆菌门、绿湾菌门为三个文库中共同拥有的菌门。湿地开发为稻田后，土壤中变形菌的数量和多样性明显增加。稻田土壤中变形菌占细菌群体的37%，毛果湿地和小叶樟湿地中变形菌分别占17%和24%。此外，稻田土壤中存在α-、β-、γ-、δ-四种变形菌纲，湿地中只存在α-、δ-两种变形菌纲。主成分及群落相似性分析结果表明，三个文库中的细菌群落总体上差异较大，毛果湿地与小叶樟湿地细菌群落组成比较接近，稻田与湿地中细菌群落相似性较低。三江湿地中酸杆菌数量偏低不同于其它湿地生态系统，说明三江湿地中存在着独特的细菌组成。厚壁菌门在稻田以及毛果湿地中数量波动不大，却远高于小叶樟湿地。小叶樟湿地中的疣微菌明显高于稻田和毛果湿地。此外，在稻田和毛果湿地中发现了浮霉菌，小叶樟湿地中未发现；稻田和小叶樟湿地中发现了拟杆菌，毛果湿地中未发现。因此，我们认为植被以及湿地类型（稻田属于人工湿地）会影响土壤中细菌群落结构及多样性。
通过对三江湿地（毛果、小叶樟）以及稻田生态系统中的T4型细菌病毒群落的g23基因序列构建系统进化树并结合UniFrac分析在湿地以及稻田水体中发现了g23基因序列的新组。同时发现，湿地生态系统中的T4型细菌病毒多样性不同于水生生态系统和旱地黑土，而与稻田生态系统中的T4型细菌病毒的多样性相似。UniFrac分析表明，湿地土壤中的T4型细菌病毒明显不同于水体中的T4型细菌病毒。随着时间和季节的变化，T4型细菌病毒的群落结构也有所变化，且水体中T4型细菌病毒群落结构的变化大于土壤中。通过与其它生态系统中的T4型细菌病毒进行比较发现，湿地生态系统中T4型细菌病毒的环境异质性大于湖泊、旱地黑土以及稻田土壤。
To illustrate the effects of the exploitation on bacterial community structure anddiversity in Sanjiang wetlands, and explore the diversity characteristic of T4-type phages inwetlands and paddy ecosystem as well, Molecular biology techniques were used to investigatethe community of bacteria and T4-type phages.
Bacterial16S rRNA gene clone libraries were constructed, and300clones were obtainedfrom three libraries (paddy, Carex lasiocarpa and Deyeuxia platyphylla wetlands) in total.Unique phylotypes were defned as OTUs with≤97%16S rRNA gene sequences similarity,and we obtained70,75,75OTUs from paddy, Carex lasiocarpa and Deyeuxia platyphyllawetlands, respectively. The coverages of clone libraries were49%,44%and42%, whichexplain approximately half of the total bacterial species in the study sites. Shannon index andspecies richness index Schao1show that bacterial diversity in wetlands is higher than that inpaddy. Proteobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Acidobacteria,Planctomycetes, Bacteroidetes, Chloroflexi and Armatimonadetes were identified among the300clones. And Proteobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Acidobacteria,Chloroflexi were the shared bacterial Phylums among the three libraries. Significant increasein the number and diversity of proteobacteria in the soil has been found after the exploitationfrom wetlands to paddy. Composition analysis result shows that the proportion ofProteobacteria is37%,17%and24%in paddy, Carex lasiocarpa and Deyeuxia platyphyllawetlands, respectively. There are4Proteobacteria classes (α-, β-, γ-, δ-)in paddy, while only2classes(α-, δ-) in wetlands. Results of principal component and community similarity analysisindicate that bacterial communities from the three libraries is divergence in general, bacterialcommunities in Carex lasiocarpa wetland is similar to that in Deyeuxia platyphylla wetland,similarity of bacterial communities between paddy and wetlands is relatively low. The lowerproportion of Acidobacteria obtained in this study inferred that the bacterial communities inwetlands in Sanjiang plain were unique and differed with those found in other wetlandsaround the world. We concluded that vegetation and wetland type will affect the diversity ofbacterial communities. The number of Firmicutes didn't fluctuate obviously between paddyand Carex lasiocarpa wetland, however, much higher than that in Deyeuxia platyphyllawetland. And Verrucomicrobia in Deyeuxia platyphylla wetland is much higher than that inpaddy and Carex lasiocarpa wetland. In our study, Planctomycetes was found in paddy andCarex lasiocarpa wetland, which didn't exist in Deyeuxia platyphylla Wetland. We also foundBacteroidetes in paddy and Deyeuxia platyphylla wetland, which didn't exist in Carex lasiocarpa wetland.
Based on the analysis of Phylogenetic tree and UniFrac, We found novol group of g23sequences in waters in the wetlands and the paddy field. The distribution of g23sequences inthe wetlands was distinctly differed with those from aquatic environments and upland blacksoils, but similar with those from the paddy fields. Unifrac analysis shows that the T4-typebacterial viruses between soil and water in wetland ecosystem changed significantly. Greatertemporal change of T4-type phage communities in waters than in soils was discovered in thisstudy. Heterogeneous of T4-type phage communities in the wetlands is much more than thosein lake freshwater, upland black soils and paddy field soils.
引文
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