新疆两典型微咸水湖水体免培养古菌多样性

Archaea diversity in water of two typical brackish lakes in Xinjiang

微咸水湖是湖泊演化过程中的一个重要中间状态,以新疆两典型微咸水湖-赛里木湖和柴窝堡湖水为研究对象,采用微孔滤膜收集菌体,SDS-酚-氯仿抽提法直接提取湖水总DNA,利用古菌16S rRNA基因通用引物进行PCR扩增,分别构建两湖古菌16S rRNA基因克隆文库。用限制性内切酶HaeⅢ对随机挑选的阳性克隆子进行酶切分型,分别得到7个和8个可操作分类单元(Operational Taxonomic Units,OTUs),两文库覆盖率均大于98%。BLAST比对和系统发育分析表明赛里木湖全部克隆子归属于泉古菌门(Crenarchaeota),97%的克隆子与不同环境免培养氨氧化泉古菌有较高的序列相似性(>97%)。柴窝堡湖水古菌归为3个门:Thaumarchaeota(81.2%)、广古菌门(Euryarchaeota)(13%)和泉古菌门(Crenarchaeota)(5.8%),81.2%的克隆子与具有氮代谢功能的氨氧化古菌纯培养物具有较高的序列相似性(97%—98%),13%的克隆子与已分离到的产甲烷古菌序列同源性大于97%。研究发现新疆微咸水湖可能存有大量新划分的古菌Thaumarchaeota门类群、可培养氨氧化及产甲烷古菌类群,两典型微咸水湖泊中古菌类群多样性较低且群落组成差异大。

Brackish lakes are important intermediate links in the evolution of lakes with water salinity between 1 and 35 g/L. The salt content of lacustrine water is gradually increased due to drought, and water salinity in the lakes is accelerating in recent years in northwest arid and semi-arid regions of China. Microbial diversity in freshwater and saline environments has been well studied, however, little is known about the microbial community composition and distribution in brackish lake. Sayram Lake and Chaiwopu Lake in Xinjiang are representative brackish lakes, and belonged to mountain lakes and mountain-basin lakes respectively, with low-salt, weakly alkaline, high altitude and other geological features. These conditions have a profound effect on the ecology of the lake, and may be expected to affect the composition and activity of the microbial community of the lake. In order to investigate the composition and diversity of archaea in Sayram Lake and Chaiwopu Lake water, in August 2010, water samples were collected and transported to the laboratory within 4 h to minimize changes in the microbial population. The water samples were divided into two parts, one was used to physicochemical analyses by physical and chemical test center （Xinjiang Academy of Agricultural Science, China）. The rest of water samples （1000 mL） were pressure filtered through Millipore Sterivex filter cartridges （0.22μm-pore size） to collect microbial biomass for subsequent DNA extraction. Environmental DNA was directly extracted with SDS-phenolchloroform method from the water samples, and 16S rRNA gene was amplified by PCR with archaea-specific primers.Randomly selected positive clones were identified by Restriction Fragment Length Polymorphism （RFLP） with restriction endonuclease Hae 11, and the unique RFLP patterns were used for sequencing, alignment and constructing 16S rRNA gene phylogenetic tree. In total, 72 and 69 positive clones were selected from the libraries of Sayram and Chaiwopu lake-water respectively, 7 and 8 Operational Taxonomic units （OTUs） were obtained. BLAST analysis indicated that all clones from Sayram were affiliated to the phylum Crenarchaeota, the 80TUs from Chaiwopu were classified into 3 phyla： Crenarchaeota （5.8%）, Thaumarchaeota （ 81. 2% ）, Euryarchaeota （ 13% ）. Of these, Thaumarchaeota was the dominant group. Additionally, most clones that derived from the two libraries showed high similarity to ammonia-oxidizing archaea with 97% to 98% of sequence identity. 81.2% of clones from Chaiwopu Lake are grouped into the phylum Thaumarchaeota, which are a newly-proposed phylum of the Archaea, containing so far four species and all of them are chemolithoantotrophic ammonia-oxidizers and may play important roles in biogeochemical cycles. 97% clones of Sayram Lake have high similarity to the uncultured ammonia-oxidizing Crenarchaeota from soil and underground radioactive hot springs. Besides, 13 percent of Chaiwopu Lake clones showed more than 97% of 16S rRNA gene sequences similarity to pure cultured methanogenic strains that isolated from Micro lagoons and lagoon sediments. Coverage C values of the two clone libraries were 99% and 98 % respectively, indicating that most of the estimated archaeal diversity was sampled. Results of this study showed that the archaea diversity of the two lakes was comparatively low and archaea compositions are significantly different, but a large fraction of them might involve in nitrogen metabolism or methanogenesis.