内蒙古湿地植物根际细菌及氨氧化功能菌群的多样性

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1、密 级： 学校代码：10075 分类号： 学 号： 20091065 理学硕士学位论文 内蒙古湿地植物根际细菌及氨氧化功能 菌群的多样性 内蒙古湿地植物根际细菌及氨氧化功能 菌群的多样性 学位申请人： 刘 莹 指 导 教 师 ： 孙 磊 副教授 学 位 类 别 ： 理学硕士 学 科 专 业 ： 微生物学 授 予 单 位 ： 河北大学 答 辩 日 期 ： 二 一二年六月 Classified Index: CODE: 10075 U.D.C.: NO: 20091065 A Dissertation for the Degree of M. Science Diversity of Plant

2、Rhizosphere Bacteria and Ammonia Oxidizing Microorganism in Inner Mongolia Wetlands Candidate: Liu Ying Supervisor: Associate Prof. Sun Lei Academic Degree Applied for: Master of Science Specialty: Microbiology University: Hebei University Date of Oral Examination: June, 2012 摘 要 摘 要 本文采用分离培养方法对内蒙古河

3、套灌区天然湿地和退化湿地的芦苇根际细菌多 样性进行了分析，采用变性梯度凝胶电泳（DGGE）技术对内蒙古河套灌区人工湿地所 种植的芦苇和香蒲两种植物的根际氨氧化细菌（AOB）和氨氧化古菌（AOA）多样性及 动态变化进行了研究。 利用 R2A 培养基，采用分离培养法从天然湿地和退化湿地的芦苇根际一共分离出 99 株可培养细菌。16S rRNA 基因系统发育分析的结果显示，分离自天然湿地芦苇根际 的 45 株细菌分属于变形菌门（44.44%）、厚壁菌门（42.22%）和放线菌门（13.33%）3 大类群，其中，变形菌门为最优势类群，芽孢杆菌属（42.2%）为最优势菌属；分离自 退化湿地芦苇根际的 4

4、4 株细菌分属变形菌门（20.45%）、厚壁菌门（47.73%）及放线 菌门（31.82%）3 大类群，其中厚壁菌门为最优势类群，芽孢杆菌属（40.9%）为最优 势菌属。 研究结果表明， 天然湿地和退化湿地芦苇根际细菌都具有较丰富的物种多样性， 但天然湿地与退化湿地芦苇根际细菌群落结构存在一定差异。 通过 DGGE 技术对人工湿地芦苇和香蒲两种植物的根际 AOB 的 amoA 基因的多样 性进行了分析。DGGE 图谱中的主要条带的序列分析结果显示，芦苇和香蒲根际土壤样 品中 AOB 群落大多数为未培养细菌，变形菌门 -变形菌纲为优势类群，且这些 AOB 与亚硝化单胞菌属和亚硝化螺菌属的 AOB

5、 同源性相近。DGGE 图谱遗传多样性分析结 果显示， 内蒙古河套灌区人工湿地植物根际土壤的 AOB 群落多样性因不同的植被类型、 不同取样时间和土壤基质的不同存在一定差异。方差分析显示植被类型对 AOB 的多样 性指数影响极显著； 不同取样时间对AOB的多样性指数影响显著； 不同土壤基质对AOB 的多样性指数影响不显著，这可能是由于尽管构成人工湿地的土壤基质不同，但根系周 围的土壤理化特性相似， 从而导致不同样地在同一时间相同植物的根际氨氧化细菌群落 多样性差别不大。 通过 DGGE 技术对人工湿地芦苇和香蒲两种植物的根际 AOA 的 amoA 基因的多样 性进行了分析。DGGE 图谱中的主

6、要条带的序列分析结果显示，芦苇和香蒲根际土壤样 品中 AOA 群落都属于泉古菌门，大多数为未培养古菌，大部分测序菌种与土壤中已经 研究发表的AOA同源性相近， 个别的菌种属于水环境和海洋底泥中的AOA类群。 DGGE 摘 要 II 图谱遗传多样性分析结果显示， 不同的因素对内蒙古河套灌区人工湿地根际土壤的AOA 群落多样性的影响差异与 AOB 分析结果一致，主要受植被类型的影响。 关键词 湿地 根际 氨氧化细菌 氨氧化古菌 变性梯度凝胶电泳 多样性 Abstract III Abstract The diversity of culturable bacteria of reed (Phrag

7、mites australis) rhizosphere from natural wetlands and degraded wetlands in Inner Mongolia Hetao irrigated region was investigated in this study. The rhizosphere ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) community composition and dynamic changes of two plants, reed and cat

8、tail(Typha orientalis Presl), in constructed wetlands of the Inner Mongolia Hetao irrigated region were examined by denaturing gradient gel electrophoresis (DGGE). A total of 99 bacterial strains with different colony characteristics were isolated on R2A medium: 45 isolates from natural wetlands， 44

9、 isolates from degraded wetlands. Bases on 16S rRNA gene sequence analysis, 45 rhizosphere isolates from natural wetlands were grouped into three groups, Proteobacteria (44.44%), Firmicutes (42.22%), and Actinobacteria (13.33%). The dominant group was Proteobacteria. The dominant genus was Bacillus

10、(42.22%) and Pseudomonas (22.2%); 44 rhizosphere isolates from degraded wetlands were belonged to three groups, Proteobacteria (20.45%), Firmicutes (47.73%), and Actinobacteria (31.82%). The dominant group was Firmicutes. And the genus Bacillus (40.91%) was the predominant genus, which included 18 b

11、acterial isolates. The results indicated that the diversity of rhizosphere bacteria from wetlands in Inner Mongolia Hetao irrigated region was abundant and the rhizosphere bacterial communities between natural wetlands and degraded wetlands had differences. Diversity of gene amoA in rhizosphere AOB

12、of the reed and cattail in the constructed wetlands was analyzed by using DGGE. Sequences of excised DGGE bands showed that the major group was uncultured bacteria in rhizosphere AOB community, which similar to the genera Nitrosomonas and Nitrosospira, and the dominat group was Betaproteobacteria. B

13、ased on PCR-DGGE, distinct differences in AOB communities were revealed in relation to vegetation types, sampling time and types of soil. The results of variance analysis showed that the influence of vegetation types was extremely significant; the influence of different sampling time was significant

14、; the influence of soil types was not significant. This may be due to different soil matrix in the constructed wetlands, but the physico-chemical characteristics Abstract IV of soil around the plant roots were similar. So the diversity index of AOB of the same plant in the different sampling sites a

15、t the same sampling time had no evident difference. Diversity of gene amoA in rhizosphere AOA of the reed and cattail in the constructed wetlands was analyzed by using DGGE. Sequences of excised DGGE bands showed that the major group was uncultured archaea in rhizosphere AOA community, and the domin

16、at group was Crenarchaeota. Most of them are close to the soil AOA that had been studied, individual species belonged to AOA groups from the water environment and the Marine sediment. The analysis results of PCR-DGGE were consistent with the AOB analysis results, that distinct differences in AOA communities were mainly affected by vegetation types. Keywords wetland rhizosphere ammonia oxidizin