淡水富营养型湖泊沉积物亚硝酸还原酶基因(nirS)的多样性和系统发育

【目的】以亚硝酸盐还原酶基因(nirS)为分子标记,探讨富营养化湖泊武汉东湖沉积物中NirS类反硝化细菌群落的多样性及系统发育,并分析环境因子对群落分布的影响。【方法】在武汉东湖4个典型子湖郭郑湖、汤菱湖、团湖和庙湖采集沉积物样品,测定环境参数;提取沉积物中微生物群落基因组DNA,分别构建4个子湖的反硝化微生物的nirS基因文库,利用限制性片段长度的多态性分析(Restriction Fragment LengthPolymorphism,RFLP)技术初步分群,确定各群的代表菌株并测定其nirS基因序列;利用DOTUR软件计算各群落多样性和丰富度指数,以Neighbor-Joining法构建供试菌与参比菌的系统发育树。【结果】环境参数测定结果表明东湖4个子湖中庙湖沉积物总氮(TN)和氨态氮(NH 4+-N)含量最高,团湖最低,郭郑湖沉积物中NO 3-浓度最高。基于NirS序列的生物多样性和丰富度分析表明团湖生物多样性和丰富度指数最高而庙湖各项指数均较低。各子湖供试序列及其代表序列综合RFLP聚类分析表明,武汉东湖沉积物中NirS类反硝化微生物种群具有丰富的多样性。NJ系统发育分析表明东湖沉积物NirS类反硝化菌群可分成3个较大群体(群I-III)。群I占总群体的67.7%,广泛分布于不同的生态环境;来自郭郑湖代表菌的81%分布于群I,而庙湖的代表菌中65%分布于群II。比较分析发现来自于东湖和人工湿地两种生境的NirS群落间具有较高的相似性。【结论】武汉东湖淡水富营养型湖泊沉积物中亚硝酸还原酶基因(nirS)具有丰富的多样性。东湖沉积物中TN、NH 4+和NO 3-的浓度可能是影响NirS类反硝化微生物多样性和空间分布的重要因素之一。

Phylogeny diversity of the nitrite reductase gene (nirS) in the sediments of the eutrophic East Lake, Wuhan

Abstract: [Objective] The study aims to investigate the phylogeny diversity of the denitrification bacteria communities in the sediments of the eutrophic East Lake, Wuhan based on nitrite reductase gene (nirS) restriction fragment length polymorphism (RFLP) method and sequencing analysis, and to analyse community variation according to the environment parameters. [Methods] We collected the sediment samples from the four typical sub-lake of East Lake in Wuhan, Guozheng Lake, Tangling Lake, Tuan Lake and Miao Lake, and measured the environmental parameters appropriately. After extracted the genomic DNA from the sediment, four nirS gene clone libraries were successfully constructed. The operation taxonomy units (OTUs) were determined by RFLP method and the representative fragment of every OTU was sequenced. The diversity, richness and evenness statistics of the NirS-like communities were calculated by using DOTUR software. Neighbour-joining phylogenetic tree was constructed basing on the amino acid sequences of NirS from the East Lake sediments and reference sequences retrieved from the GenBank database. The relationship between tested NirS communities and the references from different environments was also discussed. [Results] Environmental parameters showed that Miao Lake sediment contains the highest amount of total nitrogen (TN) and NH4+-N, while the Tuan Lake sediments contains the lowest amount. Among the four sub-lakes, Tuan Lake harbours the highest diversity and richness of NirS-like denitrifiers, while the denitrifiers in Miao Lake was the lowest. Phylogenic analyses suggested that sedimentary NirS-like denitrifiers in the East Lake could be distributed into three groups, Group I to III. Group I accounts for 67.7% of all tested communities. Eighty-one percent of sequences from Guozheng Lake were clustered into Group I, while 67.7% of sequences from Miao Lake were clustered into Group II. Comparative analysis of communities from East Lake and artificial wetland found there are phylogenetically related. [Conclusion] There are diverse and abundant NirS-like denitrifiers inhabited in the sediments of East Lake, Wuhan. The diversity indices and spatial distribution of these communities are affected by the content of TN, NH4+ and NO3- nutrients in the sediments.